Antibody-modified barium titanate nanoparticles and preparation method and application thereof

Abstract

The present invention belongs to the field of material synthesis and biomedicine technology, and specifically relates to an antibody-modified barium titanate nanoparticle, and a preparation method and application thereof. The present invention synthesizes piezoelectric barium titanate nanoparticles by a hydrothermal method, and coats polydopamine on the surface and modifies antibodies to obtain αCD56‑PDA@BTO nanoparticles. The nanoparticles obtained by the above preparation method are small and uniform in size, with a particle size of about 150 nm, and the material is piezoelectric. The antibody-modified nanoparticles have the ability to target NK cells. Under ultrasonic stimulation, αCD56‑PDA@BTO nanoparticles can increase the activation level and effector function of NK cells. In vitro and in vivo experiments have confirmed that they have good anti-tumor ability and are expected to be used in the treatment of solid tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of material synthesis and biomedicine, and specifically relates to antibody-modified barium titanate nanoparticles and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In recent years, the number of new cancer cases and deaths has shown a rapid growth trend. Clinically, tumors are generally treated through surgery, radiotherapy, chemotherapy, etc., but there are unavoidable tumor recurrence and other side effects. Immunotherapy is to curb tumor development by enhancing or activating the body's specific immune response, thereby achieving the purpose of radical cure of tumors. It has become the most promising tumor treatment method.

[0004] Natural killer (NK) cells are a promising tool for cancer immunotherapy. NK cells are an important component of the human body's innate immunity. Because they are not restricted by MHC and tumor antigens, they can respond quickly to the external environment without pre-sensitization, and play an important role in the body's anti-tumor process. NK cells act directly on tumor cells mainly through cytotoxic pathways and the production of cytokines to achieve the purpose of rapid killing of tumor cells; and can cooperate with other immune cells in the body to produce a sustained anti-tumor immune response. However, the hypoxic and low-nutrient conditions in the tumor microenvironment increase the degree of NK cell exhaustion, decrease cell function, and reduce the number of cells. Unlike hematological tumors, the migration and infiltration abilities of NK cells in solid tumors are reduced, and the cytotoxic function is weakened, making the effect of NK cell adoptive transfer therapy unsatisfactory. Therefore, the reduction of NK cell function and the reduction of cell migration and infiltration in solid tumors are the main obstacles to the use of NK cells for anti-tumor treatment. Developing a strategy and program that can enhance the anti-tumor function of NK cells in the tumor microenvironment will have important clinical application value.

[0005] In recent years, mechanical signals generated by cell mechanical stimulation and response have been fully demonstrated to play an important role in the cell life cycle. Cell mechanical signals can not only effectively regulate cell spreading, migration, proliferation, differentiation and other behaviors, but also play a decisive role in the body's embryonic development, tissue formation, disease development and other critical biological processes. In the process of immune cells recognizing and killing tumor cells, mechanical signals are generated after immune cells come into contact with tumor cells, which in turn triggers the movement of immune cell actin, causing immune cells to produce nanoscale receptor organizations and induce cell polarization, form immune synapses, control the secretion of cytokines and kill tumor cells. Cell electrical signals also exist in the cell life cycle and play an important role. Electrical signals can affect biological processes such as cell proliferation, apoptosis, differentiation, and cell function by controlling calcium ion influx, cytoskeleton movement, and cell plasma membrane polarization. Therefore, the design of joint activation of NK cells based on mechanical signals and electrical signals acting on the surface of NK cells to improve cell function is expected to provide a new strategy for the treatment of solid tumors. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an antibody-modified barium titanate nanoparticle and its preparation method and application. Based on the piezoelectricity, biocompatibility and small size of barium titanate nanoparticles that are easy to carry in cells, and combined with the targeting of antibodies and the advantages of external field ultrasound to provide a controllable signal source, the present invention prepares a nanoparticle that can target the surface of NK cells and generate mechanical signals and electrical signals under the action of external field ultrasound to synergistically activate the function of NK cells. In vitro and in vivo experiments have confirmed that it has good anti-tumor ability and is expected to be used in the treatment of solid tumors.

