A nano boron delivery agent, its preparation method and application

Abstract

The present invention discloses a nano-boron delivery agent and its preparation method and application. The nano-boron delivery agent h-10BN-PG nanoparticles provided by the present invention have the characteristics of being easy to prepare, non-toxic, good biocompatibility, high 10B content, good dispersibility and stability in physiological solutions, etc. The h-10BN-PG nanoparticles can inhibit protein adsorption and non-specific uptake by macrophages, and have good "stealth" characteristics, thus avoiding rapid clearance by the MPS, and their blood circulation time is up to 8.5 hours. The h-10BN-PG nanoparticles can accumulate efficiently in CT26 tumor tissues through the EPR effect and can be further endocytosed by tumor cells; the concentration of 10B in tumors can reach 8.8% ID / g or 102.1 ppm. BNCT mediated by h-10BN-PG nanoparticles can completely eradicate subcutaneous CT26 tumors with only one injection of nanoparticles and one neutron irradiation.

Description

Technical Field

[0001] The present invention relates to a nano boron delivery agent, a preparation method thereof and an application thereof, belonging to the technical field of biomaterials. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is a binary targeted tumor therapy method that has received wide attention in recent years. BNCT treatment requires the delivery of a sufficient amount of boron-10 ( 10 10B) to tumor tissues first, and then the tumor site is irradiated with thermal neutrons. 10 The 10B atoms collide with thermal neutrons to generate a nuclear fission reaction, producing α particles with a high linear energy transfer and recoil 7 7Li, thereby killing tumor cells. BNCT treatment requires that the boron content per gram of tumor tissue 10 10B is not less than 20 mg or not less than 10 9 per 10 10B atom in each tumor cell. However, the two small molecule boron delivery agents currently used clinically, namely 4-dihydroxyboranilophenylalanine (BPA) and sodium dodecaborane disulfide (BSH), do not have strong tumor targeting, and have a short retention time in tumors. The lack of boron delivery agents with high tumor accumulation has become one of the bottlenecks restricting the clinical application of BNCT. Boron-containing nanoparticles can carry a large number of 10 10B atoms, and can target and enter tumor tissues through the Enhanced Permeability and Retention (EPR) effect, so they have received more and more attention in recent years.

[0003] Hexagonal boron nitride (hBN) is a ceramic material with a layered structure similar to graphite, commonly known as "white graphite". In recent years, researchers have prepared nano hBN with different morphologies, including nanosheets, nanotubes, hollow nanospheres and nanoparticles, etc. Nano hBN not only has good biocompatibility and a super high specific surface area, but also can be slowly hydrolyzed and metabolized in vivo, so it has broad application prospects in the biomedical field. Since a single nano hBN contains a large number of boron atoms and can enter and retain in tumor tissues through the EPR effect, nano hBN is also a very promising boron delivery agent for BNCT.

[0004] Inorganic nanoparticles cannot be stably dispersed in physiological solutions and therefore must be appropriately surface-modified before they can be applied in the biomedical field. Currently, inorganic nanoparticles are usually surface-modified with hydrophilic polymers to improve their dispersibility and stability in aqueous solutions and extend their circulation time in the blood, thereby increasing the likelihood of their penetration into tumor tissues. Covalently grafting polyglycerol (PG) onto the surface can not only greatly increase the hydrophilicity of inorganic nanoparticles, but also its excellent "stealth" property can inhibit the recognition and clearance by the mononuclear phagocytic system (MPS), prolonging the circulation time of the nanoparticles in the blood, so that the nanoparticles can enter tumor tissues by means of the enhanced permeability and retention (EPR) effect.

