IBNPs coated SiO2 nanoparticles and controllable preparation method and application thereof

Abstract

The invention discloses IBNPs (at) SiO2 nanoparticles and a controllable preparation method and application thereof. The method comprises the following steps: by taking iron and boron precursors and functional reagents as raw materials, synthesizing monodispersed iron borate nanoparticles by adopting a high-temperature thermal decomposition method; the size of the nano-particles is controlled by adjusting reaction parameters, so that the iron borate nano-particles with proper width and length are obtained; and carrying out SiO2 surface modification on the purified iron borate nano-particles, so as to prepare the IBNPs coated SiO2 nano-particles with good colloidal stability and biocompatibility. The IBNPs coated SiO2 nano-particles have long residence time at a tumor part, and the distribution and metabolism condition monitoring of a medicine in a body can be realized by virtue of the visual function of the IBNPs coated SiO2 nano-particles, so that the IBNPs coated SiO2 nano-particles can be used as a medical magnetic resonance imaging contrast agent and boron delivery agent. The novel boron delivery agent is helpful for providing a new diagnosis and treatment strategy for a boron neutron capture therapy technology.

Description

Technical Field

[0001] The present invention belongs to the application field of magnetic resonance imaging and boron neutron capture therapy, and specifically relates to an IBNPs@SiO2 nanoparticle and a controllable preparation method and application thereof. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is an emerging binary targeted therapy for tumor treatment. After nearly 90 years of development, this technology has proven to be the most effective treatment for melanoma and glioma. The successful implementation of BNCT technology is inseparable from the excellent performance of boron delivery agents. At present, there are two main types of clinically accessible boron delivery agents, namely sodium borocaptate (BSH) and 4-dihydroxyboronophenylala-nine (BPA).

[0003] However, clinical and scientific research results show that BSH has been proven to be inefficient and expensive due to its lack of receptor-mediated tumor targeting ability, while BPA has problems such as poor solubility, unstable boronic acid groups, and insufficient retention time in tumors. An ideal boron delivery agent should have high solubility, good tumor targeting ability, and stable boron content. In addition, the visualization function can realize the distribution of drugs in the body and the monitoring of metabolism. Therefore, there is still a long way to go to develop a visual boron delivery agent with excellent performance.

[0004] Iron borate nanoparticles (IBNPs) have attracted much attention due to their unique magnetic and electrical properties. In recent years, they have shown remarkable potential in the fields of catalysis and energy. At the same time, their applications in the biomedical field, especially in boron neutron capture therapy technology, remain to be developed. In order to prepare high-quality IBNPs, people usually use a combination of chemical and physical methods. However, the products prepared by these methods are usually not suitable for biomedical applications due to factors such as poor particle size uniformity, complex preparation process and long time consumption.

[0005] In contrast, high-temperature thermal decomposition provides a simpler, more controllable, and more uniform preparation method for nanoparticles. A large number of reported works have shown that the high-temperature thermal decomposition method can be used to prepare a variety of functional inorganic nanomaterials, which also highlights the high efficiency of the high-temperature thermal decomposition method in preparing high-quality functional inorganic nanomaterials. Summary of the invention

[0006] Based on this, the present invention provides an IBNPs@SiO2 nanoparticle and a controllable preparation method and application thereof, aiming to achieve the effect of inhibiting tumors while realizing magnetic resonance imaging in vivo, namely, visualized boron neutron capture therapy (BNCT).

[0007] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0008] A controllable preparation method of IBNPs@SiO2 nanoparticles comprises the following steps:

[0009] Step 1) using an iron precursor, a boron precursor and a functional reagent thereof as raw materials, and synthesizing monodisperse iron borate nanoparticles (IBNPs) by high temperature thermal decomposition method under closed conditions;

[0010] Step 2) During the high temperature thermal decomposition process, the size of the synthesized nanoparticles is controlled by adjusting reaction parameters including reaction temperature, reaction time or reaction concentration, thereby obtaining iron borate nanoparticles of suitable width and length;

[0011] Step 3) The purified iron borate nanoparticles are modified on the surface of SiO2 to form an inorganic nano-coating structure on the surface of the iron borate nanoparticles, thereby finally preparing IBNPs@SiO2 nanoparticles with good colloidal stability and biocompatibility.

[0012] Furthermore, the iron precursor is an iron salt (organic iron complex). Preferably, the iron salt is ferric acetylacetonate.

[0013] Further, the boron precursor is an organic boron small molecule. Preferably, the organic boron small molecule is synthesized by organic synthesis of n-hexanol and boronic acid-10 in toluene, the molar ratio of n-hexanol to boronic acid-10 is 3:1, the reflux temperature is 140° C., and the reflux time is 6 hours.

[0014] Furthermore, the functional agents are oleic acid and oleylamine.

[0015] Furthermore, the molar ratio of iron salt, organic boron small molecule, oleic acid and oleylamine is 1:1:(5-7):(5-7).

[0016] Furthermore, the adjustment range of the high-temperature thermal decomposition reaction temperature is 200-400° C. During the high-temperature thermal decomposition process, the length of the iron borate nanoparticles is controlled to be 5.3-54.8 nm and the width is controlled to be 2.5-22.4 nm by adjusting the reaction temperature parameters.

[0017] Furthermore, the adjustment range of the high-temperature thermal decomposition reaction time is 0.5 to 10 hours. By adjusting the reaction time parameters, the length of the iron borate nanoparticles is controlled to be 5.3 to 65.8 nm and the width is controlled to be 2.8 to 9.9 nm.

[0018] Furthermore, the concentration of the high-temperature thermal decomposition reaction is adjusted within a range of 1 to 20 mmol / L. By synchronously adjusting the concentration parameters of the iron precursor and the boron precursor, the length of the iron borate nanoparticles is controlled to be 7.3 to 17.0 nm and the width to be 8.3 to 8.5 nm.

[0019] Furthermore, the molar ratio of the iron borate nanoparticles to the inorganic nano-coating structure is (8-10):1.

[0020] Furthermore, the reaction temperature of the iron borate nanoparticles and the inorganic nano-coating structure is 15 to 37° C., and the reaction time is 15 to 48 hours.

[0021] An IBNPs@SiO2 nanoparticle is prepared by using the controllable preparation method of the IBNPs@SiO2 nanoparticle.

[0022] The above-mentioned IBNPs@SiO2 nanoparticles are used as medical magnetic resonance imaging contrast agents and boron delivery agents.

[0023] The above-mentioned IBNPs@SiO2 nanoparticles are used as medical magnetic resonance imaging contrast agents and boron delivery agents for melanoma.