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

[0008] In a first aspect, the present invention provides an antibody-modified barium titanate nanoparticle, wherein the nanoparticle comprises polydopamine-barium titanate nanoparticle and an antibody modified on the surface thereof, wherein the antibody is selected from one of αCD56 and αNK1.1.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned antibody-modified barium titanate nanoparticles, comprising the following steps:

[0010] S1, Ba(OH)2 . 8H2O and TiO2 are mixed and reacted to obtain barium titanate nanoparticles (BTO);

[0011] S2, dispersing dopamine hydrochloride into the solution and incubating it with barium titanate nanoparticles to obtain polydopamine-modified barium titanate nanoparticles (PDA@BTO);

[0012] S3. Dispersing the polydopamine-modified barium titanate nanoparticles into a solution for incubation, and then adding antibodies for co-incubation to obtain antibody-modified barium titanate nanoparticles.

[0013] Preferably, in step S1, the Ba(OH)2 . The mass of 8H2O is 9.0~10.0 g, and the mass of TiO2 is 0.5~1.0 g.

[0014] Preferably, in step S1, the heating temperature of the reaction is 200-220° C., and the heating time is 36-60 h.

[0015] Preferably, in step S1, the mixture after the reaction needs to be cooled, washed and dried in sequence; wherein the washing refers to washing and centrifuging with HCl and ultrapure water; preferably, the concentration of HCl is 0.1-0.2 M.

[0016] Preferably, in step S2, the mass of the barium titanate nanoparticles is 0.1-0.5 g; and the concentration of the dopamine hydrochloride is 0.01-0.02 M.

[0017] Preferably, in step S2, the dopamine hydrochloride is dispersed in a Tris-HCl solution; further preferably, the concentration of the Tris-HCl solution is 0.01-0.05 M, and the volume of the Tris-HCl solution is 100-500 mL.

[0018] Preferably, in step S2, the co-incubation temperature is room temperature, and the co-incubation time is 4 to 8 h.

[0019] Preferably, in step S3, the mass of the polydopamine-modified barium titanate nanoparticles is 10-50 μg; and the mass of the antibody is 10-200 μg.

[0020] Preferably, in step S3, the polydopamine-modified barium titanate nanoparticles are dispersed in a mixed solution of EDC and Sulfo-NHS, and then the pH value of the mixed solution is adjusted to be incubated under neutral conditions, and then the antibody is added to the mixture and incubated to obtain antibody-modified polydopamine-barium titanate nanoparticles.

[0021] Further preferably, the molar ratio of EDC to Sulfo-NHS is 0.5-1.5:0.5-1.5, more preferably 1:1.

[0022] The third aspect of the present invention provides the use of the antibody-modified barium titanate nanoparticles described in the first aspect in the preparation of drugs for tumor targeting and treatment.

[0023] Preferably, the drug is a drug that enhances the activation level of NK cells or a drug that enhances the anti-tumor function of NK cells.

[0024] Preferably, the tumor includes lung cancer, melanoma, glioma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, blood tumor, lymphoma; further, it is melanoma.

[0025] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0026] (1) The present invention uses the non-contact mechanical signals and electrical signals generated by piezoelectric materials and acting on the surface of NK cells to improve the function of NK cells and enhance the anti-tumor effect of NK cells, providing a new treatment method for regulating NK cells in adoptive immune cell therapy;

[0027] (2) The αCD56-PDA@BTO nanoparticles provided by the present invention can be targetedly loaded on the surface of NK cells, and promote the activation level and functional effect of NK cells through the synergistic effect of mechanical and electrical signals. In vivo and in vitro experiments have confirmed that they have good anti-tumor ability and are expected to be used in the treatment of solid tumors;

[0028] (3) The barium titanate nanoparticles prepared by the present invention are a kind of functional conversion material. Due to their excellent piezoelectricity, they can convert mechanical signals into electrical signals. With the help of the energy source provided by the external ultrasonic device, ultrasonic treatment is performed at a fixed position in the human body, which can effectively regulate the local area of ​​NK cells.

