A nanodiagnosis and treatment preparation as well as a preparation method and application thereof

Abstract

The present application belongs to the technical field of nanodiagnosis and nanotherapy preparation, and particularly relates to a kind of nanodiagnosis and nanotherapy preparation and its preparation method and application.The present application realizes the closed loop of "boron drug delivery-treatment-evaluation of curative effect" by using M1 macrophage exosome membrane as a biomimetic shell, and gold nanoparticles with surface modification of carborane and fluorescent polypeptide sequence as nanocomposite core, solves the problems that traditional preparations are difficult to guarantee sufficient concentration of boron drug delivery and lack of instant feedback of cell apoptosis level during treatment in boron neutron capture therapy.The present application not only optimizes the delivery efficiency of boron drugs, makes the boron drugs aggregate around the cell nucleus, significantly improves the curative effect of boron neutron capture therapy in glioma treatment, but also monitors the treatment effect in real time through the recovery mechanism of fluorescent signal, thereby solving the problem of lag in clinical efficacy evaluation, and has important clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanotherapeutic agents, specifically relating to a nanotherapeutic agent, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common primary intracranial tumors of the central nervous system, characterized by poor prognosis, high invasiveness, and high recurrence rates. High-grade gliomas, especially glioblastomas, are insensitive to surgery, radiotherapy, chemotherapy, immunotherapy, and gene therapy; even with combined treatment, the 5-year survival rate remains below 5%. The main reason is the difficulty in completely removing infiltrating tumor cells without damaging normal brain tissue, necessitating new, highly effective, and low-toxicity strategies.

[0003] Boron neutron capture therapy (BNCT) is a novel radiotherapy modality. Its principle is to stabilize isotopes. 10 When B is irradiated by low-energy thermal neutrons or ultrathermal neutrons, it undergoes a capture-fission reaction, producing high-energy, short-range alpha particles and... 7 Li 3+ The particles (with ranges of approximately 9 μm and 4 μm, respectively) have a killing range limited to the scale of a single cell (~20 μm) and have minimal impact on surrounding normal tissues, thus offering a unique advantage in the treatment of gliomas.

[0004] To advance the clinical translation of boron-containing drug therapy (BNCT), two core issues urgently need to be addressed: First, the development of highly selective boron drug delivery systems. This requires boron-containing drugs to accumulate in sufficient doses within tumor cells. 10 B. The boron drug must remain within the tumor tissue for a sufficient time to achieve a tumor-to-normal tissue boron concentration ratio of 3-4:1, thus ensuring treatment selectivity. Secondly, precise measurement of radiation dose and assessment of efficacy are crucial. Currently, tumor recurrence and radiation necrosis of normal tissue still occur in BNCT clinical practice, mainly because it is difficult to monitor boron drug distribution and predict efficacy in real time during surgery, resulting in the inability to individually adjust the radiation dose. Therefore, achieving targeted and efficient boron drug delivery for gliomas and precise assessment of BNCT efficacy are current research priorities and challenges. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the present invention aims to provide a nano-therapeutic formulation, its preparation method, and its application. This formulation achieves a closed loop of "boron drug delivery-treatment-efficacy evaluation" through a single formulation, aiming to solve the problems in boron neutron capture therapy where traditional formulations cannot guarantee sufficient concentration of boron drug delivery and lack real-time feedback on the level of cell apoptosis during treatment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a nanotherapeutic agent comprising a biomimetic shell and a nanocomposite core; wherein the biomimetic shell is an M1 macrophage exosome membrane, and the nanocomposite core comprises gold nanoparticles and surface-modified carborane and fluorescent polypeptide sequences; wherein the fluorescent polypeptide sequences can be specifically recognized and cleaved by apoptosis markers.

[0007] A second aspect of the present invention provides a method for preparing the nanotherapeutic agent described in the first aspect, comprising the following steps: The pH of the gold nanoparticle colloidal solution was adjusted, fluorescent peptides and carborane were added, and the solution was frozen and incubated in the dark. After thawing at room temperature and purification, surface-functionalized gold nanoparticles AuBC were obtained. Pre-cooled M1 macrophage exosomes (mEVs) were mixed with AuBC solution, placed in an ice bath, sonicated, incubated at low temperature overnight, and purified to obtain surface-functionalized gold nanoparticles AuBC@mEVs coated with M1 exosome membranes, which are nanotherapeutic agents.

[0008] A third aspect of the present invention provides the application of the nanotherapeutic agents described in the first aspect in the preparation of antitumor drugs, wherein the tumor includes head and neck tumors, brain tumors, and melanoma; Preferably, the tumor is a glioma; More preferably, the tumor is a glioblastoma.

[0009] A fourth aspect of the present invention provides the application of the nanotherapeutic agents described in the first aspect in evaluating the efficacy of boron neutron capture therapy by monitoring changes in fluorescence signals to assess the efficacy of boron neutron capture therapy.

[0010] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The nanotherapeutic formulation provided by this invention can significantly improve the efficacy of BNCT in the treatment of gliomas and provide real-time assessment feedback for the treatment process. It not only optimizes the delivery efficiency of boron drugs, enabling them to accumulate around the cell nucleus and enhance intracellular efficacy, but also... 10 B atom loading; and through the fluorescence signal recovery mechanism, it releases fluorescence signals in situ during BNCT, allowing for real-time monitoring of treatment effects, thereby solving the problem of delayed efficacy assessment in clinical practice, and has important clinical application prospects.