[0024] The present invention discloses the characterization of the morphological properties of water-soluble IBNPs@SiO2 nanoparticles, as well as their toxicity at the cellular level and cellular uptake. Experimental results show that when the boron-10 concentration is 60 μg / mL or less, there is almost no toxicity to B16F10 cells. Cellular uptake shows that the concentration of boron-10 gradually increases with the extension of incubation time. Compared with the requirements of boron neutron capture therapy technology for boron-10, the concentration of boron-10 in cells is sufficient to support the inhibition of cancer cell growth. Confocal fluorescence microscopy also tracked the passive targeting of IBNPs@SiO2 into cells, indicating that nanoparticles can be passively targeted into cells for subsequent boron neutron capture therapy.

[0025] The present invention discloses the imaging capability of water-soluble IBNPs@SiO2 nanoparticles in subcutaneous melanoma. The results show a significant bright imaging effect in the tumor area. Signal analysis shows a significant contrast enhancement at the tumor site, which peaks immediately after injection of water-soluble IBNPs@SiO2 nanoparticles and continues to enhance for up to 8 hours. This extended tumor site imaging time window facilitates accurate tumor diagnosis.

[0026] In order to evaluate the killing ability of water-soluble IBNPs@SiO2 nanoparticles on cells after entering cells at the cellular level, the present invention discloses the cell viability of melanoma cells that have taken up nanoparticles after thermal neutron irradiation. The results show that compared with the control group, after thermal neutron irradiation, the viability of melanoma cells is significantly reduced, and the more nanoparticles taken up, the lower the cell viability.

[0027] The invention discloses the proliferation ability of melanoma cells after thermal neutron irradiation. The results show that compared with the control group, the viability and proliferation ability of melanoma cells after taking nanoparticles and thermal neutron irradiation is significantly reduced.

[0028] The present invention discloses an evaluation of the killing ability of water-soluble IBNPs@SiO2 nanoparticles on cells after entering cells at the animal level. The results show that melanoma was significantly inhibited in mice after thermal neutron irradiation after administration. During the subsequent monitoring period, the weight of the mice did not change significantly. Compared with the control group, the survival period of mice after administration after thermal neutron irradiation was significantly prolonged, which fully confirms that IBNPs@SiO2 nanoparticles have a good inhibitory effect on melanoma.

[0029] The IBNPs@SiO2 nanoparticles of the present invention can meet the following characteristics in the application of boron neutron capture therapy: (1) having good T1 magnetic resonance imaging effect; (2) being rich in boron-10 element; (3) having good crystallinity of the nanoparticles and maintaining good crystallinity after phase transfer; (4) being easily taken up and accumulated by tumor cells and having a long retention time in the tumor; and (5) having a high content of intracellular boron-10 element, which meets the boron content requirement of boron neutron capture therapy.

[0030] The beneficial effects of the present invention are:

[0031] The IBNPs@SiO2 nanoparticles of the present invention not only have a long residence time at the tumor site, but also their visualization function can realize the distribution of drugs in the body and the monitoring of metabolism, thus solving the problems of poor solubility, unstable boric acid groups and insufficient residence time in the tumor of existing clinically accessible small molecule boron delivery agents.

[0032] The present invention demonstrates that the particle size of IBNPs@SiO2 nanoparticles can be controlled and that the synthesis of IBNPs@SiO2 nanoparticles of different sizes can be achieved by optimizing the reaction conditions. The surface-modified IBNPs@SiO2 nanoparticles not only have good colloidal stability and biocompatibility, but also have good T1 magnetic resonance imaging capabilities.

[0033] Cell and animal neutron irradiation experiments confirmed that the IBNPs@SiO2 nanoparticles of the present invention have a good inhibitory effect on malignant melanoma. This new boron delivery agent is expected to provide a new diagnostic and therapeutic strategy for boron neutron capture therapy technology.

[0034] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 a is a schematic diagram of synthesizing IBNPs in Example 1 of the present invention;

[0037] Figure 1 b- Figure 1 e is the TEM, SAED and HRTEM images of IBNPs prepared in Example 1 of the present invention;

[0038] Figure 1 f- Figure 1 g is the XPS (Fe 2p and b1 s) sub-spectrum of IBNPs prepared in Example 1 of the present invention;

[0039] Figure 1 h is the XRD spectrum of IBNPs prepared in Example 1 of the present invention.

[0040] Figure 2 a- Figure 2 d is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 250° C. in Example 1 of the present invention;

[0041] Figure 2 e- Figure 2 h is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 280° C. in Example 1 of the present invention;

[0042] Figure 2 i- Figure 2 l is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310°C in Example 1 of the present invention.

[0043] Figure 3 a- Figure 3d is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310° C. for 1 hour in Example 1 of the present invention;

[0044] Figure 3 e- Figure 3 h is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310° C. for 4 hours in Example 1 of the present invention;

[0045] Figure 3 i- Figure 3 l is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310° C. for 7 hours in Example 1 of the present invention;

[0046] Figure 3 m- Figure 3 p is the TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310° C. for 10 hours in Example 1 of the present invention.

[0047] Figure 4 a- Figure 4 d is the TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized with 2 mmol / L Fe(acac)3 at 310°C for 4 hours in Example 1 of the present invention;

[0048] Figure 4 e- Figure 4 h is the TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized with 10 mmol / L Fe(acac)3 at 310°C for 4 hours in Example 1 of the present invention;

[0049] Figure 4 i- Figure 4 l is the TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized using 20 mmol / L Fe(acac)3 at 310°C for 4 hours in Example 1 of the present invention.

[0050] Figure 5 a is a typical low magnification transmission electron microscope (TEM) image of the water-soluble IBNPs@SiO2 nanoparticles synthesized in Example 2 of the present invention;

[0051] Figure 5 b is a magnified TEM image of the water-soluble IBNPs@SiO2 nanoparticles synthesized in Example 2 of the present invention. The nanoparticles are composed of nanorods as the core and a layer of silicon dioxide coated on the outer layer to form a core-shell structure;

[0052] Figure 5 c is a HRTEM image of the water-soluble IBNPs@SiO2 nanoparticles synthesized in Example 2 of the present invention;

[0053] Figure 5 d is the SAED image of the water-soluble IBNPs@SiO2 nanoparticles synthesized in Example 2 of the present invention;

[0054] Figure 5 e is a dynamic light scattering curve of the water-soluble IBNPs@SiO2 nanoparticles synthesized in Example 2 of the present invention in ultrapure water, and the inserted photo is a macroscopic dispersion of the nanoparticles in the aqueous solution;

[0055] Figure 5 f is a graph showing the cell viability results of B16F10 cells in Example 2 of the present invention after 24 hours in the presence of different concentrations of IBNPs@SiO2 nanoparticles (n=5);

[0056] Figure 5 g is a graph showing the boron uptake results of B16F10 cells at the same boron dose and at different incubation times in Example 2 of the present invention (n=3);

[0057] Figure 5 h is a representative confocal microscopy image of B16F10 cells incubated with IBNPs@SiO2 nanoparticles for 0 and 24 hours, respectively, in Example 2 of the present invention (scale bar, 10 μm);

[0058] Figure 5 i- Figure 5 j is the MRI images of tumor-bearing mice at different time points after intratumoral injection and the signal distribution curve of the tumor site in Example 2 of the present invention.

