M-coated PLGA-10BX compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery, and particularly relates to an M-coated PLGA-10BX compound as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing 10B-enriched boron nitride (h-10BN); extracting a cell membrane of the macrophage RAW264.7; polylactic acid-glycolic acid copolymer (PLGA) nano particles loaded with h-10BN are prepared; and finally, the bionic nano platform M (at) PLGA-10BN based on the macrophage membrane is prepared. The bionic nano-platform prepared by the invention is uniform in particle size and morphology, has relatively high biological safety, and has no obvious damage to various tissues and visceral organs. The compound can be used as a general platform for boron compound delivery, can also be used as an immune activator, is suitable for intravenous injection administration, and expands the application of boron neutron capture therapy (BNCT) in treatment of glioblastoma (GBM).

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, specifically relating to an M@PLGA- 10 BX complex, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the central nervous system, and their highly aggressive and recurrent biological characteristics pose a major challenge to clinical treatment. According to the World Health Organization (WHO) pathological grading system, gliomas are classified into grades I-IV, with glioblastoma (GBM, WHO grade IV) being the most aggressive subtype, resulting in a median survival of less than 15 months and an extremely poor prognosis. The current standard treatment regimen involves surgical resection combined with postoperative concurrent chemoradiotherapy (primarily temozolomide). However, due to the inherent heterogeneity and invasive growth characteristics of the tumor, as well as drug resistance and the presence of the blood-brain barrier (BBB), the clinical benefits of traditional treatments are very limited. Given the persistent difficulty in improving the survival rate of glioma patients, developing novel treatment strategies has become an urgent need in the field of neuro-oncology.

[0003] Boron neutron capture therapy (BNCT) is a binary targeted radiotherapy technique based on nuclear reactions, proposed by Professor Locher, with its development dating back to the mid-20th century. As a promising cancer treatment, BNCT officially received clinical approval in Japan in 2020, demonstrating unique advantages, particularly in the treatment of refractory tumors such as gliomas. The treatment mechanism of this technology is highly specific: when tumor cells selectively take up boron neutrons containing... 10 Compound B, after being subjected to low-energy neutron irradiation, is stable. 10 The boron nucleus can capture a neutron and transform into an excited state. 11 The B atom nucleus then immediately undergoes a nuclear fission reaction, releasing alpha particles with high linear energy transfer (LET). 4 He) and recoil lithium core ( 7 (Li). These high-energy particles have an effective action range (4-9 μm), comparable to the diameter of a single cell. They can form dense ionization tracks within the cell, inducing various forms of damage, such as DNA base damage, single-strand and double-strand breaks, ultimately leading to tumor cell death. Compared to traditional radiotherapy, BNCT has three significant advantages: First, its cell-killing effect is independent of oxygen concentration, achieving uniform killing in hypoxic tumor tissue; second, this technology is effective against tumor cells at all cell cycles, including G0-phase cells resistant to conventional treatment; and most importantly, BNCT's selective killing properties allow it to precisely eliminate tumor cells while maximally protecting surrounding normal tissues. These characteristics make BNCT a highly attractive new option in the treatment of GBM.

[0004] However, the clinical application of BNCT still faces major challenges. Currently, only two boron-containing drugs, boron phenylalanine (BPA) and mercapto borane (BSH), have been approved for clinical BNCT treatment. BPA can be selectively taken up by tumor cells through the L-type amino acid transporter (LAT), but its rapid metabolism and insufficient tissue enrichment limit the therapeutic effect; BSH has good water solubility and low toxicity, but it has poor pharmacokinetic properties. For GBM, the presence of the BBB is an important reason for the poor efficacy of BNCT. The BBB is a precise structure composed of brain microvascular endothelial cells, pericytes, basement membrane and astrocyte end feet, which strictly regulates the entry and exit of substances into the brain parenchyma through a variety of highly expressed receptors and transporters. Existing boron-containing drugs are difficult to effectively penetrate this barrier, resulting in a low concentration of B in tumor tissues 10 The concentration of B is difficult to reach the treatment threshold (20 μg / g), and the tumor / normal tissue (T / N) and tumor / blood (T / B) ratios are not ideal, which seriously restricts the therapeutic effect of BNCT. However, due to the high expression of multiple receptors and transporters by the BBB, it provides a potential physiological basis for the delivery of boron-containing compounds using receptor-mediated endocytosis (RMT) and carrier-mediated transport (CMT) strategies.

[0005] In recent years, to overcome the limitations of clinically approved boron drugs, researchers have been working to develop boron-rich nanocarriers. Among them, inorganic boron-containing nanoparticles are considered as efficient delivery carriers, such as boron carbide (B4C), boron phosphate (BPO4), carborane, gadolinium boride, and boron nitride (BN), etc. These boron nanoparticles significantly increase the boron content in tumors and exhibit good therapeutic effects in various animal models. However, due to the high uptake rate of this type of boron drugs in the reticuloendothelial system, which can lead to accumulation toxicity, most of them are difficult to apply in clinical practice. It is worth noting that BN stands out due to its degradability and biological safety. Previous studies have confirmed that it is easily metabolized by water molecules to form small molecular compounds such as boric acid. Larger size BN nanomaterials usually have the advantage of passive targeting through the enhanced permeability and retention (EPR) effect, but their ability to penetrate the BBB and penetrate deep into tumor tissues is limited, which poses a huge challenge to the therapeutic effect.

[0006] Therefore, it is highly desirable to design a strategy that allows boron nitride to accumulate and fully penetrate in tumors while passing through BBB. Nanotechnology provides unique opportunities for targeted drug delivery, imaging, and therapeutic diagnosis, which has completely changed the field of tumor therapy. Encapsulating various therapeutic drugs in nanocarriers can improve the solubility, stability and bioavailability of bioactive substances, and achieve controllable sustained-release drug delivery, thereby reducing the frequency of drug administration and reducing systemic toxicity. The Food and Drug Administration (FDA) approved polylactic-co-glycolic acid (PLGA) as an excellent drug carrier due to its biodegradability, biocompatibility, controllable drug release and targeted tumor cell delivery properties. In addition, PLGA-based drug delivery systems have the advantage of flexible integration of multiple therapeutic agents, which can achieve combined anticancer therapy through synergistic or complementary mechanisms. However, PLGA nanoparticles are easily intercepted by macrophages, resulting in reduced immune compatibility. This interception hinders their ability to reach the target tissue, thereby affecting their therapeutic effect. In addition, the recognition of PLGA nanoparticles by macrophages can trigger an immune response, leading to inflammation and potential adverse reactions.

[0007] In view of the limitations of traditional nanometer delivery systems, cell membrane biomimetic drug delivery systems exhibit significant advantages in the field of tumor therapy due to their unique biological properties. In particular, biomimetic carriers based on macrophage membranes can effectively prolong the blood circulation time of nanoparticles, reduce the recognition and clearance of the mononuclear phagocyte system, and specifically bind to vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells through the overexpression of integrin α4 / β1 on the membrane surface, thereby significantly enhancing the ability of nanoparticles to penetrate the BBB. Although the EPR effect caused by vascular leakage and impaired lymphatic drainage during tumor progression can partially disrupt the integrity of the BBB, the elevated interstitial fluid pressure within the tumor and the residual BBB still pose a significant barrier to drug delivery. Notably, macrophage membrane biomimetic systems not only facilitate the crossing of the BBB by nanoparticles, but also enhance their diffusion and penetration within the tumor tissue through their unique biological functions.

[0008] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present application, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0009] The present application aims to provide a biomimetic nanoplatform that can achieve precise delivery and efficient enrichment of boron 10 in glioblastoma and a preparation method thereof.

[0010] In a first aspect of the present application, a M@PLGA- 10BX complex, said M is a cell membrane of macrophage, PLGA is polylactic-co-glycolic acid, 10 BX is a boron compound, X is selected from the group consisting of: N, C, H, O.

[0011] In another preferred embodiment, said cell membrane of macrophage is selected from the group consisting of: wild type cell membrane of macrophage, modified cell membrane of macrophage.

[0012] In another preferred embodiment, said M@PLGA- 10 BX is M@PLGA- 10 BN, wherein 10 BN is boron nitride (h- 10 BN).

[0013] In another preferred embodiment, said macrophage is selected from the group consisting of: macrophage RAW264.7, macrophage J774, macrophage THP-1.

[0014] In another preferred embodiment, said M@PLGA- 10 BN complex has a diameter of 165-195 nm, preferably 170-190 nm, most preferably 175 nm-185 nm (e.g. 180 nm).

[0015] In a second aspect of the present application, there is provided a method for preparing the M@PLGA- 10 BN complex of the first aspect of the present application, said method comprising the steps of:

[0016] (1) providing a cell membrane of macrophage M (e.g. cell membrane of macrophage RAW264.7);

[0017] (2) providing h- 10 BN;

[0018] (3) providing PLGA- 10 BN;

[0019] (4) preparing the M@PLGA- 10 BN complex.

[0020] In another preferred embodiment, said step (3), the method for preparing the PLGA- 10 BN comprises the steps of:

[0021] (3a) preparing an organic phase suspension of PLGA and h- 10 BN;

[0022] (3b) subjecting the organic phase suspension in (3a) to low temperature sonication to form an organic phase of emulsifier;

[0023] (3c) adding an aqueous phase to the organic phase in (3b) followed by continued low temperature sonication to form a uniform emulsion;

[0024] (3d) solidifying the nanoparticles and volatilizing the organic phase;

[0025] (3e) centrifuging to remove excess aqueous phase and collecting the nanoparticles PLGA- 10 BN.

[0026] In another preferred embodiment, in the step (3a), the organic phase is selected from the group consisting of dichloromethane, acetone.

[0027] In another preferred embodiment, in the step (3c), the aqueous phase is selected from the group consisting of polyvinyl alcohol (PVA), Tween-80.

[0028] In another preferred embodiment, in the step (3d), the nanoparticles are solidified by magnetic stirring.

[0029] In another preferred embodiment, in the step (3d), a solidifying agent (such as isopropanol, ethanol, tert-butanol) is added to the emulsion and the nanoparticles are solidified by magnetic stirring.

[0030] In another preferred embodiment, the method further comprises: (3f) resuspending the PLGA- 10 BN in deionized water.

