Triple targeting 10B4C nanosheet as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines and nano materials, in particular to a triple targeting 10B4C nanosheet as well as a preparation method and application thereof. According to the technical scheme, the triple targeting 10B4C nanosheet comprises a 10B4C nanosheet and a targeting peptide modified on the surface of the 10B4C nanosheet, the targeting peptide comprises an NGR peptide, an NLS peptide and a PD-L1 targeting peptide. The preparation method comprises the following steps: carrying out ball milling treatment on 10B4C powder to obtain 10B4C nanosheets; the preparation method comprises the following steps: carrying out surface functionalization on a 10B4C nanosheet by using glycidol to obtain a 10B4C-PG nanosheet; the 10B4C-PG nanosheet, an NGR peptide, an NLS peptide and a PD-L1 targeting peptide are subjected to a coupling reaction in the presence of a coupling agent, and the triple targeting 10B4C nanosheet is obtained. The main purpose of the invention is to provide a new choice for the BNCT boron delivery agent.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biomedicine and nanomaterial technology, and particularly relates to a triple-targeting 10 B4C nanoplatelets, a preparation method therefor, and an application thereof. BACKGROUND

[0002] Boron Neutron Capture Therapy (BNCT) is a promising binary targeting radiotherapy strategy. Its working principle is based on the nuclear fission of 10 B after capturing thermal neutrons, producing high linear energy transfer (LET) α particles and 7 Li recoil nuclei. The range of these particles in biological tissues is extremely short (about 5-9 μm), comparable to the diameter of a single cell, so they can accurately kill cells that undergo neutron capture reactions, while causing minimal damage to surrounding normal tissues.

[0003] However, the realization of the therapeutic effect of BNCT highly depends on the efficient and specific enrichment of boron drugs at tumor sites. An ideal BNCT boron delivery agent should meet the following conditions: (1) the concentration of 10 B in tumor tissues should not be less than 20 μg / g; (2) the concentration ratio of 10 B in tumor and normal tissues (T / N) and tumor and blood (T / B) should both be greater than 3, so as to ensure the selectivity and safety of treatment. Early BNCT clinical studies have used boric acid and its simple derivatives, but due to the inherent defects of such small molecule drugs, such as poor targeting, rapid clearance in vivo, and greater toxicity to normal tissues, their clinical application has been severely limited.

[0004] BPA is mainly enriched in some types of tumors through the amino acid transporter LAT1, but still has limited targeting ability and uneven intratumoral distribution; BSH is mainly distributed in blood vessels and is difficult to effectively penetrate the cell membrane into tumor cells, resulting in a generally low T / B ratio, limiting its application in the treatment of most solid tumors. Therefore, developing new and efficient boron delivery systems to achieve high-concentration and specific accumulation of 10 B in tumor cells is still a core challenge to further develop BNCT.

[0005] On the other hand, tumor immunotherapy, especially immune checkpoint blockade therapy targeting programmed death-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 the tumor microenvironment on T cells. However, its clinical application still faces problems such as limited response rate and easy drug resistance.

[0006] In view of the limitations of single therapy, combination therapy has become an important development direction of tumor treatment, but the existing BNCT boron delivery agents are mainly single-targeted or single-treatment functional, and lack of combination therapy type boron delivery system integrating multiple targeting and multimodal therapy. SUMMARY

[0007] In view of the problems of the prior art, the present application provides a triple-targeting 10 B4C nanosheets to solve the problem of poor targeting of existing BNCT boron delivery agents, and can also solve the problem of greater toxicity of existing BNCT boron delivery agents to normal tissues, and can also solve the problem of single targeting of existing BNCT boron delivery agents, and can also solve the problem that existing BNCT boron delivery agents cannot achieve multimodal synergistic therapy.

[0008] The technical solutions of the present application are as follows: The present application provides a triple-targeting 10 B4C nanosheets, comprising 10 B4C nanosheets and a targeting peptide modified on the surface of the 10 B4C nanosheets; The targeting peptide comprises an NGR peptide, an NLS peptide and a PD-L1 targeting peptide.

[0009] Through the synergistic effect of the NGR peptide (targeting tumor blood vessels) and the NLS peptide (targeting the cell nucleus), the 10 B4C nanosheets achieve precise delivery to the tumor cell nucleus, solving the problem of poor targeting of existing BNCT boron delivery agents. This design is expected to significantly improve the enrichment concentration of 10 B near the tumor cell nucleus, thereby meeting the high requirements of BNCT on 10 B concentration and T / N, T / B ratio. BNCT, immune checkpoint blockade therapy (achieved by the PD-L1 targeting peptide) and photothermal therapy (based on the photothermal properties of the 10 B4C nanosheets themselves) are integrated into one. Its synergistic mechanism is that the high-LET particles produced by BNCT can accurately kill tumor cells; the PD-L1 targeting peptide can block the immune checkpoint pathway and reverse immune suppression; at the same time, the photothermal effect of the nanosheets under near-infrared laser irradiation can directly ablate the tumor. The three work together, and are expected to achieve the organic combination of physical killing and immune activation. The triple-targeting 10 B4C nanosheets are expected to 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.

[0010] 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.

[0011] Furthermore, the triple targeting 10 The average hydrated particle size of B4C nanosheets is 194 nm.