[0059] Figure 6 a is a schematic diagram of the scheme flow of verifying the boron neutron capture therapy of IBNPs@SiO2 nanoparticles at the cellular level in Example 3 of the present invention;

[0060] Figure 6 b is a graph showing the results of detecting the cell survival rate of B16F10 cells under different conditions for 24 hours using the CCK-8 method in Example 3 of the present invention (n=5);

[0061] Figure 6 c is a graph showing the results of detecting the clone formation of B16F10 cells cultured in a 6-well plate under different conditions using the crystal violet staining method in Example 3 of the present invention (n=3);

[0062] Figure 6 d is a diagram showing the quantitative counting result of the number of cell clones cultured one week later in Example 3 of the present invention, where the number of cells in each cell colony is guaranteed to be greater than 50;

[0063] Figure 6 e is the treatment regimen for tumor-bearing C57BL / 6J mice in Example 3 of the present invention;

[0064] Figure 6 f is an experimental device mold for the thermal neutron irradiation experiment in Example 3 of the present invention;

[0065] Figure 6 g is the changes of head and neck tumors in different groups of mice at different times in Example 3 of the present invention;

[0066] Figure 6 h is a graph showing the average change in tumor volume of each group of mice in Example 3 of the present invention (n=4);

[0067] Figure 6 i is a graph showing the average body weight of each group of mice at a specified time point in Example 3 of the present invention; Figure 6 j is a survival curve diagram of each group of mice at a specified time point in Example 3 of the present invention. DETAILED DESCRIPTION

[0068] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0069] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0070] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, ie, should be interpreted as "including, but not limited to."

[0071] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0072] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0073] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0074] The present invention first provides a controllable preparation method of IBNPs@SiO2 nanoparticles, comprising the following steps:

[0075] Step 1) using an iron precursor, a boron precursor and a functional reagent thereof as raw materials, and synthesizing monodisperse iron borate nanoparticles (IBNPs) by high temperature thermal decomposition method under closed conditions.

[0076] Among the iron precursors, boron precursors and their functional reagents, some are existing compounds and some are self-synthesized compounds.

[0077] The iron precursor is an existing compound, which is composed of an iron salt (organic iron complex), preferably iron acetylacetonate.

[0078] The functional agent is also an existing compound, consisting of oleic acid and oleylamine.

[0079] The boron precursor is a self-synthesized compound obtained by organic synthesis of n-hexanol and boric acid-10 in toluene. Preferably, the molar ratio is 3:1, the reflux temperature is 140° C., and the final product is obtained by reflux for 6 hours.

[0080] The molar ratio of iron salt, organic boron small molecule, oleic acid and oleylamine is 1:1:(5-7):(5-7).

[0081] The ratios of the iron precursor, the boron precursor and the functional reagent in the above reaction all follow the above preferred molar ratio.

[0082] In the present invention, there are many methods for synthesizing IBNPs, mainly a combination of physical and chemical methods. For example:

[0083] The iron borate nanoparticles are prepared by combining a hydrothermal method with high-temperature calcination;

[0084] The iron borate nanoparticles were prepared by ball milling and high temperature calcination.

[0085] Firstly, the intermediate product is synthesized by the sol-gel method, and then the final target product is prepared by ball milling and high-temperature calcination.

[0086] The IBNPs synthesized by the above method may have good application effects for specific applications reported in the literature (there is no special requirement for the uniformity of nanoparticles), but in the present invention, some improvements are made, and a high-temperature thermal decomposition method is used to prepare iron borate nanomaterials with uniform nanoparticles and high crystallinity. Characterization results show that the high-temperature thermal decomposition method can effectively prepare iron borate nanoparticles with certain magnetic properties, high crystallinity, good dispersibility, and adjustable nanoparticle size.

[0087] Step 2) During the high temperature thermal decomposition process, the size of the synthesized nanoparticles is controlled by adjusting reaction parameters including reaction temperature, reaction time or reaction concentration, thereby obtaining iron borate nanoparticles with suitable width and length.

[0088] In the present invention, the particle size of IBNPs can be adjusted during the synthesis process of high temperature thermal decomposition method. IBNPs of different widths and lengths can be obtained by adjusting the reaction temperature, reaction time or reaction concentration in the experiment. Specifically:

[0089] By adjusting the temperature parameters, preferably, the particle length of IBNPs can be controlled to be 5.3-54.8 nm, and the particle width can be controlled to be 2.5-22.4 nm;

[0090] By adjusting the time parameters, preferably, the particle length of IBNPs can be controlled to be 5.3-65.8 nm, and the particle width can be controlled to be 2.8-9.9 nm;

[0091] By adjusting the concentration parameters, preferably, the particle length of IBNPs can be controlled to be 7.3-17.0 nm, and the particle width can be controlled to be 8.3-8.5 nm.

[0092] The particle size is the size under transmission electron microscopy.

[0093] Preferably, the reaction temperature of the high temperature thermal decomposition is 200-400°C.

[0094] Preferably, the reaction time of high temperature thermal decomposition is 0.5 to 10 hours.

[0095] Preferably, the concentration of the reactants is adjusted within a certain concentration range under the premise that the molar ratio of the iron salt to the organic boron small molecule is 1:1. Taking the iron salt as an example, preferably, its concentration is adjusted to 1-20 mmol / L, and the concentration of the organic boron small molecule changes with the molar ratio of the iron salt.

[0096] It should be noted that when adjusting the concentration, the concentration of the functional reagent should be kept constant. When adjusting the reaction temperature and time, other conditions should remain the same.

[0097] Step 3) The purified iron borate nanoparticles are modified on the surface of SiO2 to form an inorganic nano-coating structure on the surface of the iron borate nanoparticles, thereby finally preparing IBNPs@SiO2 nanoparticles with good colloidal stability and biocompatibility.

[0098] The present invention preferably uses IBNPs with intermediate particle sizes for surface inorganic nano-coating modification. Preferably, the molar ratio of the iron borate nanoparticles to the surface inorganic nano-coating modification is (8-10):1.

[0099] Preferably, the reaction temperature of the iron borate nanoparticles and the surface inorganic nano-coating modifier is 15 to 37° C., and the reaction time is 15 to 48 hours.

[0100] The present invention also provides IBNPs@SiO2 nanoparticles prepared by the controllable preparation method of the IBNPs@SiO2 nanoparticles.