[0005] Li et al. prepared hBN nanosheets by pyrolysis at high temperature and then further assembled them into hBN nanoparticles (BNNPs) for BNCT treatment of 4T1 breast cancer in mice (ACS Nano, 2019, 13, 13843–13852). However, since the boron element in BNNPs is at natural abundance, the 10 10B with BNCT activity only accounts for 19.60%, and the tumor accumulation ability is insufficient, requiring multiple injections to increase the 10 10B concentration in the tumor, resulting in an unsatisfactory in vivo BNCT efficacy and the inability to completely eliminate the tumor. Nakamura et al. used DSPE-PEG2000-coated BN nanotubes (BNNT-DSPE-PEG2000) to carry out BNCT treatment of B16 melanoma cells in mice. Similarly, due to the low 10 10B abundance in the nanotubes, the BNCT efficacy was poor (Bioorg. Med. Chem. Lett., 2015, 25, 172–174). Kaur et al. used boric acid ([[]] 10 10B(OH)3) with a high 10 10B abundance (99%) and ammonia water as raw materials to prepare 10 10B-rich hBN nanoparticles by a solvothermal method (Mater. Lett., 2020, 259, 126832), but due to their poor dispersibility in physiological solutions, in vivo BNCT experiments could not be carried out.

[0006] In summary, the prior art still has the problem that the 10 10B abundance of most nano-hBN is relatively low, usually only at the natural abundance of boron (19.60%), while the existing 10The synthesis of hBN nanoparticles of B requires the use of ammonia water, which is highly toxic and corrosive, as a raw material, which is harmful to human health and the environment. In addition, existing nano-hBN uses hydrophilic polymers to improve its dispersibility and stability in physiological solutions. However, the connection between the polymer and nano-hBN is a non-covalent bond, which will gradually dissociate, leading to a decrease in the stability of the nanoparticles. And the existing hBN nanoparticles have insufficient ability to deliver 10 B to tumors, resulting in 10 low B content in tumors, and the BNCT treatment mediated by it cannot completely eliminate tumors. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a nano-boron delivery agent, a preparation method thereof and an application. The present invention improves the 10 abundance and content of B in the boron delivery agent, improves the dispersibility and stability of the boron delivery agent in physiological solutions, and improves the delivery of the boron delivery agent to tumors 10 efficiency of B, increases 10 the accumulation of B in tumor tissues, and obtains better BNCT efficacy.

[0008] The first object of the present invention is to provide a preparation method of a nano-boron delivery agent, which includes the following steps:

[0009] S1. After grinding and mixing the powders of boric acid and nitrogen source, under the condition of protective gas, react at 800-1200 °C for 1-10 hours. After cooling, grind the product into powder, remove the soluble impurities in the powder, and collect the nanoparticles in the insoluble components;

[0010] S2. Add the nanoparticles prepared in step S1 to water, use a surfactant to disperse the nanoparticles to obtain monodisperse nanoparticles, and then graft a hydrophilic polymer onto the monodisperse nanoparticles, and purify to obtain the nano-boron delivery agent;

[0011] Among them, the 10 abundance of B in boric acid is 19.60% - 99.99%.

[0012] Further, the 10 abundance of B in boric acid is 80.00% - 99.99%.

[0013] Further, the nitrogen source is one or more of melamine, urea, and polyimide.

[0014] Further, in step S1, removing the soluble impurities in the powder is carried out through the following steps: ultrasonically treating the powder in water, and then dialyzing with a dialysis membrane with a molecular weight cut-off of 8000-14000 Da to remove the soluble impurities in the powder.

[0015] Further, in step S1, the nanoparticles in the insoluble component are collected through the following steps: centrifuging the suspension of the insoluble component at 1500-2000 g for 4-10 minutes, collecting the milky supernatant, and removing the moisture to obtain the nanoparticles.

[0016] Further, the surfactant is one or more of polyvinylpyrrolidone, Tween, and Triton X-100

[0017] Further, the hydrophilic polymer is polyglycerol or polyethylene glycol and its derivatives.

[0018] Further, in step S2, the grafting reaction is carried out at 100-200 °C for 20-30 hours.

[0019] Further, the protective gas is nitrogen or an inert gas.