[0012] 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); The glycidol functionalized 10 Surface modification of the target peptide with B4C nanosheets yields triple-targeted peptides. 10 B4C nanosheets.

[0013] 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.

[0014] This invention also provides the above-mentioned triple targeting 10 The method for preparing 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 10The B4C-PG nanosheet, the NGR peptide, the NLS peptide and the PD-L1 targeting peptide are coupled in the presence of a coupling agent to obtain the triple-targeting 10 The B4C nanosheet.

[0015] The preparation method mainly includes three steps of ball milling treatment, surface functionalization and coupling reaction, has the advantages of simple preparation process without complicated operation process. 10 The B4C powder is prepared into 10 The B4C nanosheet is beneficial to 10 The surface functionalization modification of the B4C nanosheet and the coupling with other substances. The NGR peptide, the NLS peptide and the PD-L1 targeting peptide are integrated on 10 The surface of the B4C-PG nanosheet, and finally the triple-targeting 10 The B4C nanosheet has the triple-targeting function, can accurately recognize and enrich tumor cells, improves the treatment effect, reduces the damage to normal tissues, and solves the problem of the large toxicity of the existing BNCT boron delivery agent to normal tissues.

[0016] Further, the S1 comprises: 10 The B4C powder is added into ultrapure water, ball milled, first centrifuged, the supernatant is collected, filtered to obtain a filtrate, first dried to obtain 10 The B4C nanosheet; Further, the S2 comprises: 10 The B4C nanosheet is added with propylene glycol, ultrasonically dispersed in a water bath, and then functionalized, cooled to room temperature, added with ultrapure water, ultrasonically dispersed, second centrifuged, the supernatant is collected, which is the crude product of the propylene glycol functionalized 10 The B4C nanosheet, the crude product is dispersed, third centrifuged, the precipitate is collected, washed, and the product is obtained, second dried to obtain 10 The B4C-PG nanosheet.

[0017] Further, the S3 comprises: 10 The B4C-PG nanosheet, the NGR peptide, the NLS peptide, the PD-L1 targeting peptide, the coupling agent and the catalyst are dissolved in a reaction solvent to perform a coupling reaction.

[0018] After the coupling reaction is completed, fourth centrifugation is performed, the precipitate is collected, resuspended, repeated for 5 times, the product is collected, third dried to obtain the triple-targeting 10 The B4C nanosheet.

[0019] The preparation method has the advantages of simple preparation process, mild conditions, reliable purification and the like, and in the presence of a coupling agent, a coupling reaction is carried out in a combined reaction solvent, so that NGR peptide (targeting tumor blood vessels), NLS peptide (targeting the nucleus) and PD-L1 targeting peptide (immune checkpoint blockade therapy) are integrated on the B4C-PG nanosheet in a high-efficiency manner 10 The B4C-PG nanosheet surface is successfully modified with a targeting peptide 10 The B4C nanosheet (triple targeting 10 The B4C nanosheet), avoids a complex process of multi-step modification, and significantly improves the preparation efficiency. Moreover, the entire reaction is carried out under mild solution conditions, effectively protecting the structure of the nanosheet and the biological activity of each peptide segment. In combination with a repeated centrifugation-resuspension washing strategy, unreacted peptides, coupling agents, catalysts and by-products are completely removed, ensuring high purity of the final product.

[0020] Further, in the S1, the planetary ball mill is used for ball milling, the rotation speed is 400-600 r / min, and the time is 20-28 h; the first centrifugation is performed at a rotation speed of 3000 r / min for 10 min; the 200-250 nm microporous filter membrane is used for filtration; and the first drying is freeze drying. Further, in the S2, the functionalization reaction is performed at 120-160 ℃ in an oil bath for 20-48 h; the second centrifugation is performed at a rotation speed of 3000 r / min for 30 min; the dispersion solvent is ultrapure water; the third centrifugation is performed at a rotation speed of 12000-16000 r / min for 2 h; and the second drying is freeze drying.

[0021] Further, in the S3, the coupling reaction is performed under inert conditions, the temperature of the coupling reaction is 35 ℃-40 ℃, and the time is 20 h-28 h; the fourth centrifugation is performed at a rotation speed of 8000 rpm for 15 min, and the molecular cut-off of the ultrafiltration centrifuge tube used is 100 kDa; the resuspension solvent is ultrapure water; and the third drying is freeze drying.

[0022] Preferably, in the S1, the agate beads are used as grinding media, the planetary ball mill is used for ball milling, the rotation speed is 500 r / min, and the time is 24 h; and the 220 nm microporous filter membrane is used for filtration.

[0023] Preferably, in the S2, the functionalization reaction is performed at 140 ℃ in an oil bath for 24 h; and the third centrifugation is performed at a rotation speed of 14000 r / min for 2 h.

[0024] Preferably, in the S3, the inert condition is nitrogen, and the coupling reaction is specifically constant temperature oscillation reaction at 37℃ for 24 hours.

[0025] Further, in the S1, the 10 The dosage ratio of B4C powder and ultrapure water is 3 g:10 mL. Further, in the S2, the 10 The dosage ratio of B4C nanosheet and propylene glycol is 0.1 g:20 mL.