[0101] The present invention verifies the characterization of the morphological properties of water-soluble IBNPs@SiO2 nanoparticles, as well as their toxicity and cellular uptake at the cellular level. The experimental results show that when the boron-10 concentration is 60μg / mL or less, there is almost no toxicity to B16F10 cells. Cellular uptake shows that the concentration of boron-10 gradually increases with the extension of incubation time. Compared with the requirements of boron neutron capture therapy technology for boron-10, the concentration of boron-10 in cells is sufficient to support the inhibition of cancer cell growth. Confocal fluorescence microscopy also tracked the passive targeting of IBNPs@SiO2 into cells, indicating that nanoparticles can be passively targeted into cells for subsequent boron neutron capture therapy.

[0102] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles in magnetic resonance imaging, specifically, they can be used as medical magnetic resonance imaging contrast agents.

[0103] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles in boron neutron capture therapy, specifically, it can be used as a medical boron delivery agent.

[0104] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles as a medical magnetic resonance imaging contrast agent and a boron delivery agent.

[0105] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles in magnetic resonance imaging of subcutaneous melanoma, specifically, it can be used as a medical magnetic resonance imaging contrast agent for subcutaneous melanoma.

[0106] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles in boron neutron capture therapy for subcutaneous melanoma, specifically, it can be used as a medical boron delivery agent for subcutaneous melanoma.

[0107] The present invention also provides the use of the above-mentioned IBNPs@SiO2 nanoparticles as a medical magnetic resonance imaging contrast agent and boron delivery agent for melanoma.

[0108] The present invention tested the imaging ability of water-soluble IBNPs@SiO2 nanoparticles in subcutaneous melanoma. The results showed a significant bright imaging effect in the tumor area. Signal analysis showed that there was a significant contrast enhancement at the tumor site, which peaked immediately after the injection of water-soluble IBNPs@SiO2 nanoparticles and continued to enhance for up to 8 hours. This extended tumor site imaging time window helps accurate tumor diagnosis.

[0109] In order to evaluate the killing ability of water-soluble IBNPs@SiO2 nanoparticles on cells after entering cells at the cellular level, the present invention tested the cell viability of melanoma cells that had taken up nanoparticles after thermal neutron irradiation. The results showed that compared with the control group, the viability of melanoma cells decreased significantly after thermal neutron irradiation, and the more nanoparticles taken up, the lower the cell viability.

[0110] In addition, the present invention tested the proliferation ability of melanoma cells after thermal neutron irradiation. The results showed that compared with the control group, the viability and proliferation ability of melanoma cells after ingesting nanoparticles and thermal neutron irradiation was significantly reduced.

[0111] In addition, the present invention tested the ability of water-soluble IBNPs@SiO2 nanoparticles to kill cells after entering cells at the animal level. The results showed that melanoma was significantly inhibited after thermal neutron irradiation in mice after administration. During the subsequent monitoring period, the weight of the mice did not change significantly. Compared with the control group, the survival period of mice after administration was significantly prolonged after thermal neutron irradiation, which fully confirmed that IBNPs@SiO2 nanoparticles have a good inhibitory effect on melanoma.

[0112] The capabilities of the above-mentioned IBNPs@SiO2 nanoparticles of the present invention are described in detail through the following examples.

[0113] Example 1

[0114] The synthesis and characterization of IBNPs are as follows:

[0115] 1. Preparation of tributyl borate (10B):

[0116] First, weigh 768.6 mg of boric acid (10B) and 3.44 mL of 1-butanol into a two-necked flask containing 30 mL of toluene, mix well, and add a clean stirrer. Then, place the two-necked flask in an oil bath and fix it so that the liquid level in the bottle is lower than the oil bath level. Select one of the two-necked flasks and add a water separator, and add a condenser on the water separator. Connect the flowing condensed water to the condenser, and the direction of the water flow follows the principle of bottom-in and top-out. After the device is built, turn on the heating table switch, adjust the speed to 400 rpm / min, the temperature to 140°C, and wait for the liquid level in the water separator to rise, which means that the water produced by the reaction is taken out by toluene. In order to make the reaction proceed to the right thoroughly, the reaction can last for 4 to 10 hours depending on the situation. After the reaction is completed, wait for the yellow liquid product to cool to room temperature and collect the product. The obtained yellow liquid product is a mixed solution of tributyl borate (10B) and toluene solution. Take a small amount and measure ICP-AES to calibrate the 10B concentration.

[0117] 2. Synthesis of rod-shaped IBNPs:

[0118] See also Figure 1 As shown in a, 0.6mmol ferric acetylacetonate, 0.6mmol tributyl borate, 4.1mmol oleic acid and 4.1mmol oleylamine are dissolved in 30mL of toluene. The resulting solution is stirred and ultrasonicated for half an hour to ensure that all raw materials are completely dissolved in toluene. Then, the prepared solution is placed in a high-temperature autoclave with a reaction temperature detector, heated to 340°C for reaction, and maintained for 4 hours. After the reaction is completed, the crude product is collected after the autoclave is cooled to room temperature, and then the product is precipitated with acetone to remove unreacted raw materials, and then it is redispersed in cyclohexane. Repeat the above operation 3 times to purify the crude product, and the final product is dispersed in cyclohexane for subsequent use.

[0119] IBNPs of different lengths and widths can be obtained by adjusting the reaction temperature, time and reaction concentration through minor adjustments to the above experimental steps.

[0120] 3. Use o-phenanthroline spectrophotometry to calibrate the iron concentration in the solution:

[0121] First is the formulation of the standard curve. Take 0, 20, 40, 80, 120, 200 μL of Fe standard solution (1000g / mL) and place them in 6 EP tubes, add 200 μL of concentrated hydrochloric acid, 1 mL of hydroxylamine hydrochloride (mass concentration 10%), 5 mL of acetic acid-sodium acetate buffer (pH=4.5, 1M,) and 2 mL of o-phenanthroline aqueous solution (mass concentration 0.15%) in sequence. After the red color is developed, add a certain volume of ultrapure water to dilute to a moderate color and let it stand for 10 minutes to fully develop the color for testing. Use an ultraviolet spectrophotometer to test its absorbance curve, take the absorbance value at 510nm as the Y value, and the concentration of the diluted iron standard solution as the X value to draw the standard curve.

[0122] 4. Determination of iron concentration:

[0123] Take 20 μL of the prepared ferric borate solution, add 200 μL of concentrated hydrochloric acid, 1 mL of hydroxylamine hydrochloride (mass concentration 10%), 5 mL of acetic acid-sodium acetate buffer (pH=4.5, 1M,), 2 mL of o-phenanthroline aqueous solution (mass concentration 0.15%) in sequence, add a certain volume of ultrapure water to dilute to a moderate color after red color appears, let it stand for 10 minutes to fully develop color, and then test with an ultraviolet spectrophotometer. Substitute the absorbance value at 510 nm into the standard curve formula to calculate the iron concentration value of the solution.