[0031] In another preferred embodiment, in the step (4), the M@PLGA- 10 BN is prepared comprising the steps of:

[0032] (4a) resuspending the macrophage membrane and the PLGA- 10 BN, respectively, to form a solution and mixing;

[0033] (4b) enveloping (4a) to prepare M@PLGA- 10 BN.

[0034] In another preferred embodiment, in the step (4a), the resuspending is resuspending in deionized water or a buffer.

[0035] In another preferred embodiment, the buffer is selected from the group consisting of PBS, physiological saline.

[0036] In another preferred embodiment, in the step (4b), the enveloping is selected from the group consisting of ultrasonic enveloping, membrane extruder enveloping.

[0037] In another preferred embodiment, in the step (4b), the ultrasonic enveloping is performed in an ultrasonic disrupter or a probe sonicator.

[0038] In another preferred embodiment, in step (4), the M@PLGA- 10 The BN comprises the steps of:

[0039] (4a) conjugating the macrophage membrane with PLGA- 10 The BNs are each resuspended in deionized water and mixed together;

[0040] (4b) subjecting the mixture in (4a) to sonication in an ultrasonic disrupter to prepare M@PLGA- 10 BNs.

[0041] In a third aspect of the present application, there is provided use of the M@PLGA- 10 BX complex of the first aspect of the present application in the preparation of a BNCT formulation and / or an immune activator.

[0042] In another preferred embodiment, the disease to be treated by the BNCT formulation is selected from the group consisting of glioblastoma (GBM), tongue cancer, melanoma, breast cancer, intestinal cancer.

[0043] In a fourth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0044] (i) the M@PLGA- 10 BX complex of the first aspect of the present application; and

[0045] (ii) a pharmaceutically acceptable carrier.

[0046] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0047] In another preferred embodiment, the pharmaceutical composition is used in the preparation of a BNCT formulation.

[0048] In another preferred embodiment, the disease to be treated by the BNCT formulation is selected from the group consisting of glioblastoma (GBM), tongue cancer, melanoma, breast cancer, intestinal cancer.

[0049] In another preferred embodiment, the carrier is selected from the group consisting of saline, buffer, glucose, water, glycerol, ethanol, or a combination thereof.

[0050] In another preferred embodiment, the BNCT formulation is a neutron therapy adjuvant.

[0051] In another preferred embodiment, the pharmaceutical composition is administered by a method selected from the group consisting of intraperitoneal injection, intravenous injection, intracranial injection.

[0052] In a fifth aspect of the present application, there is provided a pharmaceutical combination comprising:

[0053] (i) a first active ingredient, the first active ingredient comprising the M@PLGA- described in the first aspect of the present invention. 10 BX complex, or the pharmaceutical composition described in the fourth aspect of the present invention;

[0054] (ii) PD-1 antibody.

[0055] In a sixth aspect of the invention, a neutron therapy method is provided, using a pharmaceutical composition as described in the fourth aspect of the invention or a pharmaceutical combination as described in the fifth aspect of the invention.

[0056] In another preferred embodiment, the method is performed under neutron irradiation.

[0057] In another preferred embodiment, the neutron irradiation duration is 10-30 min, preferably 20 min.

[0058] In another preferred embodiment, the neutron flux during the neutron irradiation process is 1 × 10⁻⁶. 8 -1×10 9 n·cm -2 ·s -1 The preferred value is 6.96×10. 8 n·cm -2 ·s -1 .

[0059] In another preferred embodiment, the neutron irradiation is performed 0.5-3 days after intravenous injection of the pharmaceutical composition, preferably 24 hours later.

[0060] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0061] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0062] Figure 1 This is a schematic diagram of the extraction of the RAW264.7 macrophage cell membrane.

[0063] Figure 2 For enrichment 10 B's h- 10 A schematic diagram of the synthetic route for BN, where h- 10 BN is hexagonal nitriding. 10 boron.

[0064] Figure 3 Wrap h- for PLGA 10 A schematic diagram of BN, i.e., loading h- 10 BN's PLGA nanoparticles (PLGA- 10 A schematic diagram of the preparation of BN. Wherein, h- 10 BN hexagonal nitriding 10 Boron, PLGA (polylactic acid-glycolic acid copolymer), PLGA- 10 BN polylactic acid-glycolic acid copolymer nanoparticles encapsulating hexagonal nitride 10 boron.

[0065] Figure 4 PLGA- for macrophage membrane encapsulation 10 A schematic diagram of BN nanoparticles, i.e., the biomimetic nanoplatform M@PLGA- 10 A schematic diagram of BN preparation. Wherein, M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 boron.

[0066] Figure 5 for h- 10 The structural and compositional characterization results of BN are shown in Figures A and B, respectively, obtained through FITR and XPS detection. The detection results indicate that h- 10 BN.

[0067] Figure 6 for h- 10 BN, PLGA- 10 BN and M@PLGA- 10 Transmission electron microscopy (TEM) images of BN show the morphologies of the three materials. Among them, h- 10 BN: Hexagonal Nitride 10 Boron, PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron, M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 boron.

[0068] Figure 7 For PLGA- 10 BN and M@PLGA- 10 The dynamic light scattering particle size and potential results of BN demonstrate that the particle size and potential of the two nanomaterials meet the requirements. Among them, PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron, M@PLGA- 10BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 boron.

[0069] Figure 8 For PLGA- 10 BN and M@PLGA- 10 Quantitative results of BN uptake by GBM cells, in vitro and intracellular. 10 Detection of B content, M@PLGA- 10 BN was taken up by tumor cells after being co-incubated with cells for a period of time. Among them, PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron, M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron, B-10 concentration (μg / 10) 6 cells): Boron-10 concentration (µg / 10) 6 (cells).

[0070] Figure 9 The results show the in vitro blood-brain barrier crossing of PLGA-DiD and M@PLGA-DiD. A is a schematic diagram of the in vitro blood-brain barrier, and B and C are flow cytometry results, indicating that they can cross the blood-brain barrier and be taken up by tumor cells. Among them, Fomulation: nano-formulation; bEnd.3: vascular endothelial cells; GL261: glioma cells; in vitro BBB model: in vitro blood-brain barrier model.

[0071] Figure 10 For PLGA- 10 BN and M@PLGA- 10 Transmission electron microscopy (TEM) images of BN (tumor nucleus) crossing the in vitro BBB and then being taken up by GBM cells. The in vitro TEM results demonstrate that tumor cells can take up M@PLGA- 10 BN. Among them, PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron; M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 boron.

[0072] Figure 11This study investigated the in vivo targeting results of PLGA-DiD and M@PLGA-DiD for GBM, using in vivo imaging to observe the targeting ability of nanomaterials in vivo, as well as their distribution and quantitative analysis in various organs. Specifically, Free-DiD represents a lipophilic fluorescent dye; PLGA-DiD represents a lipophilic fluorescent dye encapsulated in polylactic-co-glycolic acid (PLGA) nanoparticles; and M@PLGA-DiD represents a lipophilic fluorescent dye encapsulated in PLA nanoparticles disguised as macrophage membranes. GBM (glioblastoma) was also studied.

[0073] Figure 12 For PLGA- 10 BN and M@PLGA- 10 Pharmacokinetics of BN in GBM-bearing mice and transmission electron microscopy images showing its distribution in GBM tissues. A shows the ICP-MS analysis of BN in various organs in vivo. 10 The content of B, where B is the T / N and T / B ratio, and C is the presence of nanoparticles within tumor cells as detected by transmission electron microscopy. Among these, PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron; M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron; T / N ratio: the ratio of boron 10 content in tumors to that in normal tissues; T / B ratio: the ratio of boron 10 content in tumors to that in blood.

[0074] Figure 13 For PLGA- 10 BN and M@PLGA- 10 Results of BN inhibiting GBM cell proliferation and inducing GBM cell apoptosis in vitro. A shows cell viability as detected by CCK-8 assay, and B shows tumor cell apoptosis as detected by flow cytometry. PBS: physiological saline; PBS + N physiological saline + neutron irradiation; PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron; PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron + neutron irradiation; M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron; M@PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron + neutron irradiation; PI: propidium iodide.

[0075] Figure 14 For PLGA- 10 BN and M@PLGA- 10 The anti-GBM effect of BN-mediated BNCT in vivo. A. Changes in tumor fluorescence signal observed by in vivo imaging in mice; B. Statistical analysis of fluorescence signal values; C. Changes in mouse body weight; D. Changes in mouse survival time; E. Tumor volume detected by MR in mice. PBS: physiological saline; PBS+N: physiological saline + neutron irradiation; PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron; PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron + neutron irradiation; M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron; M@PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron + neutron irradiation.

[0076] Figure 15 To detect tumor tissue proliferation and apoptosis using immunofluorescence, PLGA- 10 BN and M@PLGA- 10 The results of BN-mediated BNCT-induced GBM apoptosis and inhibition of GBM proliferation. Among them, PBS: physiological saline; PBS+N: physiological saline + neutron irradiation; PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron; PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles. 10 Boron + neutron irradiation; M@PLGA- 10 BN: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron; M@PLGA- 10 BN+N: Hexagonal nitride encapsulated in polylactic acid-glycolic acid copolymer nanoparticles disguised as macrophage membranes. 10 Boron + neutron irradiation; DAPI4',6-diamidinyl-2-phenylindole; Ki-67; Ki-67 protein; Caspase 3: caspase 3.

[0077] Figure 16 For M@PLGA- 10 Analysis results of the immune regulatory mechanism of BN-mediated BNCT production.

[0078] A. Volcano plot shows that genes in the tumor are changed, wherein, Down-regulated: down-regulated; Non-significant: no significant difference; Up-regulated: up-regulated; Log10 P value: logarithm (base 10) of P value; Log2 Fold Change: logarithm (base 2) of fold change; B. GO pathway enrichment analysis shows that immune-related pathways are changed, wherein, GO enrichment analysis: gene ontology (GO) enrichment analysis;

[0079] positive regulation of leukocyte activation: positive regulation of leukocyte activation; positive regulation of lymphocyte activation: positive regulation of lymphocyte activation; positive regulation of leukocyte cell-cell adhesion: positive regulation of leukocyte cell-cell adhesion;

[0080] regulation of leukocyte mediated immunity: regulation of leukocyte mediated immunity; positive regulation of T cell activation: positive regulation of T cell activation; activation of innate immune response: activation of innate immune response;

[0081] positive regulation of chemolaxis: positive regulation of chemolaxis;

[0082] cytokine production involved in immune response: cytokine production involved in immune response;

[0083] positive regulation of tumor necrosis factor superfamily cytokine production: positive regulation of tumor necrosis factor superfamily cytokine production;

[0084] regulation of cytokine production involved in immune response: regulation of cytokine production involved in immune response;

[0085] Cellular response to chemokines;

[0086] Positive regulation of cytokine production involved in immune response;

[0087] Positive regulation of chemokine production; immune response to tumor cells.