[0020] The second object of the present invention is to provide a nano boron delivery agent prepared by the above preparation method.

[0021] The third object of the present invention is to provide the application of the nano boron delivery agent in boron neutron capture therapy for cancer.

[0022] The beneficial effects of the present invention are:

[0023] The nano boron delivery agent h- 10 BN-PG nanoparticles prepared by the present invention have the characteristics of convenient preparation, non-toxicity, good biocompatibility, 10 high B content, good dispersibility and stability in physiological solutions, etc. h- 10 BN-PG nanoparticles can inhibit protein adsorption and non-specific uptake by macrophages, and have good "stealth" characteristics, thus avoiding rapid clearance by the MPS, and their blood circulation time is up to 8.5 hours. h- 10 BN-PG nanoparticles can efficiently accumulate in CT26 tumor tissues through the EPR effect, and can be further endocytosed by tumor cells; the 10 concentration of B in the tumor can reach 8.8% ID / g or 102.1 ppm. h- 10 BNCT mediated by BN-PG nanoparticles can completely eradicate subcutaneous CT26 tumors with only one injection of nanoparticles and one neutron irradiation. Description of the Drawings

[0024] Figure 1 is the synthesis and physicochemical property characterization of h- 10 BN and h- 10 BN-PG nanoparticles. (a) Synthesis route of h- 10 BN nanoparticles. (b) h- 10 BN and h- 10Transmission electron microscopy images of BN-PG nanoparticles. The illustration is h- 10 High-resolution transmission electron microscopy images of BN-PG nanoparticles. (c) h- 10 Powder X-ray diffraction spectrum of BN nanoparticles. (d) h- 10 BN and h- 10 Infrared spectrum of BN-PG nanoparticles. (e)h- 10 BN and h- 10 Thermogravimetric curves of BN-PG nanoparticles in nitrogen atmosphere. (f) h- 10 Particle size distribution of hydrated BN-PG nanoparticles.

[0025] Figure 2 is (a)h- 10 BN、h- 10 BN@PEG and h- 10 Adsorption characteristics of BN-PG nanoparticles on fetal bovine serum albumin. (b) h- 10 Cytotoxicity of BN-PG nanoparticles to CT26 and 3T3 cells (n=3). (c) Cytotoxicity of CT26 cells to h- 10 BN、h- 10 BN@PEG and h- 10 Uptake of BN-PG nanoparticles (n=3), **p<0.01, ***p<0.001. (d) 3T3 cells uptake of h- 10 BN、h- 10 BN@PEG and h- 10 Uptake of BN-PG nanoparticles (n=3), **p<0.01, ***p<0.001. (e) CT26 cells were treated with PBS and h- 10 Confocal fluorescence imaging of BN-PG-Cy5 co-culture. Lysosomes were stained with LysoTracker blue.

[0026] Figure 3 (a) Nude mice bearing CT26 subcutaneous tumors were injected with h- 10 In vivo fluorescence imaging before and 4, 8, 12, 24, and 48 hours after BN-PG-Cy7 nanoparticle injection. The dotted circle indicates the CT26 tumor. (b) Curve of mean fluorescence intensity of CT26 tumors over time (n = 3). (c) Ex vivo fluorescence imaging of CT26 tumors, muscle, and major organs 48 hours after injection.

[0027] Figure 4 is (a)h- 10 BN-PG and h- 10 Plasma clearance curves of BN@PEG nanoparticles (n=3). (b) 2, 6, 12, 24, and 48 hours after injection h- 10Distribution of BN-PG nanoparticles in CT26 tumor-bearing mice (n = 5). (c) After injection of h- 10 Curve of the T / N and T / B ratios of CT26 tumor-bearing mice over time after injection of BN-PG nanoparticles (n = 5). (d) After 12 hours of injection, h- 10 Distribution of BN-PEG nanoparticles in CT26 tumor-bearing mice (n = 3).