[0124] 5. Use ICP-AES to calibrate the concentration of B in the solution:

[0125] The first step is to develop a standard curve. Take a mixed standard solution (100 μg / mL, containing element B) as the standard solution to be diluted, and take a certain volume and dilute it with 3% dilute nitric acid aqueous solution to prepare gradient solutions with concentrations of 0ppm, 0.5ppm, 1ppm, 2ppm, 5ppm, and 10ppm. Use ICP-AES to test its actual B concentration and perform fitting. When the linearity is greater than or equal to 0.9999, it indicates that the standard curve is set successfully, and the B concentration calibration of the next sample can be started.

[0126] 6. Determination of B concentration:

[0127] Take 20 μL of the solution to be tested and place it in a 15 mL EP tube, add concentrated nitric acid (67%) to fully digest the sample to be tested overnight. After complete digestion, add a certain volume of ultrapure water so that the mass fraction of nitric acid in the entire solvent system is 3%, record the volume after dilution, and calculate the dilution multiple after sample digestion. After the standard curve is successfully calibrated, directly test the digested sample and read the indication. The B concentration of the sample is the reading multiplied by the dilution multiple after sample digestion.

[0128] 7. Characterization of IBNPs

[0129] The hydrophobic IBNPs and SiO2-coated IBNPs were characterized by high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), and elemental analysis using a Tecnai G2 F20 electron microscope from FEI Company, USA.

[0130] The TEM, SAED and HRTEM images of IBNPs prepared in this example are shown in Figure 2. Figure 1 b- Figure 1 As shown in e.

[0131] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 250 °C in this example are shown in Figure 2. Figure 2 a- Figure 2 As shown in d.

[0132] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 280 °C in this example are shown in Figure 2. Figure 2 e- Figure 2 h.

[0133] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310 °C in this example are shown in Figure 2. Figure 2 i- Figure 2 l shown.

[0134] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310 °C for 1 hour are shown in Figure 2. Figure 3 a- Figure 3 As shown in d.

[0135] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310 °C for 4 hours are shown in Figure 2. Figure 3 e- Figure 3 h.

[0136] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310 °C for 7 hours are shown in Figure 2. Figure 3 i- Figure 3 l shown.

[0137] The TEM, HRTEM and SAED images of the hydrophobic IBNPs synthesized at 310 °C for 10 h are shown in Figure 2. Figure 3 m- Figure 3 As shown in p.

[0138] In this example, TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized with 2 mmol / L Fe(acac)3 at 310°C for 4 hours are shown in Figure 2. Figure 4 a- Figure 4 As shown in d.

[0139] In this example, TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized with 10 mmol / L Fe(acac)3 at 310°C for 4 hours are shown in Figure 2. Figure 4 e- Figure 4 h.

[0140] In this example, TEM, HRTEM and SAED images of hydrophobic IBNPs synthesized with 20 mmol / L Fe(acac)3 at 310°C for 4 hours are shown in Figure 2. Figure 4 i- Figure 4 l shown.

[0141] The phase of the samples was characterized by XRD-6000 instrument using Cu Kα1 radiation. The XRD spectrum of IBNPs prepared in this example is shown in Figure 1 h.

[0142] X-ray photoelectron spectroscopy (XPS) measurements were performed on an ESC ALAB Xi+ instrument. The XPS (Fe 2p and b1s) sub-spectra of the IBNPs prepared in this example are shown in Figure 2. Figure 1 f- Figure 1 As shown in g.

[0143] The hydrated particle size of the prepared IBNPs@SiO2 was characterized using a nanoparticle size and zeta potential analyzer (zs90) at room temperature.

[0144] The concentrations of iron and boron were determined using the ICAP7200 using inductively coupled plasma-atomic emission spectrometry (ICP-AES). When the iron concentration is estimated to be high, ultraviolet spectroscopy can also be used to calibrate the iron concentration.

[0145] Example 2

[0146] Synthesis of IBNPs@SiO2 nanoparticles and verification of their toxicity, uptake and imaging capabilities at the cellular level:

[0147] 1. Synthesis of IBNPs@SiO2 nanoparticles:

[0148] IBNPs (calibrated for iron concentration) with a concentration of 0.33 mg / mL were dispersed in cyclohexane, 200 μL was taken for dilution, and dissolved in 800 μL of cyclohexane for later use. 35 μL of IGEPAL CO-520 was taken, dissolved in 1 mL of cyclohexane, and dissolved by ultrasonic sonication. The diluted IBNPs cyclohexane solution was slowly added dropwise to the cyclohexane in which IGE PAL CO-520 was dissolved, and stirring was continued during the addition. After that, stirring was continued for 30 minutes, 10 μL of ammonia solution and 7.5 μL of TEOs were added, and the reaction was stopped by continuing stirring for 24 hours. After the reaction was completed, a mixture of ethanol and water was added to the reaction solution, centrifuged at a speed of 9000 rpm / min, and the supernatant was discarded. Repeat the above steps three times for purification to obtain IBNPs@SiO2 nanoparticles. The purified product was dissolved in ultrapure water and stored for later use.

[0149] 2. The FITC surface modification process of aqueous iron borate nanoparticles is slightly modified based on the experimental process of silica-coated nanoparticles:

[0150] As mentioned above, the cyclohexane solution of IGEPAL CO-520 and the cyclohexane solution of iron borate nanoparticles were fully stirred and mixed, and the NH4OH aqueous solution and TEOs were added after stirring for half an hour. After the reaction system was closed and the reaction lasted for 20 hours, 40 μL of 3-aminopropyltriethoxysilane (APTES) was added to aminize the surface of the mesoporous silica. The amount of 3-aminopropyltriethoxysilane (APTES) added followed the volume ratio of TEOs to APTES of 4:1. The amination process lasted for 4 hours. After the reaction was completed, the product was precipitated with a mixed solution of anhydrous ethanol and ultrapure water (5 mL of the solution after the reaction, 15 mL of anhydrous ethanol, and 15 mL of ultrapure water), the supernatant was removed, and the reaction product was repeatedly washed with a mixed solution of anhydrous ethanol and ultrapure water to remove the organic phase and the reactants that were not completely reacted. The final precipitated product was dissolved in 13 mL of anhydrous ethanol, ultrasonically dispersed, and set aside. Under light-proof conditions, weigh 3 mg of FITC-NHS and place it in a centrifuge tube, add 1 mL of dimethyl sulfoxide (DMSO) and shake thoroughly to dissolve. Transfer the DMSO solution of fully dissolved FITC-NHS to the anhydrous ethanol containing the product and stir overnight. The reaction is complete and FITC-NHS is bonded to the amino group on the surface of silica. Take a 30k ultrafiltration tube, transfer the reaction solution to the tube, ultrafilter at a speed of 6000 rpm / min for 10 minutes each time, repeat 3 times, and observe that the filtrate is almost colorless, that is, the purification is complete. Disperse the purified product in ultrapure water and store it in a refrigerator at 4°C for subsequent use. Note: The amount of FITC and the reaction experiment should be strictly protected from light.