[0029] (4) The barium titanate nanoparticles prepared by the present invention are small in size, which is conducive to the loading and carrying of NK cells, and the antibodies targeting NK cells are connected on the surface, which can only act on the targeted NK cells and avoid interference with other immune cells, thereby regulating NK cells and improving the anti-tumor ability of NK cells in the human body;

[0030] (5) The barium titanate nanoparticles prepared by the present invention have low toxicity, and the polydopamine-coated barium titanate nanoparticles are beneficial to improving their biocompatibility and material dispersion, and the αCD56-PDA@BTO nanoparticle material is highly stable, which is beneficial for long-term regulation of NK cells. Among them, the main functions of polydopamine are: ① providing a group that can connect to antibodies, and coupling antibodies to polydopamine through chemical bonds; ② based on the piezoelectricity of the BTO material in the present invention, it can generate electrical signals, and the polydopamine thin layer is conductive, which can transmit the electrical signals generated by BTO to NK cells; ③ improving the dispersion and stability of BTO nanoparticles in the solution system. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 The scanning electron microscope images of the barium titanate nanoparticles synthesized by the hydrothermal method and the barium titanate nanoparticles modified by polydopamine in Example 1 of the present invention, wherein A is a scanning electron microscope image of the barium titanate nanoparticles, and B is a scanning electron microscope image of the barium titanate nanoparticles modified by polydopamine;

[0033] Figure 2 TEM images of barium titanate nanoparticles synthesized by the hydrothermal method and barium titanate nanoparticles modified with polydopamine in Example 1 of the present invention, wherein A is a transmission electron microscopy image of barium titanate nanoparticles, B is a transmission electron microscopy image of local barium titanate nanoparticles magnified under a high power microscope, C is a transmission electron microscopy image of barium titanate nanoparticles modified with polydopamine, and D is a transmission electron microscopy image of barium titanate nanoparticles partially coated with polydopamine magnified under a high power microscope;

[0034] Figure 3 is an X-ray diffraction pattern of the barium titanate nanoparticles and the polydopamine-modified barium titanate nanoparticles in Example 1 of the present invention;

[0035] Figure 4 The hysteresis loop diagram and butterfly curve diagram of the piezoelectric force microscope of barium titanate in Example 1 of the present invention, wherein A is the hysteresis loop diagram of the piezoelectric force microscope of barium titanate nanoparticles, and B is the butterfly curve diagram of the piezoelectric force microscope of barium titanate nanoparticles;

[0036] Figure 5 This is an oscilloscope-electrical signal diagram of the barium titanate nanoparticles coated with polydopamine in Example 1 of the present invention;

[0037] Figure 6 This is a digital photograph of the sedimentation-time of the barium titanate nanoparticles and the polydopamine-coated barium titanate nanoparticles in Example 1 of the present invention;

[0038] Figure 7 The Fourier transform infrared spectra of the barium titanate nanoparticles, the barium titanate nanoparticles modified with polydopamine, and the polydopamine-barium titanate nanoparticles modified with antibody αCD56 in Example 1 of the present invention, wherein Ⅰ is the Fourier transform infrared spectra of the barium titanate nanoparticles, Ⅱ is the Fourier transform infrared spectra of the barium titanate nanoparticles modified with polydopamine, and Ⅲ is the Fourier transform infrared spectra of the polydopamine-barium titanate nanoparticles modified with antibody αCD56;

[0039] Figure 8These are the confocal results of the targeting test of the antibody-modified barium titanate nanoparticles prepared in Example 1 of the present invention, wherein A is the confocal result of the polydopamine-barium titanate nanoparticles modified with the antibody αCD56 binding to natural killer cells and K562 cells, respectively, and B is the confocal result of the polydopamine-barium titanate nanoparticles modified with the antibody NK1.1 binding to mouse natural killer cells and Yac-1 cells, respectively;