[0088] Positive regulation of myeloid leukocyte-mediated immunity;

[0089] Adjusted p-value: Corrected p-value; Enrichment Ratio: Enrichment ratio;

[0090] Gene Count: Gene count;

[0091] C. KEGG pathway enrichment analysis revealed changes in immune-related pathways, including KEGG enrichment analysis: Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis;

[0092] Cytokine-cytokine receptor interaction; Chemokine signaling pathway.

[0093] Viral protein interaction with cytokine and cytokine receptor;

[0094] Natural killer cell mediated cytotoxicity; Phagosome;

[0095] JAK-STAT signaling pathway: JAK-STAT signaling pathway;

[0096] T cell receptor signaling pathway: T cell receptor signaling pathway

[0097] Leukocyte transendothelial migration: Leukocyte transendothelial migration

[0098] Complement and coagulation cascades: Complement and coagulation cascades

[0099] Th1 and Th2 cell differentiation: Th1 and Th2 cell differentiation

[0100] NF-kappa B signaling pathway: NF-kappa B signaling pathway

[0101] Inflammatory mediator regulation of TRP channels: Inflammatory mediator regulation of TRP channels

[0102] C-type lectin receptor signaling pathway: C-type lectin receptor signaling pathway

[0103] Toll like receptor signaling pathway: Toll like receptor signaling pathway

[0104] B cell receptor signaling pathway: B cell receptor signaling pathway

[0105] Antigen processing and presentation: Antigen processing and presentation

[0106] Gene Count: Gene Count

[0107] D. Some immune-related genes were changed, PBS+N: phosphate buffer solution+N (neutron irradiation) group; M@PLGA-1 0 BN+N: M@PLGA-1 0BN+N (neutron irradiation) group; Group: grouping; Enrichment Ratio: enrichment ratio; Adjusted p-value: corrected P value; Casp1: caspase 1; Cxcl9: chemokine (C-X-C motif) ligand 9; Cd8a: Cd8a molecule; Jchain: joining chain; Nkg7: NK cell granule protein 7; Cd36: Cd36 molecule; Slamf7: signal lymphocyte activating molecule family member 7; Clec12a: C-type lectin domain family 12 member A; IL16: interleukin 16; Clec10a: C-type lectin domain family 10 member A; Fasl: Fas ligand; Klra7: killer cell lectin-like receptor A7;

[0108] E. PD-1 and PD-L1 pathway changes, PD-L1 expression and PD-1 checkpoint pathway in cancer: PD-L1 expression and PD-1 checkpoint pathway in cancer; Running Enrichment Score: running enrichment score; NES: 1.34: standardized enrichment score: 1.34; P value: 0.017: P value: 0.017; Ranked List Metric: ranked list metric; Rank in Ordered Dataset: rank in ordered dataset.

[0109] Figure 17 M@PLGA- 10 BN-mediated anti-GBM effect of BNCT combined with PD-1 antibody. A. Live imaging to observe changes in intracranial tumors, wherein PBS: physiological saline; ICB: immunotherapy; PLGA- 10 BN+N: hexagonal boron nitride nanoparticles coated with poly (lactic-co-glycolic acid) 10 boron + neutron irradiation; M@PLGA- 10 BN+N+ICB: hexagonal boron nitride nanoparticles coated with poly (lactic-co-glycolic acid) 10 boron + neutron irradiation + immunotherapy; B. is the MR observation of tumor volume in mice, C. changes in tumor fluorescence signal in mice, photo: photon; D. is the survival of mice. DETAILED DESCRIPTION

[0110] Example implementations will now be described more fully with reference to the accompanying drawings. Example implementations, however, can be implemented in many different forms and should not be construed as limited to the implementations set forth herein; rather, these implementations are provided so that this disclosure will be thorough and complete, and fully convey the concept of example implementations to those skilled in the art.

[0111] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0112] Through extensive and in-depth research, the inventors have designed and prepared M@PLGA- for the first time. 10 BN complex, which is a carrier disguised as a macrophage membrane 10 B's biomimetic nanoplatform. M@PLGA- 10 BN is the cell membrane (M) of macrophages, polylactic acid-glycolic acid copolymer (PLGA), and enrichment. 10 Boron nitride of B ( 10 A complex formed by BN (B, N, and N), possessing a specific structure and function. This invention, M@PLGA- 10 The BN complex enables precise delivery and efficient enrichment of boron 10 in glioblastoma, and can be used to prepare BNCT agents and / or immune activators. It has high biosafety, has not caused long-term toxicity during BNCT therapy, and has demonstrated good BNCT efficacy and strong immune microenvironment remodeling capabilities.

[0113] Specifically, after intravenous administration, M@PLGA- 10 BN in the GBM region 10 The boron accumulation peaked at 53.1508 μg / g 24 hours after injection. At this point, the boron concentration at the tumor site was 3.95 times that of blood and normal brain tissue, fully meeting the requirements for BNCT treatment. Notably, the biomimetic nanoplatform exhibited a significantly prolonged residence time in GBM, providing a wider irradiation time window for clinical treatment. Furthermore, after neutron irradiation, M@PLGA- 10 BN significantly induces DNA damage and γ-H2AX focal point formation both in vivo and in vitro, thereby killing GBM, inhibiting its growth, and prolonging the survival of tumor-bearing mice. Furthermore, M@PLGA- 10 BN-based BNCT therapy can induce immunogenic cell death, thereby reversing the immunosuppressive microenvironment of GBM, manifested as CD8+. + The infiltration and proportion of T cells and M1 glioma-associated macrophages (GAMs) were significantly increased. Combining this therapy with PD-1 antibodies further enhanced the anti-tumor efficacy and improved the immune microenvironment. Based on this, the present invention was completed.

[0114] the term

[0115] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0116] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0117] As used herein, the term "comprising" or "including" can be open-ended, semi- closed, and / or closed. In other words, the term "comprising" or "including" can be used in the sense of "including, but not limited to," or "including, but not exhaustive."

[0118] M

[0119] In the present application, the term "M" refers to the cell membrane of a macrophage, which can be obtained in various ways, for example, can be extracted from the corresponding macrophage.

[0120] Typically, the cell membrane of the macrophage is selected from the group consisting of the cell membrane of a wild-type macrophage, a modified macrophage membrane.

[0121] Typically, the macrophage is selected from the group consisting of macrophage RAW264.7, macrophage J774, macrophage THP-1.

[0122] In a specific embodiment of the present application, the M is the cell membrane of macrophage RAW264.7, and the extraction method is shown in the following step (1).

[0123] Step (1):

[0124] (1a) After culturing RAW264.7 cells in a 10 cm dish to a high degree of fusion, the cells are collected using a scraper, the cell precipitate is collected by centrifugation, and the cells are washed 3 times with phosphate buffer solution (PBS);

[0125] (1b) Add hypotonic solution and lyse overnight at 4°C; the next day, perform low-temperature ultrasonication, remove the cell nuclei and organelles by centrifugation, collect the supernatant, and stand overnight at 4°C;

[0126] (1c) On the third day, remove the precipitate by centrifugation and collect the supernatant, discard the supernatant by ultracentrifugation, and wash the oily precipitate with deionized water 3 times. After washing, resuspend the precipitate in deionized water and disperse it by low-temperature ultrasonication;

[0127] (1d) Finally, use a micro-liposome extruder to extrude the membrane solution through a 1 μm polycarbonate porous membrane 30 times back and forth to remove membrane fragments with larger particle sizes, and then use the standard BCA method to quantify the concentration of the membrane.

[0128] The reaction process is shown in Figure 1 .

[0129] Preferably, the fusion degree of RAW264.7 cells in step (1) is 100%.

[0130] Preferably, the rotation speed for centrifugal collection of cell precipitate and each time for washing the cells is 250g, and the time is 4min; the concentration of PBS solution is 0.1M, and the pH is 7.4.

[0131] Preferably, the hypotonic solution in step (1) is 0.1×TM buffer, and 1mM of protease inhibitor PMSF and 1×phosphatase inhibitor are contained therein; the volume of the hypotonic solution is 20-30mL.

[0132] Preferably, the temperature for low-temperature ultrasonic dispersion in step (1) is controlled at 4-12℃, the ultrasonic power is 220W, and the time is 2h.

[0133] Preferably, the rotation speed for centrifugal removal of cell nucleus in step (1) is 3200g, the time is 10min, and the temperature is 4℃; the rotation speed for centrifugal removal of cell organelle is 20000g, the time is 20min, and the temperature is 4℃, and the centrifugal times is 2.

[0134] Preferably, the rotation speed for centrifugal removal of precipitate on the third day in step (1) is 20000g, the time is 20min, and the temperature is 4℃; the rotation speed for ultracentrifugation is 100000g, the time is 1h, and the temperature is 4℃.

[0135] Preferably, the volume of deionized water used for resuspension of oil precipitate in step (1) is 5-10mL.

[0136] Preferably, the temperature for low-temperature ultrasonic dispersion of cell membrane in step (1) is controlled at 4-12℃, the ultrasonic power is 220W, and the time is 2h.

[0137] 10 BX

[0138] In the present application, the term "BX" refers to a boron compound, wherein X is selected from: N, C, H, O. 10 BX 10 10 BN, i.e. boron nitride (h- 10 BN).

[0139] In one specific embodiment of the present application, 10 BX 10 BN, i.e. boron nitride (h- 10 BN).

[0140] h- 10 BN

[0141] In this invention, the term "h- 10 "BN" refers to enrichment. 10 Boron nitride of B can be obtained in a variety of ways, such as through laboratory synthesis.