[0151] 3. In vivo MRI was characterized using a 3.0T small animal MRI system. Imaging parameters:

[0152] T1w: TR=850ms, TE=11ms, FOV=40mm×40mm, Thickness=1mm;

[0153] T2w: TR=5000ms, TE=68ms, FOV=40mm×40mm, Thickness=1mm.

[0154] Three C57BL / 6J mice with subcutaneous tumors were randomly selected, and the average cell volume was consistent with 50-60mm 3 The mice were anesthetized with 5% isoflurane. Several volumes of silica-coated iron borate aqueous solution (Fe concentration, 600 ppm) were prepared. The imaging ability of iron borate nanoparticles in vivo was verified by intratumoral injection. The injection volume was 25 μL / mouse.

[0155] 4. Cytotoxicity assay:

[0156] B16 cells in the logarithmic growth phase were seeded in a 96-well plate at a concentration of 8×10 3 / well, 5 replicates for each concentration, and 6 drug concentrations were set. The inoculated 96-well plate was placed in a cell culture incubator at 37°C containing 5% CO2 / 95% air and incubated overnight. The next day, wait for the cells to adhere to the wall, add different concentrations (0, 50, 200, 400, 600 μg / mL) of IBNPs@SiO2 nanoparticles to the wells, and continue incubation for 24 hours. After the incubation, 1640 culture medium containing CCK8 (10% volume ratio) was added to the 6-well plate, and the cell viability was measured on a full-function microplate reader.

[0157] 5. Cell uptake experiment:

[0158] B16 cells in the logarithmic growth phase were seeded in 6-well plates at a concentration of 1×10 5 / well, 3 replicates for each concentration, 4 drug uptake time points are set, and there are three blank control wells for each time point. The inoculated 6-well plate was placed in a cell culture incubator at 37°C containing 5% CO2 / 95% air and incubated overnight. The next day, wait for the cells to adhere to the wall, add IBNPs@SiO2 nanoparticles at a concentration of 600μg / mL (Fe concentration) to the wells, continue incubation, and take a 6-well plate at 2, 6, 12, and 24 time points to digest the cells of the control and cell uptake groups with trypsin, wash them 3 times with PBS, and count them. Finally, the cells were centrifuged and concentrated, and the cell clusters were digested with concentrated nitric acid and diluted to a certain multiple to ensure that the nitric acid concentration in the solution was 3%, and its B concentration was determined by ICP-AES.

[0159] Typical low-magnification transmission electron microscopy (TEM) images show that the synthesized IBNPs@SiO2 nanoparticles have a core-shell structure, with hydrophobic IBNPs encapsulated in the center and good particle uniformity. High-resolution TEM images show that the encapsulated IBNPs have identifiable lattice fringes with a lattice spacing of 2.43 angstroms, corresponding to The selected area electron diffraction (SAED) pattern shows three clear diffraction rings, corresponding to (020) and (022) crystal planes. IBNPs@SiO2 nanoparticles have excellent water solubility, a relatively narrow particle size distribution, a single peak at 58.8 nm, and an almost unchanged particle size curve, which indicates that IBNPs@SiO2 nanoparticles have good colloidal stability at room temperature.

[0160] As a qualified boron delivery agent, its cytotoxicity and biocompatibility are the focus of investigation. The results showed that when the boron-10 concentration was 60 μg / mL or lower, there was almost no significant toxicity to B16F10 cells. This safe concentration can be used as a reference for subsequent cell uptake experiments. The cellular uptake of IBNPs@SiO2 nanoparticles was studied by inductively coupled plasma atomic emission spectrometry (ICP-AES), and B16F10 cells were incubated with IBNPs@SiO2 nanoparticles (boron-10 concentration: 60 μg / mL) and then detected. The results showed that the concentration of boron-10 in the cells increased with the extension of the incubation time. When the incubation time was 2 hours, the boron-10 concentration reached 16.11 μg / 10 7 When the incubation time was extended to 24 hours, the concentration of boron-10 tended to stabilize and reached 34.03 μg / 10 7 cells. 9 Boron-10 / cell, i.e. 166.7ng Boron-10 / 10 7Compared with 10 cells per 100 μg / mL, the intracellular boron-10 concentration was sufficient to support cancer cell inhibition experiments. Confocal fluorescence microscopy was used to track the intracellular transport of IBNPs@SiO2 nanoparticles. The results showed obvious green fluorescence emission in the cytoplasmic region of B16F10 cells, indicating that FITC-labeled IBNPs@SiO2 nanoparticles successfully entered the cytoplasm during co-incubation with B16F10 cells. To verify the imaging effect of the prepared product in vivo, T1-weighted magnetic resonance imaging (MRI) was performed after intratumoral injection of IBNPs@SiO2 nanoparticles. The images showed obvious bright imaging effects in the tumor area. Signal analysis results showed that the contrast of the tumor site was significantly enhanced, reaching a peak after injection and continuing to enhance for 8 hours. The above results fully demonstrate that the synthesized IBNPs@SiO2 nanoparticles not only have good colloidal stability and biocompatibility, but also have high boron uptake, and have great potential in boron neutron capture therapy.

[0161] See also Figure 5 As shown, Figure 5 Evaluation of cellular uptake and imaging of water-soluble IBNPs@SiO2 nanoparticles.

[0162] in,

[0163] Typical low magnification transmission electron microscopy (TEM) images of synthesized IBNPs@SiO2 nanoparticles are shown in Figure 2. Figure 5 As shown in a.

[0164] The magnified TEM image of the synthesized IBNPs@SiO2 nanoparticles is shown in Figure 5 As shown in b, the nanoparticles are composed of nanorods as the core and covered with a layer of silica on the outside to form a core-shell structure.

[0165] HRTEM images of IBNPs@SiO2 nanoparticles Figure 5 As shown in c.

[0166] SAED images of IBNPs@SiO2 nanoparticles are shown in Figure 2. Figure 5 As shown in d.

[0167] Dynamic light scattering curve of IBNPs@SiO2 nanoparticles in ultrapure water Figure 5 e shows that the inserted photograph is an aqueous solution of nanoparticles at a macroscopic level.

[0168] Cell viability of B16F10 cells after 24 h after exposure to different concentrations of silicon-coated IBNPs (n=5) Figure 5 f.