[0011] (2) The coating of the M1 type exosome membrane can not only improve the biocompatibility of nanodiagnosis and treatment, increase its targeting of glioma cells, and achieve efficient boron delivery across the blood-brain barrier, but also the co-stimulatory factors such as CD80 and CD86 carried on its surface have the potential to reshape the tumor microenvironment and change from immunosuppression to immune activation, thus forming a potential functional complement to the physical killing of BNCT. Attached Figure Description

[0012] Figure 1 Compound II in Example 1 11 B NMR characterization image.

[0013] Figure 2 This is a mass spectrometry characterization of the thiol-modified polypeptide in Example 1.

[0014] Figure 3 This is a high-performance liquid chromatogram of the thiol-modified polypeptide in Example 1.

[0015] Figure 4 This is a hydrated particle size distribution diagram of AuBC in Example 1.

[0016] Figure 5 This is an immunofluorescence staining characterization image of CD80 and CD86 in M1 macrophages in Example 1.

[0017] Figure 6 This is a particle size distribution diagram of M1 type exosomes mEVs measured by NTA in Example 1.

[0018] Figure 7 This is a hydration particle size distribution diagram of AuBC@mEVs in Example 1.

[0019] Figure 8 This is an electron microscope image of AuBC@mEVs in Example 1.

[0020] Figure 9 The image shows the results of Western blotting analysis of surface proteins of M1 type exosomes mEVs and AuBC@mEVs in Example 1.

[0021] Figure 10 The graphs show the quantitative test results of the peptides in Example 2. A represents the standard calibration curve of the thiol-modified peptide labeled with TAMRA; B represents the fluorescence measurement results of AuBC prepared in Example 1 before and after DTT treatment.

[0022] Figure 11 The graph shows the biosafety test results of AuBC@mEVs in Example 3.

[0023] Figure 12 These are confocal images of AuBC@mEVs cells incubated for different time periods in Example 4. The first column shows the magnified portion within the white box on the right.

[0024] Figure 13The images show the spatial distribution and boron concentration quantification of cells after incubation with AuBC@mEVs in Example 4. A represents the fluorescence colocalization results of AuBC@mEVs; B represents the linear fluorescence intensity distribution of a single cell; C represents the spatial distribution of AuBC@mEVs; D represents the quantitative analysis results of the spatial distribution of AuBC@mEVs; E represents the flow cytometry analysis of cells after incubation with AuBC@mEVs for different time periods; and F represents the intracellular boron-10 uptake measured by ICP-MS after incubation with different concentrations of AuBC@mEVs.

[0025] Figure 14 The figure shows the results of the correlation verification experiment between the fluorescence activation ability of AuBC@mEVs and cell apoptosis in Example 5. Among them, A is the fluorescence intensity quantification graph at different time points after AuBC@mEVs treatment; B is the ELISA quantitative results of intracellular Caspase-3 after treatment at different time points; C is the result of the linear regression equation constructed based on the average gray value and Caspase-3 protein concentration.

[0026] Figure 15 The images show the results of fluorescence signal observation and quantitative analysis using confocal microscopy (CLSM) for different treatment groups in Example 6. In this figure, A represents fluorescence imaging of different treatment groups; B represents the quantification of fluorescence intensity for different treatment groups.

[0027] Figure 16 The images show the results of immunofluorescence staining analysis of different treatment groups in Example 6. A represents fluorescence imaging of different treatment groups; B represents the quantitative statistical graph of absorbance in the colony formation experiment.

[0028] Figure 17 The images show the results of the colony formation experiment in Example 6. A shows images of cell colony formation in different treatment groups; B shows the UV absorption spectrum obtained after dissolving the cells in different treatment groups.

[0029] Figure 18 The images show the results of the Transwell invasion experiment in Example 6. In this figure, A shows images of cell invasion in different treatment groups; B shows a statistical chart of the relative number of invasive cells in different treatment groups. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0032] This invention provides a nanotherapeutic formulation comprising a biomimetic shell and a nanocomposite core. The biomimetic shell is an M1 macrophage exosome membrane, and the nanocomposite core consists of gold nanoparticles and surface-modified carborane and fluorescent peptide sequences, wherein the fluorescent peptide sequences can be specifically recognized and cleaved by apoptosis markers.

[0033] In some embodiments of the present invention, the carborane is terminally modified with a thiol group and anchored to the surface of gold nanoparticles (AuNPs) via a gold-sulfur bond (Au-S). The structure of the carborane is as follows: .

[0034] In some embodiments of the present invention, the apoptosis marker is caspase-3.

[0035] In some embodiments of the present invention, the N-terminus of the fluorescent polypeptide sequence is labeled with the 5-carboxytetramethylrhodamine fluorescent group TAMRA, with the sequence TAMRA-Gly-Trp-Glu-His-Asp-Gly-Lys-Cys-COOH. The fluorescent polypeptide sequence is anchored to the surface of gold nanoparticles (AuNPs) via a gold-sulfur bond (Au-S). Wherein, Gly stands for glycine (G); Trp for tryptophan (W); Glu for glutamic acid (E); His for histidine (H); Asp for aspartic acid (D); Lys for lysine (K); and Cys for cysteine ​​(C).