[0026] Further, in the S3, the 10 The dosage ratio of B4C-PG nanosheet, 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.

[0027] Further, the coupling agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride or N,N'-dicyclohexyl carbodiimide; the catalyst is 4-dimethylamino pyridine or 1-hydroxybenzotriazole; and the reaction solvent is anhydrous N,N-dimethylformamide.

[0028] The application also provides the above-mentioned triple-targeting 10 The B4C nanosheet or the triple-targeting 10 The application also provides the use of the B4C nanosheet in the preparation of a boron drug.

[0029] Further, the boron drug is used for boron neutron capture therapy, immune checkpoint blockade or photothermal therapy.

[0030] Further, the preparation method of the boron drug comprises the following steps: mixing the triple-targeting 10 The B4C nanosheet with a pharmaceutically acceptable carrier to prepare a preparation form suitable for tumor administration.

[0031] Further, the boron drug is used for treating tumors, and the treatment comprises at least one of boron neutron capture therapy (BNCT), immunotherapy and photothermal therapy (PTT).

[0032] Further, the boron drug is used for preparing a drug for combined treatment of tumors, and the combined treatment comprises at least two of boron neutron capture therapy, immunotherapy and photothermal therapy.

[0033] Further, when the photothermal therapy is performed, 808 nm near-infrared laser irradiation is combined.

[0034] Further, the tumor is liver cancer, lymphoma or head and neck tumor.

[0035] Furthermore, the drug is administered via intravenous injection or intratumoral injection.

[0036] When boron drugs are used to treat tumors, they can induce immunogenic cell death and activate anti-tumor immune responses.

[0037] 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.

[0038] The beneficial effects of this invention are: 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... 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The triple targeting provided by this invention 10The B4C nanoplate provides a brand-new multi-modal synergistic treatment strategy for tumor treatment, which can effectively inhibit primary tumors and induce systemic anti-tumor immune response and inhibit tumor metastasis and recurrence through synergistic effect of at least two of boron neutron capture therapy (BNCT), immunotherapy and photothermal therapy (PTT), and has important popularization and application value and clinical transformation potential in the field of tumor treatment technology. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 For 10 Hydrated particle size distribution diagram of B4C nanoplate; Figure 2 For triple targeting 10 Hydrated particle size distribution diagram of B4C nanoplate; Figure 3 For 10 B4C nanoplate and triple targeting 10 Comparison diagram of zeta potential of B4C nanoplate; Figure 4 For triple targeting 10 Transmission electron microscope morphology diagram of B4C nanoplate; Figure 5 For 10 B4C nanoplate and triple targeting 10 X-ray photoelectron spectroscopy fine spectrum analysis diagram of B4C nanoplate; Figure 6 For 10 B4C nanoplate and triple targeting 10 Comparison diagram of infrared spectrum of B4C nanoplate; Figure 7 For triple targeting 10 Hydrated particle size of B4C nanoplate in phosphate buffer solution changes with time diagram for 1-7 days; Figure 8 For triple targeting 10 Photothermal heating curve diagram of B4C nanoplate in phosphate buffer solution, wherein the concentration of triple targeting 10 B4C nanoplate in phosphate buffer solution is 0 mg / mL, 2 mg / mL and 5 mg / mL; Figure 9 For triple targeting 10 Photothermal cycle stability diagram of B4C nanoplate in phosphate buffer solution; Figure 10 For triple targeting 10 B4C nanoplate in phosphate buffer solution infrared thermal imaging diagram of different concentrations; Figure 11 For triple targeting 10 Hemolysis experiment result diagram of B4C nanoplate of different concentrations; Figure 12 The hemolysis rate results chart of the experimental group, PBS group and H2O group; Figure 13 The 0.1 M calcium chloride aqueous solution, PBS aqueous solution and different concentrations of triple-targeting 10 The coagulation effect comparison chart of B4C nanosheets at different time points; Figure 14 The Scc25 cell treated with different concentrations of triple-targeting 10 The cell survival rate chart of Scc25 cells treated with B4C nanosheets for 24 h, 48 h and 72 h; Figure 15 The Scc25 cell treated with different concentrations of triple-targeting 10 The live and dead staining fluorescence chart of Scc25 cells treated with B4C nanosheets for 48 h; Figure 16 The NC group, NCT group, PTT group, BNCT+ 10 B4C group and BNCT+PTT+ 10 The cell survival rate chart of cells treated with B4C nanosheets for 24 h, 48 h and 72 h; Figure 17 The NC group, NCT group, PTT group, BNCT+ 10 B4C group and BNCT+PTT+ 10 The live and dead staining fluorescence chart of cells treated with B4C nanosheets; Figure 18 The NC group, NCT group, PTT group, BNCT+ 10 B4C group and BNCT+PTT+ 10 The apoptosis chart of cells treated with B4C nanosheets; Figure 19 The NC group, NCT group, PTT group, BNCT+ 10 B4C group and BNCT+PTT+ 10 The cell clone formation chart of cells treated with B4C nanosheets. DETAILED DESCRIPTION

[0044] The application will be further described in the following examples without limiting the application in any way. All other examples that can be derived by those skilled in the art without creative work based on the specific examples in the present application shall fall within the scope of the present application.