[0040] Fig. 9 The live / dead staining result graphs of natural killer cells and mouse natural killer cells after the αCD56-PDA@BTO nanoparticles prepared in Example 1 of the present invention were co-incubated with natural killer cells, and the αNK1.1-PDA@BTO nanoparticles were co-incubated with mouse spleen natural killer cells, wherein A is a live / dead staining result graph of natural killer cells after the αCD56-PDA@BTO nanoparticles were co-incubated with natural killer cells, and B is a live / dead staining result graph of mouse spleen natural killer cells after the αNK1.1-PDA@BTO nanoparticles were co-incubated with mouse spleen natural killer cells;

[0041] Fig.10 The cell viability graph of natural killer cells after the prepared αCD56-PDA@BTO nanoparticles are combined with natural killer cells and the cell viability level graph of mouse natural killer cells after the prepared αNK1.1-PDA@BTO nanoparticles are combined with mouse natural killer cells, which are statistically analyzed using trypan blue staining in Example 1 of the present invention, wherein A is the cell viability graph of natural killer cells after the prepared αCD56-PDA@BTO nanoparticles are combined with natural killer cells, and B is the cell viability level graph of mouse natural killer cells after the prepared αNK1.1-PDA@BTO nanoparticles are combined with mouse natural killer cells;

[0042] Fig.11 The figures are result graphs of natural killer cells killing different tumor cells in different effector-target ratio systems after the αCD56-PDA@BTO nanoparticles prepared in Example 1 of the present invention are combined with natural killer cells, wherein A is a graph showing the killing of K562-Luc cells at an effector-target ratio of 0.5:1; B is a graph showing the killing of K562-Luc cells at an effector-target ratio of 1:1; C is a graph showing the killing of HepG2-Luc cells at an effector-target ratio of 0.5:1; D is a graph showing the killing of HepG2-Luc cells at an effector-target ratio of 1:1; E is a graph showing the killing of U251-Luc cells at an effector-target ratio of 0.5:1; and F is a graph showing the killing of U251-Luc cells at an effector-target ratio of 1:1;

[0043] Fig.12This is a graph showing the killing of B16-Luc cells by mouse natural killer cells after the αNK1.1-PDA@BTO nanoparticles prepared in Example 1 of the present invention are combined with mouse natural killer cells;

[0044] Fig.13 Figures showing the ELISA results of tumor necrosis factor α and interferon γ of the effector function of natural killer cells after the antibody-modified barium titanate nanoparticles prepared in Example 1 of the present invention bind to natural killer cells, wherein A is a graph showing the secretion of tumor necrosis factor α in the supernatant detected after co-culture of natural killer cells loaded with αCD56-PDA@BTO nanoparticles with HepG2 cells, B is a graph showing the secretion of interferon γ in the supernatant detected after co-culture of natural killer cells loaded with αCD56-PDA@BTO nanoparticles with HepG2 cells, C is a graph showing the secretion of tumor necrosis factor α in the supernatant detected after co-culture of mouse spleen natural killer cells loaded with αNK1.1-PDA@BTO nanoparticles with B16 / F10 cells, and D is a graph showing the secretion of interferon γ in the supernatant detected after co-culture of mouse spleen natural killer cells loaded with αNK1.1-PDA@BTO nanoparticles with B16 / F10 cells;

[0045] Fig.14 Figures 1 and 2 are tumor volume graphs, mouse tumor growth curve graphs, and mouse survival graphs of each group of mice in a tumor inhibition experiment on the sixth day after the αNK1.1-PDA@BTO nanoparticles prepared in Example 1 of the present invention were combined with mouse spleen natural killer cells, wherein A is a tumor volume graph of each group of mice in the tumor inhibition experiment on the sixth day, B is a tumor growth curve graph of each group of mice in the tumor inhibition experiment, and C is a survival graph of each group of mice in the tumor inhibition experiment;