[0036] In some embodiments of the present invention, the molar ratio of carborane modified on each gold nanoparticle to fluorescent peptide chain is 2.1-2.5:1. The inventors have discovered that when the molar ratio of carborane modified on each gold nanoparticle to fluorescent peptide chain is controlled at 2.1:1-2.5:1, it not only provides the necessary components for BNCT treatment... 10 The high boron loading and the ability to maintain optimal quenching density on the surface of gold nanoparticles ensure signal sensitivity.

[0037] Preferably, the molar ratio of carborane modified on each gold nanoparticle to fluorescent peptide chain is 2.3:1.

[0038] In this invention, an M1 macrophage exosome membrane is used as a biomimetic shell to encapsulate a nanocomposite core. Based on the inherent transendothelial transport mechanism of M1 cells, effective penetration of the blood-brain barrier can be achieved. Simultaneously, the co-stimulatory factors such as CD80 and CD86 carried on the surface of the M1 macrophage exosome membrane possess the potential to reshape the tumor microenvironment and shift from immunosuppression to immune activation, thus potentially complementing the physical killing effect of BNCT.

[0039] In some embodiments of the present invention, the hydrated particle size of the nanotherapeutic agent is mainly distributed in the range of 120-160 nm.

[0040] This invention also provides a method for preparing nanotherapeutic agents, comprising the following steps: The pH of the gold nanoparticle colloidal solution was adjusted, fluorescent peptides and carborane were added, and the solution was frozen and incubated under light-protected conditions. After thawing at room temperature and purification, surface-functionalized gold nanoparticles (AuBC) were obtained.

[0041] Pre-cooled M1 macrophage exosomes (mEVs) were mixed with AuBC solution, placed in an ice bath, sonicated, incubated at low temperature overnight, and purified to obtain surface-functionalized gold nanoparticles (AuBC@mEVs) coated with M1 exosome membranes, which are nanotherapeutic agents.

[0042] In this invention, AuBC is prepared using a freeze-thaw method, which improves the efficiency of surface functionalization modification.

[0043] The preparation process of this invention is simple, and a balance between treatment and monitoring is achieved through specific component ratios. Compared with formulations without exosome protection or using inorganic boron nitride, the AuBC@mEVs prepared in this invention exhibit higher biostability and in-situ therapeutic feedback performance.

[0044] In some embodiments of the present invention, the pH of the gold nanoparticle colloidal solution is adjusted to 7.2-7.4 so that the charge state of the amino acid side chains in the peptide chain is most favorable for the formation of gold-sulfur bonds.

[0045] Preferably, the molar ratio of carborane, fluorescent peptide, and Au NPs is 100:300:1.

[0046] In some embodiments of the present invention, when preparing AuBC, the temperature for freezing incubation is -20 ℃ to -35 ℃, and the time is 25-35 min.

[0047] Preferably, when preparing surface-functionalized gold nanoparticles, the freeze-incubation temperature is -20 °C and the time is 30 min. This allows the ligands to accumulate and efficiently couple to the surface of the gold nanoparticles during the freeze-incubation process.

[0048] In some embodiments of the present invention, when preparing AuBC, the purification process adopts low-temperature high-speed centrifugation at a temperature of 3-5 ℃, a rotation speed of 9000-11000 rpm, and a centrifugation time of 8-12 min. The supernatant is discarded, and the precipitate is redispersed with 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES).

[0049] Preferably, when preparing surface-functionalized gold nanoparticles, the purification process uses low-temperature high-speed centrifugation at a temperature of 4°C, a rotation speed of 10,000 rpm, and a centrifugation time of 10 min.

[0050] In some embodiments of the present invention, the particle ratio of pre-cooled M1 macrophage exosomes (mEVs) to AuBC solution is 1:8-12, and the ice bath standing time is 8-12 min.

[0051] Preferably, the ratio of pre-cooled M1 macrophage exosomes (mEVs) to AuBC solution is 1:10, and the ice bath standing time is 10 min.

[0052] In some embodiments of the present invention, the ultrasonic power is 25-35 W and the duration is 5-10 min. Preferably, the ultrasonic power is 30 W and the duration is 5 min, using a pulse mode with an on for 2 seconds and an off for 5 seconds.

[0053] In some embodiments of the present invention, the low-temperature incubation temperature for preparing AuBC@mEVs is 3-5 °C, preferably 4 °C.

[0054] In some embodiments of the present invention, the synthetic route of the carborane compound II is as follows:

[0055] In some embodiments of the present invention, the method for preparing the carborane compound II includes the following steps: o-Carborane, propylene oxide, and n-butyllithium were dissolved in tetrahydrofuran and reacted under heating and stirring. Hydrochloric acid and deionized water were added, and the mixture was extracted, dried, and filtered. The solution was then concentrated under reduced pressure to obtain compound I.

[0056] Under an inert gas atmosphere, compound I, triphenylphosphine, N-bromosuccinimide (NBS), and dichloromethane were mixed and reacted with stirring at room temperature. Sodium bicarbonate solution was added, and stirring continued at room temperature. Extraction and drying yielded a purified intermediate.