[0045] The specific techniques or conditions not specified in the following examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0046] The reagents or instruments used in the following examples are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0047] The Chinese name of NGR peptide is asparagine-glycine-arginine peptide, the Chinese name of NLS peptide is nuclear localization signal peptide, and the Chinese name of PD-L1 targeting peptide is PD-L1 programmed death ligand-1 targeting peptide.

[0048] Example 1 Triple targeting 10 Preparation of B4C nanosheets Triple targeting 10 The preparation method of B4C nanosheets is as follows: (1) 10 Preparation of B4C nanosheets: Weigh 3.0 g 10 B4C powder is dispersed in 10 mL ultrapure water, a number of maroon beads with a diameter of about 2 mm are added, and they are transferred to a 50 mL maroon ball mill tank together, and the filling degree is controlled to be about 50%. Then the maroon ball mill tank is fixed in the planetary ball mill, and the rotation speed is 500 r / min. Ball mill for 24 h. The dispersion liquid after ball milling is centrifuged at 3000 r / min for 10 min, the supernatant is collected, and is filtered through a 220 nm microporous filter membrane. The obtained filtrate is freeze-dried to obtain 10 B4C nanosheets.

[0049] (2) 10 Preparation of B4C-PG nanosheets: In a 50 mL round-bottom flask, 0.1 g of 10 B4C nanosheets prepared in step (1) and 20 mL of glycidol (epoxypropanol) are uniformly dispersed by water bath ultrasonic dispersion, and then reacted in an oil bath at 140°C for 24 h. After the reaction is completed, the system is cooled to room temperature, 20 mL of ultrapure water is added, and ultrasonic dispersion is performed again. The dispersion liquid is centrifuged at 3000 r / min for 30 min, and the supernatant, i.e. the crude product of epoxypropanol functionalized 10 B4C nanosheets, is collected. The obtained crude product is redispersed in ultrapure water, and centrifuged at 14000 r / min for 2 h. The supernatant is discarded, and the precipitate is collected and washed for 3 times to completely remove free glycerol. Finally, the product is freeze-dried to obtain 10 B4C-PG nanosheets, which are stored at 4°C for standby.

[0050] (3) Triple targeting 10 Preparation of B4C nanosheets: Weigh 100 mg of 10B4C-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.

[0051] Example 2 Triple Targeting 10 Preparation of B4C nanosheets Triple targeting 10 The specific steps for preparing B4C nanosheets are as follows: (1) 10 Preparation of B4C nanosheets: 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.

[0052] (2) 10 Preparation of B4C-PG nanosheets: 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. 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.

[0053] (3) Triple targeting 10 Preparation of B4C nanosheets: 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.

[0054] Example 3 Triple Targeting 10 Preparation of B4C nanosheets Triple targeting 10 The specific steps for preparing B4C nanosheets are as follows: (1) 10 Preparation of B4C nanosheets: 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.

[0055] (2) 10Preparation of B4C-PG nanosheets: In a 50 mL round-bottom flask, 0.1 g of B4C nanosheets prepared in step (1) was dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF) and 0.1 g of glycerol was added into the flask. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycerol-functionalized B4C nanosheets were obtained. 10 B4C nanosheets were dispersed in 20 mL of glycidol (3-oxirane propanol) and ultrasonically dispersed for 10 min. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycidol-functionalized B4C nanosheets were obtained. 10 B4C nanosheets were dispersed in 20 mL of glycidol (3-oxirane propanol) and ultrasonically dispersed for 10 min. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycidol-functionalized B4C nanosheets were obtained. 10 B4C-PG nanosheets were prepared by the method described in step (2). The product was stored at 4 °C for later use.

[0056] (3) Triple-targeting B4C nanosheets 10 Preparation of B4C-PG nanosheets: In a 50 mL round-bottom flask, 0.1 g of B4C nanosheets prepared in step (1) was dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF) and 0.1 g of glycerol was added into the flask. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycerol-functionalized B4C nanosheets were obtained. 10 In a 50 mL round-bottom flask, 0.1 g of B4C nanosheets prepared in step (1) was dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF) and 0.1 g of glycerol was added into the flask. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycerol-functionalized B4C nanosheets were obtained. 10 In a 50 mL round-bottom flask, 0.1 g of B4C nanosheets prepared in step (1) was dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF) and 0.1 g of glycerol was added into the flask. The mixture was stirred at 140 °C for 24 h. After the reaction, the mixture was cooled to room temperature and 20 mL of ultrapure water was added into the flask. The mixture was ultrasonically dispersed for 10 min and then centrifuged at 3000 r / min for 30 min. The supernatant was collected and the glycerol-functionalized B4C nanosheets were obtained.

[0057] Detection analysis The triple-targeting B4C nanosheets prepared in Example 1 were subjected to detection analysis. 10 The triple-targeting B4C nanosheets prepared in Example 1 were subjected to detection analysis.

[0058] 1. Systematic characterization of triple-targeting B4C nanosheets 10 1. Systematic characterization of triple-targeting B4C nanosheets (1) Particle size and zeta potential: The raw material B4C nanosheets were measured by dynamic light scattering method. 10B4C 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.

[0059] (2) Morphology and structure: 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.

[0060] (3) Stability and photothermal properties: 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.