[0142] As is well known to those skilled in the art, there are various preparation methods to obtain "h- 10 BN".

[0143] In a specific embodiment of the present invention, the h- 10 The synthesis method of BN is shown in step (2) below.

[0144] Step (2):

[0145] (2a) enrichment 10 After the boric acid and melamine of B are thoroughly ground and mixed in an agate mortar, they are transferred to a specially made corundum crucible.

[0146] (2b) Place the crucible in the reaction zone of the tube furnace and heat it to 900°C under continuous nitrogen protection. React at a constant temperature for 3 hours to complete the nitriding process.

[0147] (2c) After the reaction is complete, allow the mixture to cool naturally to room temperature to obtain an ivory-white blocky product. Fine grinding is then used to obtain crude h- 10 BN powder;

[0148] (2d) The powder was then resuspended in deionized water and subjected to low-temperature sonication to h- 10 BN was fully dispersed, and then small molecule byproducts were removed by dialysis;

[0149] (2e) Finally, centrifuge the dialysate and collect the solution containing h- 10 The milky white supernatant of BN was freeze-dried and stored in a desiccator for later use.

[0150] The reaction process is as follows Figure 2 As shown.

[0151] Preferably, the enrichment in step (2) 10 In boric acid of B 10 The abundance of B was 99.43%; the molar ratio between boric acid and melamine was 6:1.

[0152] Preferably, the dimensions of the specially made corundum crucible in step (2) are 60mm×30mm×15mm.

[0153] Preferably, the flow rate of nitrogen in step (2) is 100 mL / min.

[0154] Preferably, the temperature rise rate during heating in step (2) is 10°C / min.

[0155] Preferably, the volume of deionized water used to resuspend the powder in step (2) is 30-40 mL; the power of the low-temperature ultrasonic is 900 W, the time is 4 h, and the temperature is 4℃.

[0156] Preferably, the molecular weight cut-off of the dialysis bag used in step (2) is 8-14 kDa, and the dialysis time is not less than 48 h; the centrifugal speed of the dialysate is 1811 g, and the time is 5 min.

[0157] PLGA- 10 BN

[0158] In the present application, the term "PLGA- 10 BN" refers to PLGA (poly-lactic-co-glycolic acid) nanoparticles loaded with h- 10 BN. It can be obtained in various ways, for example, it can be synthesized by laboratory preparation.

[0159] Typically, the preparation / synthesis method of the PLGA- 10 BN is shown in step (3), which includes the following steps:

[0160] (3a) preparing an organic phase suspension of PLGA and h- 10 BN;

[0161] (3b) subjecting the organic phase suspension in (3a) to low-temperature acoustic shock to form an organic phase of emulsifier;

[0162] (3c) adding an aqueous phase to the organic phase in (3b), and then continuing low-temperature acoustic shock to form a uniform emulsion;

[0163] (3d) solidifying the nanoparticles and volatilizing the organic phase;

[0164] (3e) removing the excess aqueous phase by centrifugation, and collecting the nanoparticles PLGA- 10 BN.

[0165] In another preferred embodiment, the organic phase in step (3a) is selected from the group consisting of dichloromethane, acetone.

[0166] In another preferred embodiment, the aqueous phase in step (3c) is selected from the group consisting of polyvinyl alcohol (PVA), Tween-80.

[0167] In another preferred embodiment, in step (3d), the nanoparticles are solidified by magnetic stirring.

[0168] In another preferred embodiment, in step (3d), a solidifying agent (such as isopropyl alcohol, ethanol, tert-butyl alcohol) is added to the emulsion and the nanoparticles are solidified by magnetic stirring.

[0169] In another preferred embodiment, the method further includes: (3f) placing the PLGA- 10 BN was resuspended in deionized water.

[0170] In a specific embodiment of the present invention, the PLGA- 10 The preparation / synthesis method of BN is shown in step (3) below.

[0171] Step (3):

[0172] (3a) Weigh out PLGA and dissolve it in dichloromethane using ultrasound, then add h- 10 BN was also dispersed evenly using ultrasound.

[0173] (3b) The above suspension was acoustically vibrated at low temperature to form the organic phase of the emulsion using an acoustic vibrator;

[0174] (3c) Add the aqueous phase of polyvinyl alcohol (PVA) to the organic phase, and then continue low-temperature acoustic vibration to form a uniform emulsion;

[0175] (3d) Isopropanol was added to the emulsion and magnetically stirred to solidify the nanoparticles and volatilize harmful dichloromethane;

[0176] (3e) Subsequently, excess PVA was removed by centrifugation and PLGA nanoparticles were collected. 10 BN;

[0177] (3f) Finally, PLGA- 10 BN was resuspended in deionized water.

[0178] The reaction process is as follows Figure 3 As shown.

[0179] Preferably, in step (3), the PLGA has a specification of 50 / 50-COOH, a molecular weight of 15000, and a concentration of 25 mg / mL in dichloromethane.

[0180] Preferably, in step (3), h- 10 The concentration of BN in dichloromethane is 2.5 mg / mL.

[0181] Preferably, in step (3), when forming the organic phase of the emulsifier, the power of the acoustic vibrator is 63W, the acoustic vibration time is 2min (5s working, 5s stopping), and the acoustic vibration temperature is 4℃; when forming a uniform emulsifier, the power of the acoustic vibrator is 52W, the acoustic vibration time is 2min (5s working, 5s stopping), and the acoustic vibration temperature is 4℃.

[0182] Preferably, in step (3), the concentration of PVA is 4%, and the volume ratio of PVA to the organic phase is 4:1.

[0183] Preferably, in step (3), the concentration of isopropanol is 2%, and the volume ratio of isopropanol to emulsifier is 1:1.

[0184] Preferably, the magnetic stirring time in step (3) is 12 hours and the stirring temperature is 25°C.

[0185] Preferably, in step (3), the rotation speed for centrifuging to remove excess PVA is 2500 rpm and the centrifugation time is 5 min; the rotation speed for centrifuging to collect nanoparticles is 11000 rpm and the centrifugation time is 30 min.

[0186] M@PLGA- 10 BX

[0187] In this invention, the term "M@PLGA-" is used. 10 "BX" refers to the macrophage membrane camouflage carrier. 10 B's biomimetic nanoplatform. Specifically, M@PLGA- 10 BX is the cell membrane (M) of macrophages, polylactic acid-glycolic acid copolymer (PLGA), and enrichment. 10 Boron compounds of B 10 BX (e.g.) 10 A complex formed by BN, B, and B, with a specific structure and function, can be prepared in a variety of ways.

[0188] In a specific embodiment of the present invention, M@PLGA- 10 BX is M@PLGA- 10 BN.

[0189] M@PLGA- 10 BN

[0190] In this invention, the term "M@PLGA-" is used. 10 BN” “M@PLGA- 10 "BN complex", "biomimetic nanoplatform", and "nanoplatform" all have the same meaning, referring to the carriers disguised as macrophage membranes. 10 B's biomimetic nanoplatform. Specifically, M@PLGA- 10 BN is the cell membrane (M) of macrophages, polylactic acid-glycolic acid copolymer (PLGA), and enrichment. 10 Boron nitride of B ( 10 A complex formed by BN, B, and B, with a specific structure and function, can be prepared in a variety of ways.

[0191] Typically, the M@PLGA- 10 The BN preparation / synthesis method is shown in step (4), including the following steps:

[0192] (4a) Introduce macrophage membranes with PLGA- 10 BN was resuspended separately to form solutions before being mixed;

[0193] (4b) Coating the mixture from (4a) (e.g., ultrasonic coating) to prepare M@PLGA- 10 BN.

[0194] In another preferred embodiment, in step (4a), resuspension is performed in deionized water or a buffer solution.

[0195] In another preferred embodiment, the buffer solution is selected from the group consisting of PBS and physiological saline.

[0196] In another preferred embodiment, in step (4b), the coating method is selected from the group consisting of: ultrasonic coating, membrane extruder coating.

[0197] In another preferred embodiment, in step (4b), the ultrasonic encapsulation is performed in an ultrasonic disruptor or a probe-type ultrasonic processor.

[0198] In a specific embodiment of the present invention, the M@PLGA- 10 The preparation / synthesis method of BN is shown in step (4) below.

[0199] Step (4):

[0200] (4a) Introduce macrophage membranes with PLGA- 10 After each BN was resuspended in deionized water, they were mixed together.

[0201] (4b) The mixture from (4a) was subjected to ultrasonic encapsulation in an ultrasonic disruptor to prepare M@PLGA- 10 BN.

[0202] The reaction process is as follows Figure 4 As shown.

[0203] Preferably, in step (4), the macrophage membrane and PLGA- 10 The mass ratio between B and N is 2:1.

[0204] Preferably, the ultrasound program in step (4) is set as follows: power 324W, working time 99s, stopping time 10s, total duration 2min, and temperature 4℃.

[0205] In this invention, the M@PLGA- 10 The diameter of the BN complex is 165-195 nm, preferably 170-190 nm, and most preferably 175 nm-185 nm (e.g., 180 nm).

[0206] BNCT preparations

[0207] In the present invention, the term "BNCT agent" refers to a key targeted therapeutic drug with a very unique mechanism of action. It is not cytotoxic by itself, but rather acts as a "sensitizer". After boron-10 is enriched in tumor cells, by irradiating a beam of thermal or epithermal neutrons, which causes little damage to the human body, boron nuclei will capture neutrons and then undergo nuclear fission, producing high linear energy transfer α particles and lithium-7 particles. These heavy particles have a very short range (about the diameter of a cell), which can accurately destroy tumor cells rich in boron-10, while causing little damage to the surrounding normal tissues. Therefore, an ideal BNCT agent must have high tumor targeting and high tumor / normal tissue uptake ratio, which is the decisive factor for its efficacy and safety.

[0208] Applications

[0209] The present invention provides M@PLGA- 10 BX complexes (e.g. M@PLGA- 10 BN) for use in the preparation of a BNCT agent and / or an immune activator.

[0210] The present invention provides M@PLGA- 10 BX complexes (e.g. M@PLGA- 10 BN) for use in the preparation of a BNCT agent and / or an immune activator.