[0057] Under an inert gas atmosphere, the intermediate, thiourea, and ethanol were mixed and heated to reflux. After cooling to room temperature, sodium hydroxide solution was added and stirred at room temperature; then hydrochloric acid was added. The mixture was extracted, dried, and filtered; concentrated under reduced pressure to give compound II.

[0058] This invention also provides the application of nanotherapeutic agents in the preparation of antitumor drugs. The tumors include head and neck tumors, brain tumors, and melanoma.

[0059] In some embodiments of the present invention, the tumor is a glioma.

[0060] Preferably, the tumor is a glioblastoma.

[0061] The nanotherapeutic agents can effectively enter cells, enabling the accumulation of boron drugs around the cell nucleus. This maximizes the overlap between the ionization track and nuclear DNA, thereby enhancing the efficacy of boron neutron capture therapy.

[0062] The nanotherapeutic agents enable intracellular... 10 The boron atom loading is 6 times higher than the generally accepted critical threshold for BNCT treatment, providing a sufficient dosage basis for clinical treatment administration.

[0063] This invention also provides the application of a nanotherapeutic agent in evaluating the efficacy of boron neutron capture therapy. The efficacy of boron neutron capture therapy is assessed by monitoring changes in fluorescence signals.

[0064] Before BNCT treatment, the nanotherapeutic agent exhibits extremely low fluorescence signal because the emission spectrum of TAMRA fluorescent molecules overlaps with the absorption spectrum of AuNPs, resulting in initial fluorescence signal quenching by the core layer. After BNCT treatment, the secondary particles generated by the nanotherapeutic agent induce apoptosis. The activated apoptosis protease acts as a "trigger switch," releasing fluorescence in situ. The recovery of fluorescence signal is positively correlated with the activation ratio of Caspase-3, indicating that the recovery of fluorescence signal from the nanotherapeutic agent can serve as an effective indicator for real-time treatment assessment, solving the problem of delayed efficacy evaluation in clinical practice.

[0065] The nanotherapeutic agents described herein are used to simultaneously evaluate the apoptotic response of tumor cells during BNCT neutron irradiation by measuring the enhancement of fluorescence signals. In this invention, the ultra-high quenching efficiency of AuNPs on fluorescent molecules ensures that the background signal is close to zero; and under BNCT, the rapid activation of intracellular Caspase-3 enzyme triggers signal recovery, with its fluorescence intensity gain exhibiting a strict mathematical linear relationship with neutron dose and cell death rate.

[0066] In some embodiments of the present invention, unless otherwise specified, the raw materials can be prepared or purchased using conventional methods in the art.

[0067] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0068] Example 1: 1. Preparation of mercapto-modified carboranes: The synthetic route for thiol-modified carboranes is shown below.

[0069] Specifically, the steps include the following: (1) Dissolve 1.0 mmol of o-carborane, 1.0 mmol of propylene oxide and 1.0 mmol of n-butyllithium in 10 mL of tetrahydrofuran and stir at 30 °C for 8 h. Then add 1.0 mmol of hydrochloric acid and 10 mL of deionized water to the reaction system and extract three times with 20 mL of dichloromethane. Combine all the dichloromethane organic phases after extraction, add excess anhydrous sodium sulfate (supersaturated solid-liquid coexistence state) to the organic phase for drying, and let stand at room temperature for 30 min for drying (drying endpoint: no obvious clumps when shaking the system, the inorganic desiccant is loose and flowing like sand, and the organic phase is clear and transparent); filter to remove the solid desiccant and collect the filtrate. The filtrate was transferred to a rotary evaporator, and the water bath temperature was set to 35 ℃ and the vacuum pressure to 0.06-0.08 MPa for vacuum concentration and rotary evaporation. During the rotary evaporation process, the liquid state in the flask was observed. When there was no obvious droplet condensation, only a small amount of oily solid remained on the flask wall, and no flowing solvent layer, the pressure was maintained for another 5 min to completely remove the residual dichloromethane solvent, and the target compound I was obtained. The product yield of this step was 50%.

[0070] (2) Under an argon atmosphere, 1.0 mmol of compound I prepared in step (1), 1.2 mmol of triphenylphosphine, and 1.2 mmol of N-bromosuccinimide (NBS) were added to a 100 mL reaction flask, followed by 30 mL of dichloromethane to obtain a mixed solution. The mixture was stirred at room temperature for 3 h, and then 5 mL of 10 mol / L sodium bicarbonate solution was added to quench the neutralization reaction. The system was acidic, and stirring was continued at room temperature for 0.5 h. 20 mL of dichloromethane was added for extraction, and all organic phases were separated and collected. The combined dichloromethane organic phases were transferred to a vacuum drying oven and dried at room temperature and atmospheric pressure for 2 h (drying endpoint: no solvent droplets in the flask, no flowing liquid phase on the flask wall, and no change in mass). The purified intermediate 1-(3-bromopropyl)o-carborane was obtained, with a yield of 60%.