[0028] Figure 5 Is (a) h- 10 Confocal fluorescence imaging of the infiltration of BN-PG-Cy5 nanoparticles into three-dimensional CT26 cell spheroids over time. (b) Fluorescence imaging of tumor sections of CT26 tumor-bearing mice at different time points after injection of h- 10 BN-PG-Cy5 nanoparticles.

[0029] Figure 6 Is (a) Curve of the CT26 tumor size of mice in each group of BNCT treatment experiments over time. Thermal control (short irradiation) and BNCT (short irradiation) groups n = 3; other groups n = 6; **p < 0.01, ***p < 0.001, and NS (no statistical difference). (b) Photos of mice in the cold control, thermal control (standard irradiation), injection control, and BNCT (standard irradiation) groups 24 days after treatment. (c) Curve of the body weight change of mice in each group within 24 days. Detailed implementation manners

[0030] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0031] Example 1: Synthesis of h- 10 BN nanoparticles

[0032] h- 10 BN nanoparticles are synthesized from boric acid rich in 10 B ([[]] 10 B abundance: 99.63%) and melamine as 10 B source and nitrogen source respectively by a pyrolysis method. As shown in a of Figure 1 : Boric acid rich in 10 B (0.366 g, 6 mmol) and melamine (0.126 g, 1 mmol) powders are thoroughly ground. The obtained mixture is transferred to a corundum boat, and then placed in a horizontal tube furnace and purged with N2 (flow rate 100 mL / min). It is heated to 900 °C at a heating rate of 10 °C / min and maintained for 3 hours. After cooling, the crude h- 10The BN bulk was ground into powder, then sonicated in water for 4 hours, and then dialyzed (MWCO: 8000 - 14000 Da) to remove soluble impurities. The resulting suspension was centrifuged at 1811 g for 5 minutes, and the milky supernatant containing h- 10 BN nanoparticles was collected for further use.

[0033] Transmission electron microscopy (TEM) images showed that the average diameter of h-BN nanoparticles was ~8 nm, but large aggregates were easily formed on the TEM substrate ( Figure 1 in b). h- 10 High-resolution transmission electron microscopy (HRTEM) of h- 10 BN showed the (002) crystal plane of h- Figure 1 BN with an interlayer spacing of 0.33 nm ( 10 in b, inset). h- 10 BN nanoparticles contained 20 - 30 layers, corresponding to a thickness of 6.6 - 9.9 nm. X-ray diffraction (XRD) ( Figure 1 in c) indicated that h- 10 BN nanoparticles were well-crystallized, and the diffraction peaks at 26.7°, 41.6°, and 43.8° corresponded to the (002), (100), and (101) crystal planes of hexagonal boron nitride, respectively. h- 10 The Fourier transform infrared spectroscopy (FTIR) of h- -1 BN nanoparticles had a broad absorption band from 3000 to 3700 cm Figure 1 which was attributed to the stretching vibrations of O-H and N-H bonds ( -1 in d). In addition, there were two strong FTIR peaks at 1397 and 797 cm 10 which corresponded to the stretching and bending vibrations of B-N, respectively. Dynamic light scattering (DLS) results showed that h- 10 BN nanoparticles were easily aggregated in water, with an average hydrodynamic diameter of 36.4 nm. The surface potential of h- 10 BN nanoparticles was -32.2 ± 5.2 mV.

[0034] Example 2: Synthesis of h- 10 BN-PG nanoparticles

[0035] h- 10 BN nanoparticles could be stably dispersed in pure water due to the high negative charge and a large number of hydrophilic groups on their surface, but were easily aggregated and precipitated in physiological solutions such as phosphate-buffered saline (PBS). Therefore, it was necessary to modify the surface of h- 10 BN nanoparticles to improve their dispersibility and colloidal stability under physiological conditions and promote their application in the biomedical field.