[0211] Pharmaceutical compositions

[0212] The present invention also provides a pharmaceutical composition comprising the above-mentioned M@PLGA- 10 BX complexes (e.g. M@PLGA- 10 BN) and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, with a pH of about 5-8, preferably a pH of about 6-8, although the pH value can vary depending on the nature of the substance being formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, intracranial, or topical administration.

[0213] The pharmaceutical composition of the present invention can be directly used for precise targeting of intracranial tumors (such as GBM glioblastoma). In addition, other therapeutic agents can also be used simultaneously.

[0214] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned M@PLGA- 10 BX complexes (e.g. M@PLGA-10 BN) and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing dextrose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the M@PLGA- 10 BX complex (e.g., M@PLGA- 10 BN) of the present application can also be used with other therapeutic agents.

[0215] When using the pharmaceutical composition, a safe and effective amount of M@PLGA- 10 BX complex (e.g., M@PLGA- 10 BN) is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dose should also take into account the route of administration, the patient's health status, and the like, which are within the skill of the skilled physician.

[0216] Compared with the prior art, the beneficial effects of the present application include:

[0217] (1) The biomimetic nanoplatform prepared by the present application has uniform particle size and morphology, and the characteristics of the macrophage membrane wrapping the PLGA- 10 BN core can be clearly seen.

[0218] (2) The biomimetic nanoplatform prepared by the present application has high biological safety, does not cause long-term toxicity when performing BNCT therapy, and the main organs including heart, liver, spleen, lung, and kidney maintain normal tissue structure, and no pathological changes such as inflammatory infiltration, necrosis, or fibrosis are observed.

[0219] (3) The biomimetic nanoplatform prepared by the present application exhibits good BNCT efficacy and strong immune microenvironment remodeling ability.

[0220] (4) The biomimetic nanoplatform prepared by the present application is superior to many boron compounds in terms of manufacturability. It can be modularly self-assembled by simple ultrasonic, the process is simple, the cost is controllable, and it has the potential for large-scale production.

[0221] (5) The platform has high expandability: on the one hand, different boron compounds can be replaced, and on the other hand, "customized" delivery can be achieved by modifying the macrophage membrane to meet diverse clinical needs.

[0222] The present invention will be further explained below with reference to specific embodiments.

[0223] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0224] Example 1

[0225] A method for preparing a biomimetic nanoplatform capable of precise delivery and efficient enrichment of boron 10 in glioblastoma:

[0226] (1) Extraction of RAW264.7 macrophage cell membrane

[0227] according to Figure 1 The diagram illustrates the process of culturing RAW264.7 cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number: CL-0190) in 10cm round dishes until approximately 100% confluence. Cells were then collected from 20 round dishes using a scraper, centrifuged at 250g for 4 min, and the supernatant was discarded. The cell pellet was washed three times with 0.1M PBS (pH 7.4). 30mL of hypotonic buffer (containing 1mM of protease inhibitor PMSF and 1× phosphatase inhibitor (Saiwell Biotech, catalog number: G2007-1ML)) was added to lyse the cells overnight at 4°C. The next day, the cell lysis buffer was sonicated at 12°C for 2 h (sonication power: 220W). The lysis buffer was then centrifuged at 4°C and 3200g for 10 min to remove cell nuclei. The supernatant was centrifuged at 20,000g for 20 min (twice) at 4℃ to remove organelles, and then left to stand overnight at 4℃. On the third day, the supernatant was centrifuged at 20,000g for 20 min at 4℃ to further remove precipitate. The supernatant was then ultracentrifuged at 100,000g for 1 h at 4℃. The supernatant was then discarded, and the oily precipitate at the bottom of the ultracentrifugation tube was washed three times with deionized water. After washing, the oily precipitate was resuspended in 10 mL of deionized water and dispersed by sonication in a water bath at 12℃ for 2 h (ultrasonic power 220W). Finally, the membrane solution was squeezed back and forth 30 times under a 1 μm polycarbonate porous membrane using a micro liposome extruder to remove larger membrane fragments. The membrane concentration was then quantified using the standard BCA method.

[0228] (2) Enrichment 10 B's h-10 Synthesis of BN

[0229] According to the synthesis route, 0.366 g of enriched boronic acid (6 mmol) with B (99.43% abundance) and 0.126 g of melamine (1 mmol) were thoroughly mixed by grinding in a marble mortar and then transferred to a specially designed corundum crucible (60 mm x 30 mm x 15 mm). The crucible was placed in the reaction zone of a tube furnace and heated to 900 °C at a programmed heating rate of 10 °C / min under continuous nitrogen protection (flow rate 100 mL / min). The nitridation process was completed after 3 h of isothermal reaction. After the reaction was completed, it was naturally cooled to room temperature to obtain ivory white blocky product, which was finely ground to obtain crude h- 10 BN powder. Then the powder was thoroughly dispersed by 4 h of ultrasonic treatment (power 900 W) in an ultrasonic crusher at 4 °C, and then dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cut-off of 10 kDa to remove small molecular by-products. Finally, the dialysate was treated at a centrifugal force of 1811 g for 5 min, and the milky white supernatant containing h- 10 BN was collected. The product was freeze-dried for 24 h and stored in a desiccator for use.

[0230] (3) Preparation of PLGA- 10 BN

[0231] According to the schematic diagram, 50 mg of PLGA (specification 50 / 50-COOH, Mw=15000) was dissolved in 2 mL of dichloromethane to accelerate the dissolution of PLGA in the ultrasonic cleaner. After the PLGA was completely dissolved in dichloromethane, 5 mg of h- 10 BN was added, and the h- 10 BN was uniformly dispersed in the ultrasonic cleaner. The power of the sonicator was set to 63 W, and the emulsion was formed by sonicating for 2 min (5 s on, 5 s off) in an ice bath. 8 mL of 4% PVA aqueous phase was added to the organic phase, and the power of the sonicator was set to 52 W. The uniform emulsion was formed by continuing to sonicate for 2 min (5 s on, 5 s off) in an ice bath. 10 mL of 2% isopropyl alcohol solution was added to the emulsion, and the nanoparticles were solidified by magnetic stirring at 25 °C for 12 h to volatilize the harmful dichloromethane. Then, the excess PVA was removed by centrifugation at 2500 rpm for 5 min, and finally the PLGA- 10 BN was collected and resuspended in deionized water.

[0232] (4) Preparation of M@PLGA- 10 BN

[0233] According to the schematic diagram, 50 mg of PLGA (specification 50 / 50-COOH, Mw=15000) was dissolved in 2 mL of dichloromethane to accelerate the dissolution of PLGA in the ultrasonic cleaner. After the PLGA was completely dissolved in dichloromethane, 5 mg of h- 10 BN was added, and the h- 10 BN was uniformly dispersed in the ultrasonic cleaner. The power of the sonicator was set to 63 W, and the emulsion was formed by sonicating for 2 min (5 s on, 5 s off) in an ice bath. 8 mL of 4% PVA aqueous phase was added to the organic phase, and the power of the sonicator was set to 52 W. The uniform emulsion was formed by continuing to sonicate for 2 min (5 s on, 5 s off) in an ice bath. 10 mL of 2% isopropyl alcohol solution was added to the emulsion, and the nanoparticles were solidified by magnetic stirring at 25 °C for 12 h to volatilize the harmful dichloromethane. Then, the excess PVA was removed by centrifugation at 2500 rpm for 5 min, and finally the PLGA- 10 BN was collected and resuspended in deionized water. Figure 4The diagram shows the macrophage membrane with PLGA- 10 BN was resuspended separately in deionized water to achieve final concentrations of 0.5 mg / mL and 1 mg / mL, respectively; then 400 μL of each were mixed together and subjected to ultrasonic encapsulation in an ultrasonic homogenizer to prepare M@PLGA- 10 BN (ultrasonic power 324W, working for 99s, stopping for 10s, total duration 2min, temperature 4℃).

[0234] Compare with Example 1

[0235] A method for preparing a biomimetic nanoplatform capable of precise delivery and efficient enrichment of boron 10 in glioblastoma:

[0236] h- in Example 1 10 BN was replaced with a red fluorescent probe for cell membranes (DiD), and the other steps were the same as in Example 1. The resulting PLGA nanonucleus was denoted as PLGA-DiD, and the biomimetic nanoplatform was denoted as M@PLGA-DiD.

[0237] Experimental Example 1

[0238] Regarding h- in Example 1 10 The structure and composition of BN were examined:

[0239] h- was recorded using a Bruker D8 Advance X-ray diffractometer (Cu Kα radiation, 40 kV, 40 mA). 10 BN powder X-ray diffraction (XRD) pattern. Fourier transform infrared spectroscopy (FTIR) test h- 10 BN powder was analyzed using a Nicolet iS50 spectrometer (Thermo, USA) in ATR mode. The results are as follows: Figure 5 As shown, the FTIR results indicate that h- 10 BN is between 3000 and 3700 cm -1 A broad absorption band exists within the range, attributed to the stretching vibrations of the OH and NH bonds. These results confirm the presence of h- 10 BN exhibits a large number of borate groups (-B(OH)2) and amino groups (-NH2), which may have been generated at the edges of the (002) crystal plane during water etching. Furthermore, FTIR values ​​at 1418.05 and 790 cm⁻¹... -1 Two strong peaks appear at h- 10 The stretching and bending vibrations of boron nitride (BN) were observed. XRD was used to investigate the crystal structure and phase composition of boron nitride. XRD results showed h- 10 BN nanoparticles exhibit good crystallinity, with diffraction peaks located at 26.7, 41.6, and 43.8°, corresponding to h- 10The (002), (100) and (101) crystal planes of BN.

[0240] Experimental Example 2

[0241] Regarding h- in Example 1 10 BN, PLGA- 10 BN and M@PLGA- 10 The morphology of BN was examined using transmission electron microscopy:

[0242] Take 5 μL of freshly prepared h- 10 BN, PLGA- 10 BN and M@PLGA- 10 BN was dropped onto an ultrathin carbon-coated copper mesh and evaporated until dry. After staining with 1% uranyl acetate for 30 seconds, the morphological characteristics of each formulation were obtained using a transmission electron microscope (JEM-200CX, JEOL, Japan) at an accelerating voltage of 100 kV. The results are as follows: Figure 6 As shown: h- 10 BN nanoparticles have an average diameter of approximately 8 nm, but due to their high surface energy, they form large aggregates on the transmission electron microscope grid; while compared to bare spherical PLGA- 10 Compared to BN, transmission electron microscopy images showed M@PLGA- 10 The typical spherical nucleoshell structure of BN demonstrates that the macrophage membrane successfully encapsulates PLGA- 10 BN kernel surface.