[0071] (3) Under an argon atmosphere, add 6 mmol of the intermediate prepared in step (2), 6.6 mmol of thiourea and 10 mL of ethanol to a 50 mL reaction flask, mix, and heat under reflux at 80 °C for 16 h. After cooling the reaction solution to room temperature, add 8 mL of 2 mol / L sodium hydroxide solution and stir at room temperature for 0.5 h; then add 12 mmol of hydrochloric acid to the system. Extraction was performed using dichloromethane, with 20 mL used each time, for three extractions. All dichloromethane organic phases were combined, and excess anhydrous sodium sulfate was added to maintain a supersaturated solid-liquid coexistence state for drying. The mixture was allowed to stand at room temperature for 30 min (drying endpoint: no obvious clumps when shaken, inorganic desiccant appearing loose and flowing like sand, and the organic phase clear and transparent). The solid desiccant was removed by filtration. The filtrate was transferred to a rotary evaporator, and concentrated under reduced pressure at a water bath temperature of 35 ℃ and a vacuum pressure of 0.06-0.08 MPa. The liquid state in the flask was observed during rotary evaporation. Once no obvious droplets condensed, only a small amount of oily solid remained on the flask wall, and no flowing solvent layer was observed, the reduced pressure was maintained for another 5 min to completely remove the residual dichloromethane solvent, yielding compound II. The yield of this step was 40%.

[0072] Compound II was subjected to 11 B-NMR characterization, experimental results are as follows Figure 1 As shown, this embodiment successfully prepared a thiol-modified carborane, the structure of which is shown below: .

[0073] 2. Surface functionalization of gold nanoparticles: The thiol-modified peptide was purchased from Sangon Biotech (Shanghai) Co., Ltd. The N-terminus of the thiol-modified peptide is labeled with the 5-carboxytetramethylrhodamine fluorescent group TAMRA, and the sequence is TAMRA-Gly-Trp-Glu-His-Asp-Gly-Lys-Cys-COOH. Figure 2 and Figure 3 The images show the mass spectrometry and high-performance liquid chromatography (HPLC) chromatograms of the thiol-modified polypeptide.

[0074] Monodisperse gold nanoparticles were synthesized using the classic sodium citrate reduction method. The specific procedure was as follows: 100 mL of a 0.01% (w / w) aqueous solution of chloroauric acid was heated to a vigorous boil with continuous stirring; then, 3.6 mL of a 1% (w / w) aqueous solution of sodium citrate was rapidly added to the boiling system. The reaction was maintained at boiling for 20 min until the system turned a stable wine-red color. The heat source was removed, and the solution was stirred until cooled to room temperature. The resulting colloidal solution of gold nanoparticles was filtered through a 0.22 μm filter to remove large particle aggregates and stored at 4 °C for later use. The molar concentration of the gold nanoparticles was determined using UV-Vis absorption spectroscopy at a wavelength of 524 nm, and the molar extinction coefficient was taken as 2.7 × 10⁻⁶. 8 L mol 1 cm 1 .

[0075] The synthesized gold nanoparticle colloidal solution was centrifuged, resuspended, and the pH adjusted to 7.2-7.4 to obtain a 1 nM Au NPs colloidal solution. 300 nM of a thiol-modified peptide and 100 nM of compound II were added to this colloidal solution, with a molar ratio of compound II, thiol-modified peptide, and Au NPs of 100:300:1, resulting in a mixed solution. The mixed solution was incubated at -20 °C in the dark for 30 min to allow the ligands and compound II to accumulate and efficiently couple to the surface of the gold nanoparticles during incubation. The solution was thawed at room temperature and purified by low-temperature high-speed centrifugation at 4 °C and 10,000 rpm for 10 min. The supernatant containing unreacted ligands was discarded, and the precipitate was redispersed with 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES). This purification and washing process was repeated three times to ensure complete removal of excess thiol-modified peptide and compound II. Finally, surface-functionalized gold nanoparticles (AuBC) were obtained. The prepared AuBC was stored at 4 °C in the dark.

[0076] The hydration size profile of AuBC was determined using dynamic light scattering (DLS), and the results are as follows: Figure 4 As shown, the average hydrated particle size of AuBC is approximately 22.1 nm.

[0077] 3. Preparation of AuBC@mEVs: Because THP-1 cells are highly similar to human primary monocytes in morphology and function, they were used as an experimental model. Human monocytic leukemia cells (THP-1 cells) were used as the experimental model. Treatment with phorbol 12-myristate 13-acetate (PMA) induced macrophage differentiation, followed by lipopolysaccharide (LPS) to induce M1-type polarization. M1 macrophages were cultured in insulin-transferrin-selenium (ITS) supplemented medium for 48 hours. Secreted M1 macrophage exosomes (mEVs) were collected by differential ultracentrifugation. Immunofluorescence staining images of CD80 and CD86 in M1 exosomes (mEVs) are shown below. Figure 5 As shown, this demonstrates that the exosomes obtained in this embodiment originated from M1 macrophages. The particle size distribution of M1-type exosomes (mEVs) measured by nanoparticle tracking analysis (NTA) is shown in the figure below. Figure 6 As shown, it can be demonstrated that the M1 type exosomes mEVs obtained in this embodiment have good uniformity and relatively concentrated particle size, which is an ideal exosome state.