[0036] First, polyvinylpyrrolidone (PVP) was used as a surfactant to modify h-10 The BN nanoparticles were encapsulated to prevent their aggregation, and then the obtained monodisperse h- 10 BN@PVP nanoparticles were grafted with PG. The PVP adsorbed on the surface of h- 10 BN nanoparticles was replaced by covalently grafted PG. The specific preparation steps were as follows: PVP (20 mg) and water (20 mL) were added to the h- 10 BN nanoparticle powder (100 mg), and then ultrasonicated for 8 hours. The obtained suspension was centrifuged to take the supernatant, and then rotary evaporated to remove most of the water. The obtained h- 10 BN@PVP nanoparticle dispersion was ultrasonically redispersed in 20 mL of glycidol, heated to 140 °C and maintained for 24 h. Glycidol polymerized to form polyglycerol and was grafted onto the nanoparticle surface. After cooling, 40 mL of water was added to the flask and ultrasonically dispersed. The crude product was collected by ultracentrifugation and then purified by multiple ultracentrifugation / redispersion to remove the free polymer therein, and h- 10 BN-PG nanoparticles were obtained.

[0037] h- 10 BN-PG has ultrahigh dispersibility and colloidal stability in aqueous solution, and its dispersibility in pure water > 6.0 mg 10 B / mL (equivalent to 41 mg h- 10 BN-PG / mL), and it can be stably dispersed in physiological solutions such as PBS. FTIR spectra showed that h- 10 BN-PG nanoparticles had strong absorption peaks at 3360, 2870 and 1050 cm -1 ( Figure 1 d in), corresponding to the stretching vibrations of O-H, C-H and C-O-C. Thermogravimetric analysis (TGA) showed that the content of PG in h- 10 BN-PG nanoparticles was 65%( Figure 1 e in). Due to the steric shielding effect of the PG layer inhibiting particle aggregation, h- 10 BN-PG nanoparticles were monodispersed on the TEM substrate( Figure 1 b in). DLS results showed that h- 10 the average hydrodynamic diameter of BN-PG nanoparticles was 28.2 nm, and the polydispersity index (PDI) was 0.18, proving that h- 10 BN-PG nanoparticles were monodispersed in water( Figure 1 f in). h- 10 The surface potential of BN-PG nanoparticles was -5.0 ± 3.6 mV.

[0038] In addition, DSPE-PEG 5000 encapsulated h- 10 BN nanoparticles (h- 10BN@PEG) was used in the following examples to study and compare the properties of nanoparticles.

[0039] Example 3:

[0040] First, the adsorption characteristics of h- 10 BN, h- 10 BN@PEG, and h- 10 BN-PG nanoparticles to fetal bovine serum protein were investigated. As shown in Figure 2 a, compared with unmodified h- 10 BN and h- 5000 BN nanoparticles encapsulated with DSPE-PEG 10 BN@PEG), h- 10 BN-PG exhibited more excellent anti-protein non-specific adsorption performance. Specifically, the amounts of fetal bovine serum protein adsorbed on h- 10 BN and h- 10 BN@PEG nanoparticles were 62 times and 18.6 times that on h- 10 BN-PG nanoparticles, respectively. 10 BN-PG nanoparticles were 62 times and 18.6 times that on h-

[0041] The cytotoxicity of h- 10 BN-PG nanoparticles against mouse CT26 colon cancer cells and 3T3 fibroblasts was studied using the CCK-8 method. As shown in Figure 2 b, even after treatment with a high concentration (500 μg 10 B / mL) of h- 10 BN-PG nanoparticles for 24 h, the activities of CT26 cells and 3T3 cells did not decrease significantly, indicating that the nanoparticles had good cytocompatibility.