[0046] Fig.15 This is a comparison chart of indicators of the tumor inhibition experiment after the αNK1.1-PDA@BTO nanoparticles prepared in Example 1 of the present invention are combined with mouse spleen natural killer cells, wherein A is a comparison chart of the proportion of tumor-infiltrating natural killer cells, B is a comparison chart of interferon γ expression, C is a comparison chart of lysosome-associated membrane protein 1 expression, and D is a comparison chart of tumor necrosis factor α expression. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0048] Glossary: ​​PBS solution is phosphate buffered saline, NK92 cells are natural killer cells from patients with malignant non-Hodgkin's lymphoma, Ba(OH)2 .8H2O is barium hydroxide octahydrate, TiO2 is titanium dioxide, HCl is hydrochloric acid, DA is dopamine hydrochloride, PDA is polydopamine, Tris-HCl solution is tris(hydroxymethylaminomethane) hydrochloride solution, EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, Sulfo-NHS is N-hydroxysulfosuccinimide, BTO is barium titanate, PDA@BTO is polydopamine-barium titanate, αCD56 is anti-CD56 antibody (α is anti), αNK1.1 is anti-NK1.1 antibody, αCD56-PDA@BTO is polydopamine-barium titanate with anti-CD56 antibody modified on the surface, αNK1.1-PDA@BTO is polydopamine-barium titanate with anti-NK1.1 antibody modified on the surface, IFN-γ is interferon γ, TNF-α is tumor necrosis factor α, and CD107a is lysosome-associated membrane protein 1.

[0049] A first typical embodiment of the present invention provides an antibody-modified barium titanate nanoparticle, wherein the nanoparticle comprises polydopamine-barium titanate nanoparticles and an antibody modified on the surface thereof, wherein the antibody is selected from one of αCD56 and αNK1.1.

[0050] A second typical embodiment of the present invention provides a method for preparing the above-mentioned antibody-modified barium titanate nanoparticles, comprising the following steps:

[0051] S1, Ba(OH)2 . 8H2O and TiO2 are mixed and reacted to obtain barium titanate nanoparticles (BTO);

[0052] S2, dispersing dopamine hydrochloride into the solution and incubating it with barium titanate nanoparticles to obtain polydopamine-modified barium titanate nanoparticles (PDA@BTO);

[0053] S3. Dispersing the polydopamine-modified barium titanate nanoparticles into a solution for incubation, and then adding antibodies for co-incubation to obtain antibody-modified barium titanate nanoparticles.

[0054] In one or more examples of this embodiment, in step S1, the Ba(OH)2 . The mass of 8H2O is 9.0~10.0 g, and the mass of TiO2 is 0.5~1.0 g.

[0055] In one or more examples of this embodiment, in step S1, the heating temperature of the reaction is 200-220° C., and the heating time is 36-60 h.

[0056] In one or more examples of this implementation mode, in step S1, the mixture after the reaction needs to be cooled, washed and dried in sequence; wherein, the washing refers to cleaning and centrifugation using HCl and ultrapure water; the concentration of the HCl is 0.1~0.2 M.

[0057] In one or more examples of this implementation mode, in step S2, the mass of the barium titanate nanoparticles is 0.1-0.5 g; and the concentration of the dopamine hydrochloride is 0.01-0.02 M.

[0058] In one or more examples of this embodiment, in step S2, the dopamine hydrochloride is dispersed in a Tris-HCl solution.

[0059] In one or more examples of this implementation mode, the concentration of the Tris-HCl solution is 0.01-0.05 M, and the volume of the Tris-HCl solution is 100-500 mL.

[0060] In one or more examples of this embodiment, in step S2, the co-incubation temperature is room temperature, and the co-incubation time is 4 to 8 h.

[0061] In one or more examples of this embodiment, in step S3, the mass of the polydopamine-modified barium titanate nanoparticles is 10-50 μg; the mass of the antibody is 10-200 μg.