[0078] A pre-cooled mEVs suspension at 4 °C was mixed with an AuBC solution at a particle number ratio of 1:10 and allowed to stand in an ice bath for 10 minutes to stabilize. Subsequently, the mixture was sonicated for 5 minutes using a probe-type ultrasonic generator at 30 W power in pulse mode (on for 2 seconds, off for 5 seconds) to obtain a mixed solution. The entire process was carried out in an ice-water bath. After incubating the mixed solution overnight at 4 °C, it was purified by centrifugation at 8500 rpm for 5 min to remove uncoated AuBC particles and mEVs fragments, obtaining M1-type exosome membrane-coated AuBCs (AuBC@mEVs).

[0079] The hydrated particle size of AuBC@mEVs was determined using dynamic light scattering (DLS), and the results are as follows: Figure 7 As shown, the hydrated particle size of AuBC@mEVs is mainly distributed in the range of 120-160 nm, making them suitable for cellular uptake.

[0080] Electron micrographs of AuBC@mEVs are shown below. Figure 8 As shown in the figure, AuBC particles and the encapsulated M1-type exosomes (mEVs) are clearly visible.

[0081] Western blotting was used to determine the surface proteins of M1 type exosomes (mEVs) and AuBC@mEVs. The results are as follows: Figure 9 As shown, markers of cell exosomes, including CD9, CD63, and TSG101, were detected in the prepared formulation AuBC@mEVs; further demonstrating that the exosome components were completely preserved after coating modification.

[0082] Example 2: Determination of the content of peptides and carboranes modified on AuBC surface: The surface density of AuBC surface-modified peptides was quantitatively analyzed using a ligand substitution method. Specifically, a standard calibration curve for the thiol-modified peptide labeled with TAMRA was pre-plotted. 10 mmol / L dithiothreitol (DTT) was added to a 1 nM AuBC solution, and the mixture was stirred at room temperature for 12 h to allow complete dissociation and release of the peptides anchored to the AuBC particle surface via thiol bonds. After centrifugation, the TAMRA concentration in the supernatant was measured using a fluorescence spectrophotometer under the same pH and ionic strength conditions. λ ex =540 nm, λ em The fluorescence intensity was measured at 580 nm. All tests were performed in triplicate, and peptide concentrations were calculated based on the standard calibration curve. The standard calibration curve for the thiol-modified peptide labeled with TAMRA is shown below. Figure 10 As shown in Figure A, Y = 68.18X + 1040, R 2 =0.9914, where X is the concentration and Y is the fluorescence intensity; the fluorescence measurement results of AuBC prepared in Example 1 before and after DTT treatment are as follows: Figure 10 As shown in B, calculations show that each AuBC prepared in Example 1 has approximately 33 peptide chains on its surface.

[0083] Quantitative determination of carborane on AuBC surface: A 1 nM AuBC sample prepared in Example 1 was digested with concentrated nitric acid, and the total boron content was determined by inductively coupled plasma mass spectrometry (ICP-MS). The average number of carborane molecules loaded on the surface of a single gold nanoparticle was calculated based on the total boron concentration and the molar concentration of the gold nanoparticles. The calculation results showed that the carborane loading of each AuBC prepared in Example 1 was approximately 77 carborane molecules.

[0084] That is, the molar ratio of carborane to fluorescent peptide chain modified on each gold nanoparticle of AuBC prepared in Example 1 is 2.3:1.

[0085] Example 3: Biocompatibility testing of AuBC@mEVs: Human glioma cell line (U251 cells) was selected and cultured at 37℃ with 5% CO2. When the cells reached 80% confluence, different concentrations of AuBC@mEVs nanoprobes were added for treatment, with the concentration range adjusted from 1 nM to 5 nM according to the AuBC@mEVs concentration. A control group without nanoprobes was used. The cytotoxicity of the AuBC@mEVs nanoprobes prepared in Example 1 was tested using the CCK-8 assay. The results are as follows: Figure 11 As shown, AuBC@mEVs did not significantly decrease cell viability at concentrations ranging from 1 nM to 5 nM, and had no obvious toxic effects on cells, demonstrating its good biosafety.

[0086] Example 4: Drug delivery capability testing of AuBC@mEVs: Human glioma cell line (U251 cells) was selected and cultured at 37℃ with 5% CO2. U251 glioma cells were cultured in culture dishes for 24 hours. When the cells reached 80% confluence, 1 nM AuBC@mEVs nanoprobes labeled with red cell membrane fluorescent dye were added and co-incubated for 0.5 h, 1 h, 2 h, and 4 h, respectively. Afterward, the cells were washed three times with PBS buffer to remove untaken nanoprobes. The cytoplasm and nuclei were then stained with FITC and Hoechst 33342 for 30 min, respectively. After staining, the cells were washed three times with PBS buffer. Cells were observed and analyzed using a laser confocal scanning microscope (CLSM).

[0087] Confocal imaging images of AuBC@mEVs cells prepared in Example 1 after incubation for different times are shown below. Figure 12 As shown, after 4 hours of incubation, the cells exhibited a strong fluorescence intensity, indicating that the AuBC@mEVs prepared in Example 1 could enter the cells well.