[0061] The photothermal performance test results are as follows: The triple target prepared in Example 1 10B4C 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.

[0062] To evaluate photothermal stability, the triple-targeted [material] prepared in Example 1 was [used]. 10 B4C 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.

[0063] 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 PBS at 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.

[0064] 2. The triple-targeted [treatment] prepared in Example 1 10 Biocompatibility evaluation of B4C nanosheets The triple target prepared in Example 1 10B4C nanoplatelets were dispersed in PBS solution to prepare different concentrations of triple targeting 10 B4C nanoplatelet solution (0-1000 μg / mL).

[0065] (1) Hemolysis experiment: Experimental group: Fresh red blood cells were incubated with different concentrations (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL) of triple targeting 10 B4C nanoplatelets for 2 h; 3 replicates were set for each concentration.

[0066] Negative control group (PBS group): Fresh red blood cells were incubated with PBS buffer for 2 h, 3 replicates were set, and the background hemolysis under physiological conditions was established.

[0067] Positive control group (H2O group): Fresh red blood cells were incubated with ultrapure water for 2 h, 3 replicates were set, and complete rupture of red blood cells was induced to determine the 100% hemolysis value.

[0068] The results of the hemolysis experiment of the experimental group, PBS group and H2O group are shown in Figure 11 As can be seen from Figure 11 , the appearance of the supernatant of all concentrations of the experimental group has no significant difference from the PBS group, and is clear and transparent with a small amount of red blood cell sediment at the bottom, indicating that the triple targeting 10 B4C nanoplatelets have good blood compatibility and biological safety.

[0069] The absorbance (OD) values of the supernatant of the experimental group, PBS group and H2O group in the hemolysis experiment were determined, and the hemolysis rate was calculated according to the formula (i): Hemolysis rate = [(OD experimental - OD negative control) / (average value of OD positive control - average value of OD negative control)] x 100% (i) OD experimental is the single-hole absorbance value of the supernatant of the experimental group, H2O group or PBS group (the single-hole absorbance value of the experimental group is used as OD experimental to calculate the hemolysis rate of the experimental group, the single-hole absorbance value of the H2O group is used as OD experimental to calculate the hemolysis rate of the H2O group, and the single-hole absorbance value of the PBS group is used as OD experimental to calculate the hemolysis rate of the PBS group); OD negative control is the absorbance value of the supernatant of the PBS group; OD positive control is the absorbance value of the supernatant of the H2O group; the average value of OD positive control is the average value of the three replicate absorbance values of the supernatant of the H2O group, and the average value of OD negative control is the average value of the three replicate absorbance values of the supernatant of the PBS group.

[0070] The hemolysis rate results of the experimental group, PBS group and H2O group are shown in Figure 12All the triple-targeting 10 B4C nanosheets showed a hemolysis rate below 1% (see Table 2) Figure 12 , which met the biological safety requirements of medical materials, indicating that the triple-targeting 10 B4C nanosheets had no significant hemolytic toxicity.

[0071] (2) Coagulation experiment: Fresh rabbit blood was taken and anticoagulated with 3.2% sodium citrate. After centrifugation, platelet-poor plasma (PPP) was obtained. 0.5 mL of triple-targeting 10 B4C nanosheet solution at different concentrations (0-1000 μg / mL) was mixed with an equal volume of PPP as the sample of the experimental group; 0.5 mL of PBS aqueous solution mixed with an equal volume of PPP was used as the sample of the negative control group, and 0.5 mL of 0.1 M calcium chloride aqueous solution mixed with an equal volume of PPP was used as the sample of the positive control group. All samples were incubated at 37°C for different times (5 min, 15 min, 30 min and 60 min), and the formation and relative size of the blood clots in each group at different times were observed and recorded by gentle rinsing. The results are shown in Figure 13 Compared with the negative control group (PBS aqueous solution), each concentration of nanosheet group did not promote the formation of obvious blood clots; compared with the significant blood clots induced by the positive control group (0.1 M calcium chloride), the nanosheet group also did not show similar pro-coagulation effect. The results showed that the triple-targeting 10 B4C nanosheets did not cause significant activation of the coagulation system within the test range, and had good hemocompatibility.

[0072] Cytotoxicity: ① CCK-8 method for detecting cell survival rate: Scc25 cells were seeded in a 96-well plate at a density of 1000 cells per well, and fresh culture medium containing different concentrations (0-1000 μg / mL) of triple-targeting 10 B4C nanosheets was added, with 5 replicate wells for each concentration, and culture medium without cells was set as a blank control group, also with 5 replicate wells. After 24 h, 48 h and 72 h of continuous culture, the 96-well plate was taken out at 24 h, 48 h and 72 h, and 10 μL of CCK-8 solution was added to each well, which was incubated in the incubator for 2 h in the dark. Then the absorbance value (OD value) of each well at 450 nm wavelength was measured using a microplate reader. The cell survival rate was calculated according to the formula (ii) as follows: Cell survival rate=[ (ODexperimental - ODblank) / (ODcontrol average - ODblank average)]x 100% (ii) ODexperimentalis the OD value of the triple-targeting10 Single well absorbance value of B4C nanoplate treatment group; OD control is the absorbance value of single well of no triple targeting 10 Single well absorbance value of B4C nanoplate cell group; OD blank is the absorbance value of single well of blank control group, the average value of OD control is the absorbance value of single well of no triple targeting 10 The average value of 5 replicate well absorbance value of B4C nanoplate cell group, OD blank is the average value of 5 replicate well absorbance value of blank control group.