[0062] In one or more examples of this embodiment, in step S3, the polydopamine-modified barium titanate nanoparticles are dispersed in a mixed solution of EDC and Sulfo-NHS, and then the pH value of the mixed solution is adjusted to be incubated under neutral conditions, and then the antibody is added to the mixture and incubated to obtain antibody-modified polydopamine-barium titanate nanoparticles.

[0063] In one or more examples of this embodiment, the molar ratio of EDC to Sulfo-NHS is 0.5-1.5:0.5-1.5.

[0064] In one or more examples of this embodiment, the molar ratio of EDC to Sulfo-NHS is 1:1.

[0065] A third typical embodiment of the present invention provides use of the antibody-modified barium titanate nanoparticles described in the first aspect in the preparation of drugs for tumor targeting and treatment.

[0066] In one or more examples of this embodiment, the drug is a drug that enhances the activation level of NK cells or a drug that enhances the anti-tumor function of NK cells.

[0067] In one or more examples of this embodiment, the tumor includes lung cancer, melanoma, glioma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, blood tumor, lymphoma; further, it is melanoma.

[0068] The present invention modifies polydopamine-barium titanate nanoparticles by using antibodies, so that the barium titanate nanomaterial can selectively target NK cells and stably bind to the surface of NK cells, generate mechanical and electrical signals under the energy source provided by external field ultrasound, thereby regulating NK cell function.

[0069] Barium titanate nanoparticles have a small size and low toxicity, which makes them easy to load on cells without affecting the viability of NK cells, and they are highly flexible. In addition, barium titanate nanoparticles have excellent piezoelectricity. Under the premise of exogenous ultrasound as an energy source, they can vibrate on the surface of NK cells to generate mechanical signals, and generate electrical signals through the piezoelectric effect, which can coordinately regulate the function of NK cells and improve their ability to kill tumor cells. The presence of polydopamine improves the biocompatibility and dispersion stability of barium titanate nanoparticles, which is conducive to the stable dispersion of materials in solution systems; the amino groups provided by polydopamine are conducive to the binding of antibodies, so that barium titanate nanoparticles have the function of targeting NK cells; and polydopamine is conductive, which can transmit the electrical signals generated by BTO nanoparticles under ultrasound to NK cells.

[0070] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0071] Example 1: This example provides an antibody-modified barium titanate nanoparticle and a preparation method thereof

[0072] 9.5 g Ba(OH)2 . 8H2O and 0.8 g TiO2 were added to the reactor in sequence, heated at 200 °C for 48 h, cooled to room temperature, washed and centrifuged with 0.1 M HCl and ultrapure water in sequence, and dried in an oven to obtain barium titanate nanoparticles (BTO);

[0073] 0.5 g of BTO nanoparticles were added to 200 mL of 0.01 M Tris-HCl solution, and 0.015 M dopamine hydrochloride was added, stirred and incubated for 6 h. Polydopamine-modified barium titanate nanoparticles (PDA@BTO) were obtained after resuspending by centrifugation.

[0074] After 20 μg of PDA@BTO nanoparticles were incubated with EDC and Sulfo-NHS solution (the molar ratio of EDC to Sulfo-NHS was 1:1) at pH 7 for 30 min, 100 μg of αCD56 antibody and αNK1.1 antibody were added to the mixture for incubation to obtain antibody αCD56 modified barium titanate nanoparticles (αCD56-PDA@BTO) and αNK1.1 antibody modified barium titanate nanoparticles (αNK1.1-PDA@BTO), respectively.

[0075] Example 2: This example provides an antibody-modified barium titanate nanoparticle and a preparation method thereof

[0076] 9.5 g Ba(OH)2 . 8H2O and 0.8 g TiO2 were added to the reactor in sequence, heated at 210 °C for 48 h, cooled to room temperature, washed and centrifuged with 0.2 M HCl and ultrapure water in sequence, and dried in an oven to obtain barium titanate nanoparticles (BTO);

[0077] 0.2 g of BTO nanoparticles were added to 100 mL of 0.01 M Tris-HCl solution, and 0.02 M dopamine hydrochloride was added, stirred and incubated for 6 h, and polydopamine-modified barium titanate nanoparticles (PDA@BTO) were obtained after resuspending by centrifugation.