[0088] The fluorescence colocalization of AuBC@mEVs prepared in Example 1 is as follows: Figure 13 As shown in Figure A, the material is distributed within the cytoplasm. Figure 13 B is a linear distribution map of intracellular fluorescence intensity, which further demonstrates the intracellular distribution characteristics of the nanoprobe, which is enriched in the cytoplasm and does not enter the nucleus.

[0089] Quantitative characterization of the spatial distribution of AuBC@mEVs prepared in Example 1 is as follows: Figure 13 As shown in Figure C, the cytoplasm is divided into three concentric ring regions (Q1–Q3) centered on the cell nucleus. Quantitative analysis results are as follows: Figure 13 As shown in Figure D, the fluorescence signal is mainly enriched in the Q2 region, which has an average distance of 4.0 μm ± 0.5 μm from the center of the cell nucleus. This distance is related to the high linear energy transfer (high LET) α particles generated during boron fission and 7 The range of Li atomic nuclei is consistent with that of boron neutron capture therapy, which is 4.5–10 μm. Therefore, the AuBC@mEVs prepared in Example 1 of this invention can achieve the accumulation of boron drugs around the cell nucleus during drug delivery, maximizing the overlap between the ionization track and nuclear DNA, thereby enhancing the efficacy of boron neutron capture therapy.

[0090] AuBC@mEVs prepared in Example 1 were labeled with a red fluorescent dye. Flow cytometry analysis of the cells after incubation for different times is shown in the figure below. Figure 13 As shown in Figure E. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the intracellular boron-10 uptake after incubation of cells with different concentrations of AuBC@mEVs (0.5–5 nM). The results are shown in Figure E. Figure 13 As shown in Figure F, the enrichment level of boron-10 increases in a dependent manner with increasing concentration of the incubating drug. At a concentration of 1 nM, the enrichment level in a single cell... 10 The boron atom loading can reach 6.18 × 10⁻⁶. 9 The number (calculated based on a natural abundance of 19.9%) is relatively close to the generally accepted critical threshold for BNCT treatment (1×10⁶ cells per single cell). 9 indivual 10 The boron enrichment was 6 times higher than that of boron atoms; at higher drug concentrations (2~5 nM), the boron enrichment was further enhanced, laying a sufficient dosage basis for clinical treatment administration.

[0091] Example 5: Validation experiment on the correlation between the fluorescence activation ability of AuBC@mEVs and apoptosis: U251 glioma cells were cultured in culture dishes for 24 hours. When the cells reached 80% confluence, they were pre-incubated with AuBC@mEVs (1 nM) for 6 hours. After washing three times with PBS, cisplatin (10 µg / mL) was added for treatment at different times (0, 3, 6, 12, and 24 hours). Subsequently, the cells were stained with Hoechst 33342 and imaged by confocal laser scanning microscopy (CLSM). ELISA quantification: After cisplatin treatment for different times, U251 cells were collected, and the expression level of intracellular active Caspase-3 was quantitatively detected using an ELISA kit.

[0092] Cells were treated with cisplatin to induce apoptosis at different degrees, and the fluorescence intensity at different time points was quantitatively analyzed as follows: Figure 14 As shown in Figure A, prolonged cisplatin-induced apoptosis (12-24 h) leads to increased intracellular fluorescence signal. The ELISA quantitative results of intracellular Caspase-3 activity after different treatment times are shown below. Figure 14 As shown in Figure B, the expression level of Caspase-3 increased in cells treated with cisplatin. A linear regression equation was constructed based on the average gray value of TAMRA confocal imaging and the Caspase-3 protein concentration measured by ELISA, as follows: Figure 14 As shown in Figure C, fluorescence intensity was highly correlated with caspase-3 expression level, Y = 0.06996X - 0.1247, with a correlation coefficient R.2 The value was 0.9877, demonstrating that the therapeutic effect of BNCT can be fed back based on the corresponding fluorescence signal.

[0093] Example 6: Testing the efficacy feedback capability of AuBC@mEVs in boron neutron capture therapy (BNCT): U251 cells were divided into 5 groups: The PBS group (Group I) served as the blank control group, receiving no drug incubation, no BNCT treatment, and only simultaneous PBS washing. The AuBC@mEVs group (Group II) incubated cells with 1 nM AuBC@mEVs for 6 hours, without BNCT treatment. The BNCT group (Group III) received BNCT treatment with a neutron source only, without drug incubation. The AuC@mEVs+BNCT group (Group IV) incubated cells with 1 nM AuC@mEVs for 6 hours, where the AuC@mEVs were prepared using the same method as in Example 1 except without the addition of carborane, and BNCT treatment was performed using a neutron source. The AuBC@mEVs+BNCT group (Group V) incubated cells with 1 nM AuBC@mEVs for 6 hours, and BNCT treatment was performed using a neutron source.

[0094] All nanomaterials were prepared to a uniform concentration of 1 nM based on gold content. After incubating cells for 6 hours, the cells were washed three times with PBS. After boron neutron capture irradiation treatment, U251 cells were reseeded into confocal culture dishes for further culture. Subsequently, the cells were fixed with 4% paraformaldehyde, and the nuclei were stained with Hoechst 33342 dye. The activation status of the probes was detected by laser confocal microscopy.