[0073] Results as shown in Figure 14 different concentrations (0~1000 μg / mL) of triple targeting 10 B4C nanoplate, the cell survival rate was maintained above 90% after 24 h, 48 h and 72 h of culture, indicating that triple targeting 10 B4C nanoplate had no obvious toxicity to Scc25 cells in the tested concentration range, and had good biocompatibility.

[0074] ②Live and dead cell staining: Scc25 cells were seeded in 6-well plates at a density of 1×10 5 cells per well, and fresh culture medium containing different concentrations (0~1000 μg / mL) of triple targeting 10 B4C nanoplate was added. After 48 h of incubation in an incubator, the culture medium was discarded, and the cells were gently washed with PBS twice. Then, an appropriate amount of 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 incubation was performed at 37 °C in the dark for 30 min. After incubation, the residual staining solution was removed by washing with PBS again. Immediately, an inverted fluorescence microscope was used for observation and image acquisition, wherein Calcein-AM-labeled live cells showed green fluorescence, and PI-labeled dead cells showed red fluorescence. Finally, the green and red fluorescence channel images under the same field of view were superimposed to obtain a Merge image, which directly showed the spatial distribution of live and dead cells.

[0075] Results as shown in Figure 15 different concentrations (0~1000 μg / mL) of triple targeting 10 B4C nanoplate, the Scc25 cells showed uniform green fluorescence, and only a small amount of red fluorescence was observed. The above results showed that the triple targeting 10 B4C nanoplate had no obvious toxicity to Scc25 cells in the tested concentration range, and had good biocompatibility.

[0076] 3. Using Scc25 cells of oral squamous cell carcinoma as a model, the in vitro anti-tumor effect of triple targeting 10 B4C nanoplate was evaluated 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.

[0077] (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.

[0078] 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. 10B4C nanosheet solution, the NC group and the NCT group were added to PBS medium, 5 duplicate wells were set for each group, and medium without cells was set as a blank control, and incubation was continued for 4-6 h to ensure triple targeting 10 B4C nanosheets were fully taken up by cells. After incubation, the NCT group and the BNCT+ 10 B4C group were irradiated with neutrons at a dose of 2600 mu; the PTT group was irradiated with 808 nm near-infrared laser at a power density of 1.5 W / cm 2 The BNCT+PTT+ 10 B4C group was first irradiated with 808 nm laser at a power density of 1.5 W / cm 2 for 10 min, and then irradiated with neutrons at a dose of 2600 mu.

[0079] (2) Evaluation of in vitro anti-tumor effect: The CCK-8 method was used to detect cell survival rate to evaluate the killing effect of triple targeting 10 B4C nanosheets on Scc25 cells.

[0080] Specific operation steps: After the cells in each group treated in step (1) were placed in an incubator and continued to be cultured for 24 h, 48 h and 72 h, respectively, the 96-well plate was taken out at 24 h, 48 h and 72 h, respectively, and the original culture medium was discarded. Then 10 μL of CCK-8 solution was added to each well, and incubation was carried out in the incubator for 2 h in the dark. The absorbance value (OD value) of each well at 450 nm wavelength was measured using an enzyme marker. The cell survival rate was calculated according to the formula as shown in formula (iii): Cell survival rate = [(OD experiment - OD blank) / (average value of OD control - average value of OD blank)] x 100% (iii) OD experiment is the single-well absorbance value of each experimental treatment group (i.e. NCT, PTT, BNCT+ 10 B4C, BNCT+PTT+ 10 B4C group), when calculating the cell survival rate of the NC group, OD experiment is the single-well absorbance value of the NC group; OD control is the single-well absorbance value of the cell group without drug (NC group); OD blank is the single-well absorbance value of the medium containing only medium (blank control) without cells; the average value of OD control is the average value of the absorbance values of the 5 duplicate wells of the cell group without drug (NC group); the average value of OD blank is the average value of the absorbance values of the 5 duplicate wells containing only medium (blank control) without cells.

[0081] The results are shown in Table 1. Figure 16 Compared with the NC group, the 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.

[0082] (3) Live / dead staining and apoptosis analysis: ① Staining of live and dead cells: Cell viability was detected using a Calcein-AM / PI double staining kit.

[0083] 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.

[0084] 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.

[0085] ② Apoptosis analysis: Apoptosis was detected by flow cytometry.

[0086] 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+ 10 The 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.

[0087] (4) Cloning experiment: To evaluate the triple-targeting 10 The effect of B4C nanoplate on the long-term proliferation ability of Scc25 cells was verified by colony formation experiment.