[0243] Experimental Example 3

[0244] For PLGA in Example 1 10 BN and M@PLGA- 10 The dynamic light scattering particle size and potential of BN were investigated:

[0245] The dynamic light scattering technique was used to accurately measure the properties of freshly prepared PLGA- 10 BN and M@PLGA- 10 BN particle size and zeta potential (Nano ZS, Malvern, UK). Results are as follows: Figure 7 As shown: PLGA- 10 The average hydrodynamic direct velocity of BN is approximately 160 nm, and the average Zeta potential is -19.8 mV; M@PLGA- 10 The average hydrodynamic particle size of BN is approximately 180 nm, and the average Zeta potential is approximately -23.0 mV. Compared to PLGA... 10 Compared to BN, its average size and charge are both increased, further indicating that the macrophage membrane has successfully coated PLGA- 10 BN surface.

[0246] Test Example 4

[0247] For PLGA in Example 1 10 BN and M@PLGA- 10 Quantitative study of BN uptake by GBM cells:

[0248] GL261 cells (common cells in this field, purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number: ZQ0932) were seeded in 6-well plates (1×10⁶ cells per well). 5 (each well), after culturing for 24 hours, add 10 PLGA- B concentration of 100 μg / mL 10 BN and M@PLGA- 10 BN nanoparticles were incubated for 24 h. The culture medium was then discarded, and the cells were washed three times with 0.1 M PBS (pH 7.4) to completely remove residual nanoparticles. Cells were then digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min to collect the cell pellet, washed twice with 0.1 M PBS (pH 7.4), and resuspended in an appropriate amount of 0.1 M PBS (pH 7.4). Cell counting was performed using an automated cell counter. The sample was treated with a mixed acid digestion method: 4 mL of concentrated nitric acid and 4 mL of hydrofluoric acid were added, and digestion was carried out at 230 °C under sealed conditions for 30 min. After cooling, the sample was transferred to a PTFE beaker and heated at 200 °C for 6 h to remove residual hydrofluoric acid, then brought to a final volume of 100 mL. The concentrations of BN nanoparticles in the solution were determined using inductively coupled plasma mass spectrometry (ICP-MS). 10 B content. Results are as follows: Figure 8 As shown: PLGA- 10 After co-incubating BN with cells for 12h and 24h, every 10 6 Intracellular average 10 The B content was 8.62 μg and 20.00 μg, respectively; while M@PLGA- 10 The boron accumulation in the BN-treated group was significantly higher, reaching 20.28 μg and 43.43 μg, respectively; these results indicate that after 24 h of incubation, both nanoparticles can induce intracellular... 10 The B content reached the conditions required for BNCT, which provides experimental evidence for the feasibility of BNCT killing GBM cells in vitro.

[0249] Experimental Example 5

[0250] The ability of PLGA-DiD and M@PLGA-DiD to cross the in vitro BBB in Control Example 1 was investigated:

[0251] Using 3μm pores (culture area 0.33cm²) 2) coated with 2% gelatin. bEnd.3 cells (conventional cells in the art, purchased from Wuhan Ponsay Life Science Co., Ltd., Cat. No. CL-059) were seeded on the Transwell at a density of 1 x 10 4 cells / well in the upper chamber (200 μL of medium), and 800 μL of medium was added to the lower chamber. The medium was replaced every 48 h, and after 14 days of culture, GL261 cells were seeded in the lower chamber at a density of 1 x 10 5 cells / well. After 24 h of culture to ensure that the cells were completely adherent, equal concentrations of PLGA-DiD and M@PLGA-DiD nanoparticle suspensions were added to the upper chamber. The experiment was terminated after incubation at 37 °C, 5% CO2for 8, 12 and 24 h, respectively; first, the medium in the lower chamber was discarded, and the cells were gently washed three times (1 mL each time) with pre-cooled 0.1 M PBS (pH 7.4), then the cells were collected by 0.25% trypsin (containing 0.02% EDTA) digestion at room temperature for 1 min, centrifuged at 1000 g for 5 min to obtain a cell pellet, and washed twice with 0.1 M PBS (pH 7.4) to remove free nanoparticles. Finally, the cells were resuspended in 0.1 M PBS (pH 7.4), and the DiD fluorescence intensity in the cells was detected using a flow cytometer. The results are shown in Fig. 6. Compared with the PLGA-DiD group, the fluorescence intensity of M@PLGA-DiD in GL261 cells was significantly increased by about 2-fold. In addition, the accumulation of nanoparticles in the cells showed a significant time-dependent increase with time. The above results indicate that M@PLGA-DiD can effectively penetrate the BBB and be efficiently taken up by GL261 cells. Figure 9

[0252] Test Example 6

[0253] The PLGA-BN and M@PLGA-BN prepared in Example 1 were investigated for their ability to cross the BBB in vitro and then be taken up by GBM cells: 10 The PLGA-BN and M@PLGA-BN prepared in Example 1 were investigated for their ability to cross the BBB in vitro and then be taken up by GBM cells: 10 The PLGA-BN and M@PLGA-BN prepared in Example 1 were investigated for their ability to cross the BBB in vitro and then be taken up by GBM cells:

[0254] After successfully constructing the in vitro BBB model according to the method in Test Example 5, GL261 cells were seeded in the lower chamber of the Transwell at a density of 1 x 10 10 The PLGA-BN and M@PLGA-BN prepared in Example 1 were investigated for their ability to cross the BBB in vitro and then be taken up by GBM cells: 10 The PLGA-BN and M@PLGA-BN prepared in Example 1 were investigated for their ability to cross the BBB in vitro and then be taken up by GBM cells: 10 ​BN nanoparticles, and continue to culture for 24 h. After the treatment, the culture medium was discarded and the cells were gently washed with pre-cooled 0.1 M PBS (pH 7.4) for 3 times to remove the non-internalized nanoparticles. Then the cells were digested with 0.25% trypsin (containing EDTA), and the cell precipitate was collected by centrifugation at 1000 rpm for 5 min and washed with 0.1 M PBS (pH 7.4) for 2 times. After that, the cell precipitate was gently resuspended in 1 mL of freshly prepared 2.5% glutaraldehyde (0.1 M PBS (pH 7.4) preparation) and fixed at room temperature for 30 min in the dark and then transferred to 4°C for overnight fixation. After subsequent standard electron microscopy sample preparation processes such as 1% osmium acid post-fixation, ethanol gradient dehydration, and epoxy resin embedding, ultrathin sections (70-90 nm) were prepared, double-stained with uranyl acetate and lead citrate, and then observed under a transmission electron microscope at an acceleration voltage of 120 kV. The results are shown in Figure 10 Fig. 6. The nanoparticles exist in the GL261 cells and are mainly located in the cytoplasm, as indicated by the red arrows. At the same time, it can also be found that the number of M@PLGA-BN in the tumor cells is significantly more than that of PLGA-BN. 10 Fig. 6. The nanoparticles exist in the GL261 cells and are mainly located in the cytoplasm, as indicated by the red arrows. At the same time, it can also be found that the number of M@PLGA-BN in the tumor cells is significantly more than that of PLGA-BN. 10 Fig. 6. The nanoparticles exist in the GL261 cells and are mainly located in the cytoplasm, as indicated by the red arrows. At the same time, it can also be found that the number of M@PLGA-BN in the tumor cells is significantly more than that of PLGA-BN. 10 Fig. 6. The nanoparticles exist in the GL261 cells and are mainly located in the cytoplasm, as indicated by the red arrows. At the same time, it can also be found that the number of M@PLGA-BN in the tumor cells is significantly more than that of PLGA-BN.

[0255] Test Example 7

[0256] The in vivo GBM targeting ability of PLGA-DiD and M@PLGA-DiD in Comparative Example 1 was investigated:

[0257] The establishment of the orthotopic GBM model was achieved by intracranial injection of 1 x 10 5 GL261-Luc luciferase-labeled cells (conventional cells in the art, Shanghai Zhijiao Xin Zhou Biotechnology Co., Ltd., Catalog No.: LZQ0065) (total volume 5 μL) at the right striatum of the mouse (2 mm from the right side, depth 2.5 mm). On the 7th day after injection, a strong autofluorescence signal was observed from the tumor cells using a small animal living imaging system (IVIS), which confirmed the successful establishment of the model. Subsequently, DiD, PLGA-DiD and M@PLGA-DiD nanoparticles were injected through the tail vein. In order to evaluate the accumulation of nanoparticles in the brain, the fluorescence signal in the brain of the mouse was dynamically monitored using IVIS at 1, 2, 4, 8, 12, 24, 48 and 72 h after injection. In addition, at 24 h after administration, the mouse was anesthetized and perfused and the main organ samples were obtained, and the same imaging system was used for quantitative analysis of the fluorescence intensity in vitro. The results are shown in Figure 11As shown, significant nanoparticle enrichment was observed in the tumor region 1 hour after M@PLGA-DiD injection, a characteristic that was significantly detected by the strong signal of the DiD fluorescent probe. Notably, nanoparticle accumulation peaked at 24 hours, followed by a gradual signal decay, but persisted for up to 72 hours. Quantitative analysis at 24 hours revealed that the fluorescence signal intensity in the tumor region of the M@PLGA-DiD group was significantly higher than that of the PLGA-DiD group and the free DiD control group.

[0258] Furthermore, in vitro fluorescence imaging results of major organs showed that M@PLGA-DiD nanoparticles exhibited lower fluorescence signals in organs such as the heart, liver, and spleen compared to PLGA-DiD, while strong fluorescence signals in the brain indicated significant enrichment of M@PLGA-DiD nanoparticles in GBM. This suggests that M@PLGA-DiD possesses a stronger GBM targeting ability. This result may stem from two aspects: firstly, the modification of the nanoparticle surface with macrophage membranes effectively constructs a protective layer for the nanoplatform, thereby reducing the likelihood of liver metabolic clearance and splenic macrophage phagocytosis, effectively avoiding non-specific distribution; secondly, the macrophage membrane itself enhances the brain targeting ability of the nanoplatform.