[0042] To study the cellular uptake of h- 10 BN-PG, h- 10 [[ID=4�]]BN@PEG, and h- 10 BN nanoparticles, the B content in CT26 cells and RAW 264.7 macrophages was quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) 24 hours after nanoparticle treatment ( 10 c and d). The h- Figure 2 BN nanoparticles without surface modification had the highest B content in CT26 and RAW 264.7 cells, probably because their poor colloidal stability in physiological solutions led to rapid aggregation and precipitation, making them easily taken up by cells at the bottom of the culture dish. Although h- 10 BN-PG and h- 10 BN@PEG nanoparticles could also be internalized by cells in a concentration-dependent manner, but compared with h- 10 BN-PG and h- 10 BN@PEG nanoparticles could also be internalized by cells in a concentration-dependent manner, but compared with h- 10Compared with BN nanoparticles, the surface modification layer significantly inhibited the cellular uptake of these nanoparticles.

[0043] In addition, the cells treated with h- 10 BN-PG showed lower 10 B content than those treated with h- 10 BN@PEG nanoparticles. The PG modification layer inhibiting non-specific cellular uptake contributed to reducing the clearance of h- 10 BN-PG nanoparticles by macrophages. h- 10 BN-PG nanoparticles were covalently linked with Cyanine5 (Cy5) to obtain h- 10 BN-PG-Cy5 nanoparticles. After treating CT26 cells with h- 10 BN-PG-Cy5 nanoparticles at a concentration of 200 μg 10 B / mL for 6 hours, the red fluorescence of h- 10 BN-PG-Cy5 was detected in the lysosomes of the cells ( Figure 2 in e), indicating that the h- 10 BN-PG-Cy5 nanoparticles internalized by the cells were mainly distributed in the lysosomes.

[0044] Example 4: Detection of nanoparticle accumulation in tumor tissues by in vivo fluorescence imaging

[0045] h- 10 BN-PG-Cy7 nanoparticles emitting near-infrared fluorescence were prepared for in vivo fluorescence imaging. h- 10 BN-PG-Cy7 was injected into BALB / c nude mice bearing CT26 tumors via the tail vein at a dose of 12 mg 10 B / kg body weight. As Figure 3 shown in a, the fluorescence intensity of the CT26 tumor increased significantly with time and reached the maximum at 12 hours after injection, indicating that h- 10 BN-PG-Cy7 nanoparticles gradually accumulated in the tumor tissue. Subsequently, the fluorescence of the tumor gradually decreased, but still remained at a relatively high fluorescence intensity until 48 hours after injection ( Figure 3 in b), indicating the long retention time of h- 10 BN-PG-Cy7 nanoparticles in the tumor. The mice were sacrificed and dissected 48 hours after injection, and in vitro fluorescence imaging was performed on the tumors and organs. As Figure 3 shown in c, the strong fluorescence of the CT26 tumor indicated that h- 10 BN-PG-Cy7 nanoparticles had obvious accumulation in the tumor tissue.

[0046] Example 5: Plasma clearance and tissue distribution experiments of nanoparticles

[0047] h-10 BN-PG and h- 10 Physiological saline dispersions of BN@PEG nanoparticles were injected into mice via the tail vein respectively. Blood samples were collected from the tail vein at different time points after injection, and then the boron content in the blood samples was measured by ICP-OES. As Figure 4 shown in a, h- 10 BN-PG and h- 10 the blood half-lives (t 1 / 2 ) of BN@PEG nanoparticles were 8.5 and 2.7 hours respectively. The longer t 10 of h- 1 / 2 BN-PG nanoparticles indicates that PG grafting is superior to polyethylene glycol coating in prolonging the blood circulation time of h- 10 BN nanoparticles. This result and the macrophage uptake result ( Figure 2 shown in d) indicate that h- 10 BN-PG nanoparticles are superior to h- 10 BN@PEG nanoparticles in terms of "stealth" performance, and can more effectively evade the clearance of the mononuclear phagocyte system (MPS), which is one of the main biological barriers hindering the delivery of nano-boron agents into tumor tissues.