[0088] Scc25 cells were divided into two groups: control group (NC group, NCT group, PTT group) and experimental group (BNCT 10 B4C group, BNCT+PTT 10 B4C group). Brief steps are as follows: the cells of each group treated in step (1) were placed in an incubator, and the cells of each group were cultured under conventional conditions for a certain period of time (usually 7-14 days), during which the culture medium was replaced regularly to maintain nutrient supply. When visible cell colonies appeared in the control group, the culture was terminated, the culture medium was discarded, and PBS was used for gentle rinsing twice. 4% paraformaldehyde solution was added to each well for fixation for 15 minutes, and after the fixation solution was discarded, 0.1% crystal violet solution was used for staining for 20 minutes. Excess staining solution was removed by slowly washing with running water, and after air-drying at room temperature, the formed cell colonies were photographed and recorded, and the number of colonies was counted using ImageJ software. The results are shown in Figure 19 , which show that the number of colonies in the experimental group is significantly reduced compared with the control group, indicating that the nanoplate can effectively inhibit the long-term proliferation and colony formation ability of Scc25 cells.

[0089] The above experimental results (see Figure 16 , Figure 17 , Figure 18 and Figure 19 ) collectively show that the triple-targeting 10 B4C nanoplate can effectively induce tumor cell death, apoptosis and inhibit its proliferation ability. Based on the known research that BNCT and radiotherapy can induce ICD, the triple-targeting 10 B4C nanoplate of the present application is expected to further induce immunogenic cell death when performing BNCT and PTT, thereby possibly activating an anti-tumor immune response.

[0090] In summary, the triple-targeting 10 B4C nanoplate and the preparation method thereof provided by the present application have the following advantages: The present application provides a triple-targeting 10 B4C nanoplate. Through the synergistic effect of NGR peptide (targeting tumor blood vessels) and NLS peptide (targeting cell nucleus), the 10 B4C nanoplate is precisely delivered to the tumor cell nucleus. This design is expected to significantly improve 10 B enrichment concentration near the tumor cell nucleus, thereby meeting the requirement of BNCT for 10High requirements of B concentration and T / N, T / B ratio. BNCT, immune checkpoint blockade therapy (achieved by PD-L1 targeting peptide) and photothermal therapy (based on 10 The photothermal performance of B4C nanosheet itself) are integrated into one. The synergistic mechanism is that high-LET particles produced by BNCT can accurately kill tumor cells; PD-L1 targeting peptide can block the immune checkpoint pathway and reverse immune suppression; at the same time, the photothermal effect of nanosheet under near-infrared laser irradiation can directly ablate the tumor. The three synergies are expected to achieve the organic combination of physical killing and immune activation. The triple-targeting 10 B4C nanosheet 10 B4C nanosheet is used as a carrier, and NGR peptide, PD-L1 targeting peptide and NLS peptide are co-modified on the surface, realizing hierarchical targeting of tumor tissue, PD-L1 and tumor nucleus. Based on this design, the nanosheet not only accurately positions BNCT to the nucleus, but also simultaneously has the functions of immune checkpoint blockade and photothermal therapy, realizing multi-modal synergistic therapy. In summary, the triple-targeting 10 B4C nanosheet integrates tumor tissue targeting, immune checkpoint targeting and nucleus targeting into a single nanoplatform, has a triple-targeting function, can realize efficient enrichment of boron drugs in the nucleus of tumor cells, and simultaneously plays a synergistic therapeutic role of immune regulation and photothermal killing.

[0091] The present application provides a triple-targeting 10 The present application provides a preparation method of triple-targeting 10 B4C nanosheet, which mainly comprises three steps of ball milling treatment, surface functionalization and coupling reaction, has the advantages of simple preparation process without complicated operation process. 10 B4C nanosheet, which is prepared from 10 B4C powder, is conducive to 10 B4C nanosheet, which is prepared from 10 B4C nanosheet, has a triple-targeting function, can accurately recognize and enrich tumor cells, improves the therapeutic effect, reduces damage to normal tissues, and solves the problem of greater toxicity of existing BNCT boron delivery agents to normal tissues.

[0092] The preparation method has the advantages of simple preparation process, mild conditions and reliable purification. In the presence of a coupling agent, a coupling reaction solvent is used for coupling reaction, and NGR peptide (targeting tumor blood vessels), NLS peptide (targeting nucleus) and PD-L1 targeting peptide (immune checkpoint blockade therapy) are integrated on 10The surface of the B4C-PG nanosheet is successfully modified with a targeting peptide 10 B4C nanosheet (triple-targeting 10 B4C nanosheet), avoids a complex process of multi-step modification, and significantly improves the preparation efficiency. Moreover, the whole reaction is carried out under mild solution conditions, effectively protecting the structure of the nanosheet and the biological activity of each peptide segment. In combination with a repeated centrifugation-resuspension washing strategy, unreacted peptides, coupling agents and by-products are completely removed, ensuring high purity of the final product.

[0093] The triple-targeting 10 B4C nanosheet has good biocompatibility (low hemolysis rate and small cytotoxicity), is stable in dispersion in a physiological environment, and has efficient and stable photothermal cycle performance, thereby providing a basis for clinical transformation. The triple-targeting 10 B4C nanosheet can effectively induce tumor cell death, apoptosis and inhibit the proliferation ability of tumor cells.

[0094] The triple-targeting 10 B4C nanosheet provides a novel multi-modal synergistic treatment strategy for tumor treatment, which synergistically enhances at least two of boron neutron capture therapy (BNCT), immunotherapy and photothermal therapy (PTT), can effectively inhibit primary tumors, and can also induce a systemic anti-tumor immune response, inhibit tumor metastasis and recurrence, and has important popularization and application value and clinical transformation potential in the field of tumor treatment technology.