[0169] Boron uptake of B16F10 cells at different incubation times under the same boron dose (n=3) Figure 5 As shown in g.

[0170] Representative confocal microscopy images of B16F10 cells incubated with IBNPs@SiO2 nanoparticles for 0 and 24 h, respectively. Figure 5 h, where the scale bar is 10 μm.

[0171] MRI images of tumor-bearing mice at different time points after intratumoral injection and signal distribution curves of tumor sites are shown in Figure 2. Figure 5 i- Figure 5 j shown.

[0172] Example 3

[0173] Verification of IBNPs@SiO2 nanoparticles for boron neutron capture therapy at the cell and animal levels:

[0174] 1. Establishment of subcutaneous melanoma animal model in C57BL / 6J mice:

[0175] Several 5-week-old C57BL / 6J mice were purchased and raised in an SPF-grade animal room for one week. After the mice adapted to the environment and stabilized, the hair on their heads and necks was shaved, and the next day, B16 cells in the logarithmic growth phase were implanted subcutaneously in their heads and necks. After trypsin digestion, the B16 cells were resuspended in PBS, centrifuged 3 times, and finally dispersed in PBS and counted. The number of tumor-bearing cells was 50w / mouse, and the tumor volume was 50μL / mouse. After 5 days of growth after the end of tumor bearing, when the tumor volume reached 50-60mm 3 Subsequent in vivo experiments can be carried out.

[0176] 2. Cell cloning experiment:

[0177] The cell cloning experiment was divided into 4 groups, namely PBS without neutron irradiation group, PBS neutron irradiation group, drug-added without neutron irradiation group, and drug-added neutron irradiation group. B16 cells in the logarithmic growth phase were inoculated in 6-well plates at a concentration of 1000 cells / well, with 3 replicates for each group. The inoculated 6-well plates were placed in a 37°C incubator containing 5% CO 2 / 95% air in a cell culture incubator for 6 days. When the cells are cloned in the wells, remove the culture medium and rinse with PBS 3 times. Add 2mL of methanol to each well to fix the cells for 10 minutes. After the cells are fixed, remove the methanol and rinse with PBS 3 times. Add crystal violet stain and stain at room temperature for 10 minutes. Finally, discard the stain and wash the well plate with ultrapure water until no stain flows out. Count the number of cell colonies in the well plate under a microscope.

[0178] 3. Cell neutron irradiation experiment:

[0179] The cell neutron irradiation experiment was divided into four drug concentration irradiation groups, 0, 5, 20, and 60 ppm (B concentration). B16 cells in the logarithmic growth phase were inoculated in a 6-well plate at a concentration of 10 7 The inoculated 6-well plate was placed in a 37°C, 5% CO 2 / 95% air in a cell culture incubator. The next day, after the cells adhere to the wall, add the corresponding concentration of drugs to each group and continue incubation for 24 hours. After the incubation, the medium containing the drug is aspirated and washed with PBS three times. The cells are digested with trypsin and washed with PBS three times to remove the unabsorbed drugs. After washing, the cells are resuspended in 1mL of 1640 culture medium and placed in a cell irradiation fixture for neutron irradiation.

[0180] 4. Animal neutron irradiation experiment:

[0181] The qualified tumor-bearing mice were randomly divided into 4 groups, namely, PBS without neutron irradiation group, PBS neutron irradiation group, drug administration without neutron irradiation group, and drug administration neutron irradiation group. There were 8 mice in each group, and the injection volume of the drug and PBS groups was 25L. The 4 groups of mice were injected with the corresponding drugs and PBS into the tumor at similar intervals. After waiting for one hour, the PBS group and drug administration group that needed neutron irradiation were neutron irradiated for 30 minutes. After the experiment, the condition, tumor volume, and body weight of the mice were monitored daily for 14 days. After the monitoring, the corresponding survival curve, tumor volume change trend line, and body weight change trend line of each group were drawn. Tumor volume calculation follows the formula: V (tumor volume) = L × W 2 / 2 (L is the length of the tumor, W is the width of the tumor).

[0182] Cell cloning technology is an effective means to demonstrate cell proliferation ability and a powerful technology to reflect cell activity. Four groups of experimental cells were selected for cloning experiments, namely, the PBS group, the B16F10 cell co-incubation group with aqueous iron borate nanoparticles (boron concentration 60ppm), the PBS neutron irradiation group, and the B16F10 cell co-incubation group with aqueous iron borate nanoparticles (boron concentration 60ppm) followed by neutron irradiation. The results showed that in the PBS and NPs groups without neutron irradiation, the cell proliferation ability was strong, and there was no significant difference between the groups, indicating that the cell uptake of nanoparticles had no significant effect on its proliferation ability. In order to explore the killing ability of thermal neutrons on cells, the PBS+N group was set up. The results showed that compared with the non-neutron group, the cell proliferation ability of the PBS+N group did not show significant differences, indicating that thermal neutrons had no significant effect on cell proliferation. However, when the cells that had taken up the nanoparticles were irradiated with neutrons, it was found that the number of cell colonies was very low, and only a few cloned cell clusters could be seen in the wells. This shows that the effect of the nanoparticles after neutron irradiation is very lethal to the cells, causing the cells to be unable to adhere to the wall and proliferate. 10 Element B plays a fundamental role and is a powerful weapon for eliminating tumor cells.

[0183] See also Figure 6 As shown in a, in order to verify the boron neutron capture therapeutic effect of iron borate nanoparticles at the cellular level, next, 4 aqueous iron borate nanoparticles with different boron concentrations (0, 5, 20, 60) were selected to incubate with B16F10 cells for 24 hours, irradiated for 3 minutes under the same neutron flux, and after waiting for the neutron experiment of the cells to end, the CCK8 kit was used to detect the activity of the cells. The results showed that in the absence of neutron irradiation, B 16F10 cells were incubated with aqueous iron borate nanoparticles with a boron concentration of 60ppm for 24 hours and still maintained 81.5% activity, indicating that the nanoparticles have good biocompatibility. However, when the B16F10 cells treated in the same way as the non-neutron group were placed under neutron beam irradiation, the cell activity changed significantly. As shown in the figure, when the boron concentration was 0ppm, the cell activity was almost unaffected by the neutron beam and had good activity. When the boron concentration was 5ppm, the cell activity instantly reached 69.2%, indicating that the iron borate nanoparticles ingested by the B16F10 cells played a role in killing cells under neutron beam irradiation. When the boron concentration of cell incubation continued to increase to 20ppm, the cell activity dropped to 22.9%. When the boron concentration of cell incubation increased to 60ppm, the cell activity was almost close to 0%. Therefore, the above cell experimental results fully demonstrate that after being taken up by cells, aqueous iron borate nanoparticles exhibit excellent tumor killing effects after being irradiated by thermal neutrons.