[0048] The tissue distribution of h- 10 BN-PG nanoparticles over time was further studied. h- 10 BN-PG nanoparticles were injected into BALB / c mice bearing CT26 tumors via the tail vein at a dose of 58 mg 10 B / kg body weight. The mice were sacrificed at 2, 6, 12, 24, and 48 hours after injection, and tumors, muscles, and major organs were collected. The 10 B content (%ID / g) was measured by ICP-OES. As Figure 4 shown in b, the 10 B content in CT26 tumors increased steadily after injection of the nanoparticles and reached a peak of 8.8% ID / g (102.1 ppm) at 12 hours after injection, and then decreased slowly from 12 to 48 hours after injection. At 48 hours after injection, the average 10 B content in the tumors was still as high as 6.5% ID / g (75.4 ppm). The change of 10 B content in tumors over time was consistent with the in vivo fluorescence imaging result in Figure 3 a. The boron delivery agent currently used in clinical BNCT is fructose-BPA (Sci. Adv. 2020, 6(4), e aaz1722). After being injected into CT26 tumor-bearing mice via the tail vein at a dose of 500 mg / kg body weight for 1 hour, the average 10 B content in the tumors was 6.6% ID / g, and it dropped to 2.2% ID / g rapidly at 6 hours after injection. In addition, h-10 The retention time of BN-PG nanoparticles in CT26 tumors was also much longer than that of the reported polyvinyl alcohol and BPA complex (PVA-BPA) (Sci. Adv. 2020, 6(4), e aaz1722). Figure 4 In c, the T / N and T / B ratios at different time points after injection of h- 10 BN-PG nanoparticles are summarized. The T / N ratio gradually increased from 2.4 at 2 h after injection to 7.6 at 12 h after injection and remained at 5.7 at 48 h after injection. Meanwhile, the T / B ratio increased from 0.1 at 2 h after injection to 1.7 at 48 h after injection.

[0049] The in vivo distribution results also showed that the uptake of h- 10 BN-PG nanoparticles by the liver and spleen was less than 15% ID / g, which was significantly different from the reported 10 B4C-PG nanoparticles (particle size of ~70 nm) that were readily taken up by the liver and spleen in large amounts. The tissue distribution of h- 10 BN@PEG nanoparticles at 24 h after injection was studied and compared with h- 10 BN-PG nanoparticles ( Figure 4 in d). The results showed that h- 10 BN@PEG nanoparticles were mainly distributed in the liver (41.8% ID / g), and the average 10 B content in CT26 tumors was only 3.4% ID / g. These data indicate that PG grafting can more effectively prevent nanoparticles from being cleared by the MPS than polyethylene glycol coating. The above results show that h- 10 The ultra-small particle size and PG modification of BN-PG nanoparticles play an important role in improving the "stealth" property and avoiding being cleared by the MPS, thus prolonging the blood circulation time and enhancing the accumulation in tumor tissues.

[0050] Example 6: Permeation characteristics of nanoparticles in CT26 three-dimensional cell spheroids and CT26 solid tumors

[0051] The permeation characteristics of h- 10 BN-PG-Cy5 nanoparticles in CT26 three-dimensional cell spheroids and CT26 solid tumors were observed by fluorescence imaging. As Figure 5 shown in a, h- 10 BN-PG-Cy5 nanoparticles at a concentration of 200 μg 10 B / mL gradually infiltrated into the interior of CT26 cell spheres over time. After 24 h of treatment, almost all cells at a depth of 35 μm in the cell spheres showed red fluorescence of h- 10 BN-PG-Cy5 nanoparticles. h- 10 BN-PG-Cy5 nanoparticles at 12 mg10 The dose of B / kg body weight was injected into CT26 tumor-bearing mice via the tail vein, and tumors were taken for pathological analysis at 6, 12, and 24 hours after injection. Fluorescence imaging showed that at 6 hours after injection, h- 10 BN-PG-Cy5 nanoparticles were mainly located in tumor blood vessels ( Figure 5 in b). At 12 hours after injection, h- 10 BN-PG-Cy5 nanoparticles extravasated through the gaps between vascular endothelial cells and entered the extracellular matrix of the CT26 tumor parenchyma. And at 24 hours after injection, h- 10 BN-PG-Cy5 nanoparticles entered tumor cells.