Claims

1. A triple targeting 10 B4C nanoplatelets characterized in that, comprising 10 B4C nanoplatelets and modification of said 10 targeting peptides on the surface of B4C nanoplatelets; The targeting peptides include NGR peptide, NLS peptide and PD-L1 targeting peptide.

2. The triple targeting of claim 1 10 B4C nanoplatelets characterized in that, The triple targeting 10 The average hydrated particle size of the B4C nanoplatelets was 194 nm.

3. The triple targeting of claim 1 10 B4C nanoplatelets characterized in that, The 10 The surface of the B4C nanosheet is modified by functionalization with epoxypropanol to obtain an epoxypropanol-functionalized 10 B4C nanosheet In the glycidol functionalized 10 Surface modification of the B4C nanoplatelets with the targeting peptide results in a triple targeting 10 B4C nanoplatelets.

4. The triple targeting of any one of claims 1-3 10 A method for preparing B4C nanoplatelets, characterized in that, The method comprises the following steps: S1, to 10 B4C powder was subjected to ball milling to obtain 10 B4C nanoplatelets; S2, functionalizing the B4C nanoplatelets with glycidol 10 B4C nanoplatelets with glycidol, i.e. 10 B4C nanoplatelets with glycidol, i.e. 10 B4C-PG nanoplatelets; S3、coupling the B4C-PG nanosheets, the NGR peptide, the NLS peptide and the PD-L1 targeting peptide in the presence of a coupling agent to obtain the triple-targeting 10 B4C-PG nanosheets, the NGR peptide, the NLS peptide and the PD-L1 targeting peptide in the presence of a coupling agent to obtain the triple-targeting 10 B4C nanosheets.

5. The preparation method according to claim 4, characterized in that, The S1, comprising: 10 B4C powder is added to ultrapure water, ball milling, first centrifugation, collecting supernatant, filtration to obtain filtrate, first drying, obtaining 10 B4C nanosheet; And / or, the S2 comprises: 10 B4C nanosheets, adding epoxy propanol, water bath ultrasonic dispersion, then functionalization reaction, cooling to room temperature, adding ultrapure water, ultrasonic dispersion, second centrifugation, collecting supernatant, namely epoxy propanol functionalized B4C nanosheets 10 B4C nanosheets, adding epoxy propanol, water bath ultrasonic dispersion, then functionalization reaction, cooling to room temperature, adding ultrapure water, ultrasonic dispersion, second centrifugation, collecting supernatant, namely epoxy propanol functionalized B4C nanosheets 10 B4C-PG nanosheets; And / or, the S3, comprising: 10 B4C-PG nanosheets, NGR peptides, NLS peptides, PD-L1 targeting peptides, coupling agents and catalysts are dissolved in a reaction solvent to carry out a coupling reaction; After the coupling reaction is completed, a fourth centrifugation is performed, the precipitate is collected, resuspended, repeated 5 times, the product is collected, a third drying is performed, and the triple-targeted 10 B4C nanoplatelets.

6. The preparation method according to claim 5, characterized in that, In the S1, the ball milling adopts a planetary ball mill, the rotating speed is 400-600 r / min, and the time is 20-28 h; the rotating speed of the first centrifugation is 3000 r / min, and the time is 10 min; the filtration uses a 200-250 nm microporous filter film; and the first drying is freeze drying; In the S2, the conditions of the functionalization reaction are that the reaction is carried out in an oil bath at 120-160 ℃ for 20-48 h; the rotating speed of the second centrifugation is 3000 r / min, and the time is 30 min; the solvent for dispersion is ultrapure water; the rotating speed of the third centrifugation is 12000-16000 r / min, and the time is 2 h; and the second drying is freeze drying; In the S3, the coupling reaction is carried out under inert conditions, the temperature of the coupling reaction is 35 ℃-40 ℃, the time is 20 h-28 h; the rotating speed of the fourth centrifugation is 8000 rpm, the time is 15 min, the molecular cut-off of the ultrafiltration centrifuge tube used is 100 kDa; the solvent for resuspension is ultrapure water; and the third drying is freeze drying.

7. The preparation method according to claim 6, characterized in that, In the S1, the 10 The B4C powder and ultrapure water were used in a ratio of 3 g: 10 mL. And / or, in the S2, the 10 The ratio of the use amount of B4C nanosheet and epoxy propanol is 0.1 g:20 mL; And / or, in the S3, the 10 The use amount ratio of B4C-PG nanosheet, 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.

8. The preparation method according to claim 7, characterized in that, The coupling agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride or N, N'-dicyclohexyl carbodiimide; the catalyst is 4-dimethylaminopyridine or 1-hydroxybenzotriazole; and the reaction solvent is anhydrous N, N-dimethylformamide.

9. The triple targeting of any one of claims 1-3 10 B4C nanoplatelets or the triple targeting prepared by the preparation method of any one of claims 4-8 10 Use of B4C nanoplatelets in the preparation of boron drugs.

10. Use according to claim 9, characterized in that, The boron drug is used for treating tumors, and the treatment comprises at least one of boron neutron capture therapy, immunotherapy and photothermal therapy.

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