[0095] 1. The efficacy of BNCT was assessed by monitoring changes in fluorescence signals. Confocal microscopy (CLSM) was used for observation and quantitative analysis of fluorescence signals. Fluorescence signal changes were observed through a fluorescence recovery assay. Results are as follows: Figure 15 As shown, where, Figure 15 A shows confocal imaging images of different groups of cells; Figure 15 B is the TAMRA confocal gray quantification map. Only the AuBC@mEVs+BNCT group showed a strong TAMRA fluorescence signal. This is because the drug formulation can effectively deliver boron-10 and induce apoptosis in cells under neutron source irradiation. It can be seen that after BNCT treatment, the AuBC@mEVs prepared in Example 1 showed a significant recovery of fluorescence signal due to Caspase-3 activation, reflecting the apoptosis process of cells.

[0096] 2. Immunofluorescence staining analysis results are as follows: Figure 16 As shown, this further validates the close correlation between fluorescence signal recovery and cell apoptosis. Among them, Figure 16 A shows the Caspase-3 immunofluorescence staining images of cells in different treatment groups; Figure 16 B is the FTIC confocal gray quantification map. Only the AuBC@mEVs+BNCT group showed a strong green fluorescence signal. This is because the cells in this group had a higher Caspase-3 expression level after treatment. It is evident that the fluorescence signal recovery of AuBC@mEVs prepared in Example 1 after BNCT treatment is positively correlated with the Caspase-3 activation ratio, demonstrating that the fluorescence signal recovery of AuBC@mEVs can serve as an effective indicator for real-time treatment assessment. The self-reporting nanoprobe AuBC@mEVs of this invention are not only used for boron drug delivery in BNCT treatment but also enable real-time monitoring of treatment efficacy through the recovery of their fluorescence signal. During treatment, a fluorescence imaging system can be used to observe changes in fluorescence intensity before and after treatment.

[0097] 3. Results of the clone formation experiment are as follows: Figure 17 As shown, where, Figure 17 A shows cell clone staining images after day 5, showing cells after different treatments. Figure 17 B represents the UV absorbance values ​​of cells from different treatment groups after crystal violet staining and dissolution; the Transwell invasion assay results are as follows. Figure 18 As shown, where, Figure 18 A shows the stained images of cells that invaded the lower layer of the chamber after five days, after different treatments. Figure 18 B is a statistical analysis of invasive cells photographed under a microscope. It can be seen that the AuBC@mEVs+BNCT (Group V) group showed the most significant inhibitory effect on cell clone survival and invasion (P<0.001 compared to the other groups). In contrast, the incomplete drug administration group with single treatment showed a significantly weakened inhibitory effect. This is because irradiation with a neutron source under sufficient boron-10 content can effectively induce apoptosis and inhibit cell proliferation and invasion.

[0098] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanotherapeutic agent, characterized in that, The nanotherapeutic agent is composed of a biomimetic shell and a nanocomposite core; the biomimetic shell is an M1 macrophage exosome membrane, and the nanocomposite core is composed of gold nanoparticles and their surface-modified carborane and fluorescent polypeptide sequences; the fluorescent polypeptide sequence can be specifically recognized and cleaved by apoptosis markers.

2. The nanotherapeutic formulation as described in claim 1, characterized in that, The carborane is terminally modified with a thiol group, and the structure of the carborane is as follows: 。 3. The nanotherapeutic formulation as described in claim 1, characterized in that, The apoptosis marker is caspase-3, and the fluorescent polypeptide sequence is TAMRA-Gly-Trp-Glu-His-Asp-Gly-Lys-Cys-COOH.

4. The nanotherapeutic formulation as described in claim 1, characterized in that, The molar ratio of carborane modified on each gold nanoparticle to fluorescent peptide chain is 2.1-2.5:

1.

5. The nanotherapeutic formulation as described in claim 4, characterized in that, The molar ratio of carborane to fluorescent peptide chain modified on each gold nanoparticle is 2.3:

1.

6. The method for preparing the nanotherapeutic agent according to any one of claims 1-5, characterized in that, Includes the following steps: The pH of the gold nanoparticle colloidal solution was adjusted, fluorescent peptides and carborane were added, and the solution was frozen and incubated in the dark. After thawing at room temperature and purification, surface-functionalized gold nanoparticles AuBC were obtained. Pre-cooled M1 macrophage exosomes (mEVs) were mixed with AuBC solution, placed in an ice bath, sonicated, incubated at low temperature overnight, and purified to obtain surface-functionalized gold nanoparticles AuBC@mEVs coated with M1 exosome membranes.

7. The preparation method according to claim 6, characterized in that, Adjust the pH of the gold nanoparticle colloidal solution to 7.2-7.

4.

8. The preparation method according to claim 6, characterized in that, The molar ratio of carborane, fluorescent peptide, and Au NPs is 100:100-300:

1.

9. The use of the nanotherapeutic formulation as described in any one of claims 1-5 in the preparation of antitumor drugs, characterized in that, The tumors include head and neck tumors, brain tumors, and melanoma; Preferably, the tumor is a glioma; More preferably, the tumor is a glioblastoma.

10. The use of the nanotherapeutic formulation according to any one of claims 1-5 in evaluating the efficacy of boron neutron capture therapy, characterized in that, The efficacy of boron neutron capture therapy is assessed by monitoring changes in fluorescence signals.

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