[0052] Example 7: BNCT treatment animal experiment

[0053] Thirty CT26 tumor-bearing mice were randomly divided into 6 groups: 1) cold control, neither injecting h- 10 BN-PG nor irradiating with neutrons (n = 6), 2) injection control, that is, only injecting h- 10 BN-PG (n = 6), 3) thermal control (standard), neutron irradiation for 12 minutes (n = 6), 4) thermal control (short irradiation), neutron irradiation for 6 minutes (n = 3), 5) BNCT (standard irradiation), h- 10 BNCT mediated by BN-PG, neutron irradiation for 12 minutes (n = 6), 6) BNCT (short irradiation), that is, h- 10 BNCT mediated by BN-PG, neutron irradiation for 6 minutes (n = 3). h- 10 The intravenous injection dose of BN-PG nanoparticles was 58 mg 10 B / kg. BNCT experiments were carried out on a reactor neutron source with a neutron flux of 5×10 9 n.cm -2 s -1 . When irradiating with neutrons, the mice were fixed with an acrylic bracket, and other parts except the tumor were covered with a thermoplastic plate containing 40 wt% 6 LiF (96% 6 Li). The neutron irradiation time was 6 minutes or 12 minutes. The day of neutron irradiation was recorded as day 0; the tumor size and body weight of all mice were measured on days 0, 3, 6, 10, 14, 17, 21, and 24.

[0054] As Figure 6 shown in a, both standard BNCT and short-irradiation BNCT could effectively inhibit the growth of CT26 tumor grafts. Standard-irradiation BNCT ultimately led to the complete regression of tumors in three tumor-bearing mice; two of the three tumor-bearing mice receiving short-irradiation BNCT also showed complete tumor regression. In contrast, the tumors in all control groups except the BNCT group grew very rapidly (Figure 6 in a) and b). As Figure 6 shown in c), there was no significant difference in body weight between the BNCT group and the control group. The above results indicate that h- 10 BN-PG nanoparticles are a safe nano-boron delivery agent and can achieve efficient BNCT treatment for tumors.

[0055] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A preparation method of a nano boron delivery agent, characterized in that, It includes the following steps: S1. After grinding and mixing the powders of boric acid and melamine, under the condition of a protective gas, react at 800 - 1200 °C for 1 - 10 hours. After cooling, grind the product into powder, remove the soluble impurities in the powder, and collect the nanoparticles in the insoluble components; S2. Add the nanoparticles prepared in step S1 into water, disperse the nanoparticles using polyvinylpyrrolidone to obtain monodisperse nanoparticles, and then graft hydrophilic polymers onto the monodisperse nanoparticles, and purify to obtain the nano boron delivery agent; the hydrophilic polymer is polyglycerol; Among them, in boric acid 10 the B abundance is 19.60% to 99.99%.

2. The preparation method according to claim 1, characterized in that, In step S1, removing the soluble impurities in the powder is carried out through the following steps: subject the powder to ultrasonic treatment in water, and after treatment, carry out dialysis using a dialysis membrane with a molecular weight cut-off of 8000 - 14000 Da to remove the soluble impurities in the powder.

3. The preparation method according to claim 1, characterized in that, In step S1, collecting the nanoparticles in the insoluble components is carried out through the following steps: centrifuge the suspension of the insoluble components at 1500 - 2000 g for 4 - 10 minutes, collect the milky supernatant, and remove the water to obtain the nanoparticles.

4. The preparation method according to claim 1, wherein In step S2, the grafting reaction is carried out at 100 - 200 °C for 20 - 30 hours.

5. The preparation method according to claim 1, characterized in that, The protective gas is nitrogen or an inert gas.

6. A nano boron delivery agent prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the nano boron delivery agent according to claim 6 in the preparation of a drug for boron neutron capture therapy of cancer.

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