[0259] Experimental Example 8

[0260] For PLGA in Example 1 10 BN and M@PLGA- 10 The pharmacokinetics of BN in GBM-bearing mice and its distribution in GBM tissues were investigated.

[0261] GBM-bearing mice were injected with PLGA- via the tail vein. 10 BN and M@PLGA- 10 BN( 10 The dose for group B was 50 mg / kg (n=3 per group). Mice were euthanized at 12, 24, and 48 hours after administration. Samples were rapidly collected from major organs including the heart, liver, spleen, lungs, kidneys, normal brain tissue, and tumor tissue, as well as blood, and wet weight was determined. Subsequently, ICP-MS was used to analyze the levels of certain substances in the organs, tissues, and blood. 10 Quantitative analysis of B content was performed. The specific procedure was as follows: 4 mL of nitric acid and 4 mL of hydrofluoric acid were added to the collected organ tissue and blood samples, respectively, and the samples were placed in a reaction vessel and digested at 230°C for 30 min. After cooling, the digest was transferred to a polytetrafluoroethylene beaker and heated at 200°C for 6 h to remove unreacted hydrofluoric acid. Finally, the solution was quantitatively transferred to a 100 mL volumetric flask, diluted to volume, and analyzed. 10 B content.

[0262] To further observe the distribution of nanoparticles within GBM tissues, tumor-bearing mice were injected with PLGA- via the tail vein. 10 BN and M@PLGA- 10 BN( 10 (Dose B was 50 mg / kg). Mice were euthanized 24 hours after administration, and tumor tissue was rapidly collected. Residual blood was washed off the surface, and the tissue was cut into rice-grain-sized pieces using a sharp blade. The samples were then fixed in 2.5% glutaraldehyde at room temperature in the dark for 30 min, followed by fixation at 4°C in the dark for 24 h. Finally, transmission electron microscopy was performed to analyze the distribution characteristics of the GBM nanoparticles. Results are as follows: Figure 12 As shown: 24 hours after administration, M@PLGA- 10 BN Group in the GBM organization 10 The content of B (48.24 μg / g) was significantly higher than that of PLGA- 10 The BN group (17.16 μg / g) far exceeded the minimum threshold (20 μg / g) required for BNCT treatment. Furthermore, M@PLGA- 10 The tumor / normal tissue (T / N) and tumor / blood (T / B) ratios in the BN group were 3.95 and 3.85, respectively, both significantly higher than those in the PLGA group. 10 BN group. Overall, M@PLGA- 10 BN fully meets the requirements of boron-containing drugs for BNCT treatment.

[0263] Experimental Example 9

[0264] For PLGA in Example 1 10 BN and M@PLGA- 10 The ability of BN to inhibit GBM cell proliferation and induce GBM cell apoptosis in vitro was investigated.

[0265] 2×10 6 GL261 cells were seeded in 10cm culture dishes. After 24 hours of cell adhesion and growth, the cells were incubated with different materials or formulations at 37°C for 24 hours. 10 The concentration of B was 100 μg / mL. After incubation, cells were washed with 0.1 M PBS (pH 7.4), digested with trypsin to collect cells, and resuspended in DMEM medium containing 10% fetal bovine serum (a standard medium in this field, purchased from Gibco, catalog number: C11995500BT). Before neutron irradiation, the treated cells were resuspended in 200 μL of medium and transferred to 1 mL centrifuge tubes. Groups requiring neutron irradiation were exposed to a neutron beam for 20 min (neutron flux 6.96 × 10⁻⁶). 8 n·cm -2 ·s -1). Neutron beam was provided by the first AB-BNCT medical demonstration device with full independent intellectual property rights installed in the Mazu campus of the Affiliated Union Hospital of Fujian Medical University. After irradiation, the cells were reseeded and cultured for subsequent in vitro efficacy evaluation experiments.

[0266] Cell viability assay: The treated cells were first collected by centrifugation at 1000 rpm for 5 min and washed three times with 0.1M PBS (pH 7.4). Then, 4x10 3 GL261 cells were seeded in a 96-well plate and cultured for 48 h. After the end of the culture, the medium was removed and the cells were gently washed once with 0.1M PBS (pH 7.4). Then, 100 μL of medium containing 10% CCK-8 reagent was added to each well and incubated in a 37°C incubator for 2 h. Finally, the absorbance value of each well was measured at 450 nm wavelength using a microplate reader, and the relative cell viability of each experimental group was calculated according to the measurement results.

[0267] Apoptosis: The treated cells were first collected by centrifugation at 1000 rpm for 5 min and washed three times with 0.1M PBS (pH 7.4). Then, 2x10 5 GL261 cells were seeded in a 6-well plate and cultured for 48 h. After the end of the culture, the medium was removed and the cells were collected by trypsinization, then collected by centrifugation at 1000 rpm for 5 min and washed three times with 0.1M PBS (pH 7.4). Finally, the cells were stained according to the operating instructions of the Annexin V-FITC Apoptosis Detection Kit (Bi Yun Tian Biological Technology Co., Ltd., Catalog No: C1062S), and the apoptosis of the cells was analyzed by flow cytometry. The results are shown in Figure 13 PLGA- 10 BN+N and M@PLGA- 10 The cell viability of the PLGA- 10 BN+N treatment group showed a time-dependent decreasing trend, which was significantly different from the control group. Notably, the M@PLGA- 10 BN+N group showed a stronger cell proliferation inhibition effect, with a cell viability significantly lower than that of the PLGA- 10 B+N group at each time point. In particular, after 72 h of culture, the cell viability of the M@PLGA- 10 BN+N group decreased to 24.50%, while the PLGA- 10 BN group still maintained at 55.03%, which further confirmed that M@PLGA- 10 BN had a stronger anti-tumor effect. Flow cytometry detection results showed that compared with the untreated control group, the PLGA- 10 BN+N and M@PLGA-The apoptosis rate was significantly increased in all BN+N treatment groups. Quantitative analysis showed that M@PLGA- 10 The apoptosis rate in the BN+N group reached 20.73%, which was lower than that in the PLGA- 10 Both the BN+N group (12.15%) and the control group (4.32%) showed statistically significant differences. These results confirm that M@PLGA- 10 BN+N mediated BNCT can most effectively induce tumor cell apoptosis.

[0268] Experimental Example 10

[0269] For PLGA in Example 1 10 BN and M@PLGA- 10 The anti-GBM effect of BN-mediated BNCT in vivo was investigated.

[0270] The orthotopic transplantation GBM model was achieved by intracranial injection of 1×10 5 GL261-Luc luciferase-labeled cells (total volume 5 μL) were established, and the injection site was the right striatum of mice (coordinates: 2 mm from the right side, 2.5 mm depth). Mouse body weight was monitored every 4 days post-injection, and tumor growth was assessed using IVIS on day 7. Successfully modeled tumor-bearing mice were randomly divided into 6 groups: PBS control group, PBS+N group, PLGA- 10 Group BN 10 B dose 50mg / kg), PLGA- 10 BN+N group, M@PLGA- 10 Group BN 10 B dose 50mg / kg) and M@PLGA- 10 Group BN+N. After 24 hours of treatment in each group, a thermal neutron flux of 6.96 × 10⁻⁶ was applied. 8 n·cm -2 ·s -1 The mice were irradiated for 20 minutes. Tumor progression was then tracked every 5 days using an IVIS in vivo imaging system. Intracranial tumor volume was assessed in individual mice using T2-weighted imaging (T2WI) sequences of 11.7T magnetic resonance imaging (MRI). The natural survival time of the mice was recorded throughout the experiment.

[0271] The results are as follows Figure 14 As shown: M@PLGA- 10 The tumor fluorescence signal in the BN+N group decreased significantly over time, while that in other groups (including PLGA- 10 The BN+N group showed a continuous increasing trend. Quantitative analysis showed that on day 15 post-irradiation, M@PLGA- 10 The relative fluorescence intensity of the BN+N group was significantly lower than that of the PBS group and the PLGA group. 10BN+N group. PLGA- 10 Although the fluorescence signal of the BN+N group was reduced compared to the PBS group, there was no statistically significant difference. This may be related to the fact that the GBM tissue 10 of the BN+N group was less than 20 μg / g.

[0272] The MRI results were consistent with the IVIS data: M@PLGA- 10 The intracranial tumor volume of the BN+N group mice was significantly reduced, while the PLGA- 10 The BN+N group and the PBS control group showed obvious tumor progression. This finding further confirmed that M@PLGA- 10 BN-mediated BNCT had a significant anti-tumor effect. Survival analysis results showed that M@PLGA- 10 The survival of the BN+N group was significantly prolonged to 50 days, showing a significant survival benefit compared to other treatment groups. This survival advantage was highly consistent with the results of tumor growth inhibition described above. In addition, M@PLGA- 10 The body weight of mice in the BN+N treatment group gradually increased and remained stable from the 10th day, while the body weight of mice in the other control groups showed a continuous downward trend. This difference in body weight change indicates that M@PLGA- 10 BN-mediated BNCT can effectively inhibit tumor growth, thereby improving the health of mice.

[0273] Test Example 11

[0274] PLGA- 10 BN and M@PLGA- 10 Investigation of BN-mediated BNCT-induced GBM apoptosis and inhibition of GBM proliferation:

[0275] Tumor-bearing mice were treated under the same treatment conditions as in Test Example 10, and brain tissue was collected after euthanasia on day 15, fixed with 4% paraformaldehyde, and paraffin-embedded for histological analysis. Brain tissue sections were treated with H&E staining after deparaffinization, and observed by VS200 slide scanner. The specific experimental procedure of immunofluorescence is as follows: paraffin sections were treated with xylene solution (3 x 10 min), anhydrous ethanol (3 x 5 min), gradient ethanol (95% ethanol 2 x 5 min, 85% ethanol 1 x 5 min, 75% ethanol 1 x 5 min) in turn, and finally washed with deionized water (2 x 5 min). Then the sections were placed in 1 x antigen retrieval solution and heated at 95°C for 20 min, and after natural cooling, washed with 0.1M PBS (pH 7.4) (2 x 5 min). After drying the sections, the tissue area was circled with an immunohistochemical pen, and then blocked with immunofluorescence blocking solution for 15 min, and then diluted primary antibody was added and incubated at 4°C overnight. After equilibration at room temperature for 30 min the next day, 0.1M PBS (pH 7.4) (3 x 5 min) was used for washing, then 0.1M PBS (pH 7.4) diluted secondary antibody was added and incubated at room temperature for 2 h, after washing with 0.1M PBS (pH 7.4) (3 x 5 min), anti-fluorescence quenching mounting medium containing DAPI was used for mounting, the edges of the coverslips were sealed with nail polish, and finally VS120 slide scanner was used for observation and analysis. The results are shown in Figure 15 10 The expression of the proliferation marker Ki-67 in the tumor tissue of the BN+N group was significantly reduced, and the fluorescence signal of the apoptosis-related protein Caspase-3 was the strongest, indicating that M@PLGA- 10 BN-mediated BNCT can most effectively induce GBM apoptosis and inhibit its proliferation in vivo.