[0184] Although the boron neutron capture therapy effect of nanoparticles has been verified at the cellular level, verification at the animal level is still essential. This experiment selected C57BL / 6J mice as the subjects for B16F10 cell implantation. The mice were 5 weeks old and the implantation site was the head and neck. One day after the hair removal of the head and neck of the mice, 500,000 B16F10 cells were injected subcutaneously with an insulin needle and grown for 1 week until they grew to 50-60 mm 3 Prepare for neutron irradiation therapy. Pick out the mice whose tumors have reached the treatment volume, and inject 25μL of 236ppm aqueous iron borate nanoparticles into the tumor 1 hour before neutron irradiation. After the drug is completely absorbed, the mice are anesthetized with emulsified isoflurane (intraperitoneal injection). Place the anesthetized mice in a mold and irradiate with the same neutron flux for 34 minutes. After cooling, take out the mice, and monitor their tumor volume, body weight, and survival status every day. The results showed that the tumors of mice in the PBS group, NPs group, and PBS+N group grew rapidly in the first 8 days, especially in the PBS group. The average tumor volume reached 1213.9mm on the 7th day after treatment. 3 . However, the tumor volume of the NPs+N group remained small after 14 days of monitoring, which was significantly different from the other three control groups, further illustrating the boron neutron capture therapy effect of aqueous iron borate nanoparticles. In addition to tumor volume, body weight and survival curves were also monitored simultaneously. The results showed that during the monitoring period, the body weight of mice in each group remained basically constant. The survival rate of mice in the NPs+N group was the highest, and the other groups died one after another after the 7th day of treatment. In general, the above cell and animal neutron irradiation experimental results fully demonstrate that the prepared IBNPs@SiO2 nanoparticles have a good inhibitory effect on malignant melanoma, and this new boron delivery agent is expected to provide a new diagnosis and treatment strategy for boron neutron capture therapy technology.

[0185] See also Figure 6 As shown, neutron irradiation experiments in cells and mice confirmed that IBNPs@SiO2 had a good inhibitory effect on melanoma.

[0186] The CCK-8 method was used to detect the cell survival rate of B16F10 cells after 24 hours under different conditions (n=5). Figure 6 As shown in b.

[0187] Crystal violet staining was used to detect the clone formation of B16F10 cells cultured in 6-well plates under different conditions (n=3). Figure 6 As shown in c.

[0188] The results of quantitative counting of the number of cell clones cultured one week later are as follows: Figure 6 As shown in d, the number of cells in each cell colony was guaranteed to be greater than 50.

[0189] Treatment regimen for tumor-bearing C57BL / 6J mice Figure 6 As shown in e.

[0190] The experimental device mold for thermal neutron irradiation experiment is as follows Figure 6 f.

[0191] The changes of head and neck tumors in different groups of mice at different times are shown in Figure 2 Figure 6 As shown in g.

[0192] The average change of tumor volume in each group of mice (n = 4) is shown in Figure 6 h.

[0193] The average body weight of mice in each group at the designated time points is shown in Figure 6 As shown in i.

[0194] The survival curves of each group of mice at designated time points are shown in Figure 2. Figure 6 j shown.

[0195] Experiments have shown that the IBNPs@SiO2 nanoparticles of the present invention can meet the following characteristics in the application of boron neutron capture therapy: (1) having good T1 magnetic resonance imaging effect; (2) being rich in boron-10 element; (3) having good crystallinity of the nanoparticles and maintaining good crystallinity after phase transfer; (4) being easily taken up and accumulated by tumor cells and having a long retention time in the tumor; and (5) having a high content of intracellular boron-10 element, which meets the boron content requirement of boron neutron capture therapy.

[0196] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A controllable preparation method of IBNPs@SiO2 nanoparticles, characterized in that: The following steps are involved: Step 1) using an iron precursor, a boron precursor and a functional reagent thereof as raw materials, and synthesizing monodisperse iron borate nanoparticles by high temperature thermal decomposition method under closed conditions; Step 2) During the high temperature thermal decomposition process, the size of the synthesized nanoparticles is controlled by adjusting reaction parameters including reaction temperature, reaction time or reaction concentration, thereby obtaining iron borate nanoparticles of suitable width and length; Step 3) The purified iron borate nanoparticles are modified on the surface of SiO2 to form an inorganic nano-coating structure on the surface of the iron borate nanoparticles, thereby finally preparing IBNPs@SiO2 nanoparticles with good colloidal stability and biocompatibility.

2. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The iron precursor is an iron salt, the boron precursor is an organic boron small molecule, the functional reagents are oleic acid and oleylamine, and the molar ratio of the iron salt, the organic boron small molecule, oleic acid, and oleylamine is 1:1:(5-7):(5-7).

3. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The adjustment range of the high-temperature thermal decomposition reaction temperature is 200-400°C. During the high-temperature thermal decomposition process, the length of the iron borate nanoparticles is controlled to be 5.3-54.8nm and the width is controlled to be 2.5-22.4nm by adjusting the reaction temperature parameters.

4. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The adjustment range of the high-temperature thermal decomposition reaction time is 0.5 to 10 hours. By adjusting the reaction time parameters, the length of the iron borate nanoparticles is controlled to be 5.3 to 65.8 nm and the width is controlled to be 2.8 to 9.9 nm.

5. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The concentration of the high-temperature thermal decomposition reaction is adjusted in the range of 1 to 20 mmol / L. By synchronously adjusting the concentration parameters of the iron precursor and the boron precursor, the length of the iron borate nanoparticles is controlled to be 7.3 to 17.0 nm and the width to be 8.3 to 8.5 nm.

6. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The molar ratio of the iron borate nanoparticles to the inorganic nanocoating structure is (8-10):

1.

7. The controllable preparation method of IBNPs@SiO2 nanoparticles according to claim 1, characterized in that: The reaction temperature of the iron borate nanoparticles and the inorganic nanocoating structure is 15 to 37° C., and the reaction time is 15 to 48 hours.

8. An IBNPs@SiO2 nanoparticle, characterized in that: The IBNPs@SiO2 nanoparticles are prepared by the controllable preparation method according to any one of claims 1 to 7.

9. Use of IBNPs@SiO2 nanoparticles prepared by the controllable preparation method of IBNPs@SiO2 nanoparticles according to any one of claims 1 to 7 or the IBNPs@SiO2 nanoparticles according to claim 8 as a medical magnetic resonance imaging contrast agent and boron delivery agent.

10. Use of the IBNPs@SiO2 nanoparticles prepared by the controllable preparation method of IBNPs@SiO2 nanoparticles according to any one of claims 1 to 7 or the IBNPs@SiO2 nanoparticles according to claim 8 as a medical magnetic resonance imaging contrast agent and boron delivery agent for melanoma.

Images