[0276] Test Example 12

[0277] The M@PLGA- 10 The immune regulation mechanism of BN-mediated BNCT was investigated:

[0278] The tumor tissues of mice treated with M@PLGA- 10 The tumor tissues of mice treated with M@PLGA- ​Total RNA was extracted using Invitrogen reagents according to standard procedures, and genomic DNA contamination was removed using DNase I (TaKaRa). mRNA was specifically enriched using Oligo dT magnetic beads, fragmented, and then first-strand cDNA was synthesized starting with random hexamer primers, followed by second-strand synthesis. Sequencing libraries were constructed through end repair, A-tailing, adapter ligation, fragment selection, PCR amplification, and purification. Library quality was assessed using a Qubit quantification system, real-time quantitative PCR, and a bioanalyzer to determine concentration, quantify effective library content, and detect fragment distribution. Qualified libraries were pooled according to effective concentration and sequencing data requirements, and then sequenced-by-synthesis (SBS) was performed using the Illumina platform. Bridge PCR amplification was performed by adding fluorescently labeled dNTPs, DNA polymerase, and adapter primers to the flow cell, and the specific fluorescence signal released upon incorporation of each dNTP was detected in real time. Sequence information was obtained through signal conversion. Raw data were analyzed using featureCounts (v2.0.6) for gene alignment and counting, and the FPKM value (expression level per million fragments of transcripts) was calculated for each gene. Differentially expressed genes were functionally annotated and enriched using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Set Enrichment Analysis (GSEA). Results are as follows: Figure 16 As shown: Compared with the PBS group, M@PLGA- 10 The BN+N group contained 2270 differentially expressed genes, of which 1907 were significantly upregulated and 363 were significantly downregulated. Further KEGG and GO enrichment analyses showed that the upregulated genes were mainly enriched in immune response-related pathways, including innate immune activation, adaptive immune regulation, and inflammatory cytokine signaling. Furthermore, the expression levels of many immune-related genes (such as Cxcl9, Cd8a, Cd36, Jchain, and NKg7) were significantly higher in the M@PLGA- 10 Significantly elevated in the BN+N group. These results indicate that M@PLGA- 10 BN+N mediated BNCT can effectively activate anti-tumor immune responses. In addition, the inventors also found that after M@PLGA-10BN+N treatment, the expression of PD-1 / PD-L1 pathway-related genes (including Lck, Cd3g, Lfng, Zap70, etc.) in tumor tissue was significantly upregulated, suggesting that this treatment may simultaneously activate immunosuppressive feedback mechanisms, thereby potentially weakening the tumor clearance ability of cytotoxic T cells.

[0279] Experimental Example 13

[0280] For M@PLGA- in Example 1 10 The anti-GBM ability of BN-mediated BNCT combined with PD-1 antibody was investigated:

[0281] The GBM model in situ was transplanted by intracranial injection of 1×10 5 GL261-Luc luciferase-labeled cells (total volume 5 μL) were injected into the right striatum of the mice (coordinates: 2 mm from the right side, depth 2.5 mm). The tumor-bearing mice successfully constructed were randomly divided into 4 groups: PBS control group, ICB monotherapy group, M@PLGA- 10 BN+N( 10 B 50mg / kg) treatment group, and M@PLGA- 10 BN+N+ICB( 10 B 50mg / kg) combination therapy group. Each group was subjected to neutron irradiation (flux 6.96×10 8 n·cm -2 ·s -1 ) for 20 min after 24 h of drug administration. After neutron therapy, the mice in the ICB treatment group and the combination therapy group were subjected to intraperitoneal injection of PD-1 antibody (5 mg / kg) every other day, for a total of 3 doses. During the treatment, the tumor growth was monitored by IVIS every 5 days, and the intracranial tumor volume of individual mice was quantitatively analyzed by T2-weighted imaging (T2WI) sequence of 11.7T MRI. The survival status of the mice was closely monitored and recorded throughout the experiment to evaluate the efficacy differences of the treatment regimens. The results are shown in Figure 17 10 The tumor fluorescence signals of the M@PLGA- 10 BN+N group and the combination therapy group showed time-dependent weakening, and the fluorescence signals of the combination therapy group completely disappeared on day 5, while those of the control group continued to increase. MRI evaluation on day 15 further confirmed that the intracranial tumor volume of the M@PLGA- 10 BN+N group and the combination therapy group was significantly reduced, and the tumor inhibition effect of the combination therapy group was more significant, while the ICB monotherapy group and the PBS group showed obvious tumor progression. These findings strongly confirmed that ICB treatment can significantly enhance the anti-tumor effect of BNCT mediated by M@PLGA- 10 BN+N. 10 The survival analysis results showed that the survival time of the mice in the M@PLGA- 10 BN+N group and the combination therapy group was significantly prolonged, and the mice in the combination therapy group were still alive on day 120.

[0282] Discussion

[0283] The nano-platform prepared in the present application is obtained by layer-by-layer covering and wrapping of h- 10 BN with richBNs are initially dispersible in deionized water due to their high negative surface charge and abundance of hydrophilic groups, but they tend to aggregate and precipitate in physiological solutions such as PBS due to the electronic shielding effect. Previous studies have used propylene glycol (PG) and polyethylene glycol (PEG) to modify h- 10 BNs to increase their dispersibility and colloidal stability under physiological conditions, thus facilitating biomedical applications. However, the above methods have the disadvantages of multiple steps and high experimental conditions.

[0284] Therefore, the inventors explored the use of PLGA high molecular nanomaterials and a modified version of the traditional oil-in-water (O / W) single emulsion evaporation technique to encapsulate h- 10 BN. Subsequently, the present application introduced macrophage membranes into the nanoplatform through simple ultrasonication, and this biomimetic strategy can help PLGA- 10 BNs to continuously cross the BBB and target GBM, thus achieving a high concentration of B at the tumor site 10 B, while significantly increasing the B concentration ratio of T / N and T / B, laying the foundation for BNCT therapy.

[0285] In addition, M@PLGA- 10 BNs not only directly kill tumor cells, but also release tumor antigens and activate systemic immune responses by inducing immunogenic cell death, which provides a theoretical basis for the combined application of immunotherapy.

[0286] In summary, the biomimetic nanoplatform M@PLGA- 10 BN prepared by the present application shows good application prospects as a BNCT preparation, and the innovative strategy of combining immunotherapy not only provides a new idea for improving the efficacy of BNCT in treating GBM, but also has important clinical translation value.

[0287] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The application is intended to cover any variations, uses or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice in the art to which the application pertains. The specification and examples are to be regarded as exemplary only, and the true scope and spirit of the application are indicated by the appended claims.

Claims

1. An M@PLGA- 10 BX composite characterized in that, M is the cell membrane of macrophages, PLGA is polylactic acid-glycolic acid copolymer, 10 BX is a boron compound, X is selected from: N, C, H, O.

2. The M@PLGA- of claim 1, 10 BX complex, characterized in that, The M@PLGA- 10 BX is M@PLGA- 10 BN, wherein 10 BN is boron nitride.

3. The M@PLGA-10BX complex of claim 1, wherein, The macrophage is selected from the group consisting of macrophage RAW264.7, macrophage J774, macrophage THP-1.

4. The M@PLGA- 10 BN complex of claim 2, wherein the preparation method is characterized by, The method comprises the steps of: ​ (1) providing a cell membrane M of a macrophage (2) provide h- 10 BN; (3) providing PLGA- 10 BN; (4) Preparation of M@PLGA- 10 BN composites.

5. The production method according to claim 4, wherein In step (3), the PLGA- 10 The method for preparing BN comprises the following steps: (3a) Preparation of PLGA and h- 10 Organic phase suspension of BN; (3b) subjecting the organic phase suspension in (3a) to low-temperature sonication to form an organic phase of emulsifier; (3c) adding an aqueous phase to the organic phase in (3b) and then continuing low-temperature sonication to form a uniform emulsifier; (3d) solidifying the nanoparticles and volatilizing the organic phase; (3e) centrifugation to remove excess aqueous phase and collect the nanoparticle PLGA- 10 BN.

6. The production method according to claim 4, wherein In the step (4), M@PLGA is prepared 10 The BN includes the steps of: (4a) combining macrophage membranes with PLGA 10 BN were mixed after each was resuspended to form a solution; (4b) encapsulating the mixture from (4a) to produce M@PLGA- 10 BN.

7. The production method according to claim 4, wherein In the step (4), M@PLGA is prepared 10 The BN includes the steps of: (4a) Macrophage membranes were mixed with PLGA- 10 BN were each resuspended in deionized water and mixed together; (4b) subjecting the mixture from (4a) to ultrasonication encapsulation in an ultrasonic disrupter to produce M@PLGA- 10 BN.

8. The M@PLGA- of claim 1 or 2 10 Use of the BX complex in the manufacture of a BNCT formulation and / or an immune activator.

9. Use according to claim 8, wherein the compound is ###0002### The disease to be treated by the BNCT preparation is selected from the group consisting of glioblastoma (GBM), tongue cancer, melanoma, breast cancer, intestinal cancer.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (i) the M@PLGA of claim 1 or 2 10 BX composite; and (ii) a pharmaceutically acceptable carrier.

11. A drug combination, characterized in that, comprises: (i) a first active ingredient comprising M@PLGA as described in claim 1 or 2 10 BX complex, or a pharmaceutical composition as described in claim 9; (ii) a PD-1 antibody.

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