[0078] After 20 μg of PDA@BTO nanoparticles were incubated with EDC and Sulfo-NHS solution (the molar ratio of EDC to Sulfo-NHS was 1:1) at pH 7 for 30 min, 100 μg of αCD56 antibody and αNK1.1 antibody were added to the mixture for incubation to obtain antibody αCD56 modified barium titanate nanoparticles (αCD56-PDA@BTO) and αNK1.1 antibody modified barium titanate nanoparticles (αNK1.1-PDA@BTO), respectively.

[0079] Example 3: This example provides an antibody-modified barium titanate nanoparticle and a preparation method thereof

[0080] 9.5 g Ba(OH)2 . 8H2O and 0.8 g TiO2 were added to the reactor in sequence, heated at 200 °C for 60 h, cooled to room temperature, washed and centrifuged with 0.15 M HCl and ultrapure water in sequence, and dried in an oven to obtain barium titanate nanoparticles (BTO);

[0081] 0.5 g of BTO nanoparticles were added to 100 mL of 0.01 M Tris-HCl solution, and 0.01 M dopamine hydrochloride was added, stirred and incubated for 8 h, and polydopamine-modified barium titanate nanoparticles (PDA@BTO) were obtained after resuspending by centrifugation.

[0082] After incubating 10 μg of PDA@BTO nanoparticles with EDC and Sulfo-NHS solution (the molar ratio of EDC to Sulfo-NHS was 1:1) at pH 7 for 30 min, 100 μg of αCD56 antibody and αNK1.1 antibody were added to the mixture for incubation to obtain antibody αCD56 modified barium titanate nanoparticles (αCD56-PDA@BTO) and αNK1.1 antibody modified barium titanate nanoparticles (αNK1.1-PDA@BTO), respectively.

[0083] Example 4: This example provides an antibody-modified barium titanate nanoparticle and a preparation method thereof

[0084] 9.4 g Ba(OH)2 . 8H2O and 0.75 g TiO2 were added to the reactor in sequence, heated at 200 °C for 48 h, cooled to room temperature, washed and centrifuged with 0.1 M HCl and ultrapure water in sequence, and dried in an oven to obtain barium titanate nanoparticles (BTO);

[0085] 0.5 g of BTO nanoparticles were added to 200 mL of 0.01 M Tris-HCl solution, and 0.02 M dopamine hydrochloride was added, stirred and incubated for 4 h, and polydopamine-modified barium titanate nanoparticles (PDA@BTO) were obtained after resuspending by centrifugation.

[0086] After incubating 10 μg of PDA@BTO nanoparticles with EDC and Sulfo-NHS solution (the molar ratio of EDC to Sulfo-NHS was 1:1) at pH 7 for 30 min, 200 μg of αCD56 antibody and αNK1.1 antibody were added to the mixture for incubation to obtain antibody αCD56 modified barium titanate nanoparticles (αCD56-PDA@BTO) and αNK1.1 antibody modified barium titanate nanoparticles (αNK1.1-PDA@BTO), respectively.

[0087] Example 5: This example provides an antibody-modified barium titanate nanoparticle and a preparation method thereof

[0088] 9.5 g Ba(OH)2 .8H2O and 0.8 g TiO2 were added to the reactor in sequence, heated at 200 °C for 56 h, cooled to room temperature, washed and centrifuged with 0.1 M HCl and ultrapure water in sequence, and dried in an oven to obtain barium titanate nanoparticles (BTO);

[0089] 0.2 g of BTO nanoparticles were added to 200 mL of 0.01 M Tris-HCl solution, and 0.015 M dopamine hydrochloride was added, stirred and incubated for 5 h, and polydopamine-modified barium titanate nanoparticles (PDA@BTO) were obtained after resuspending by centrifugation.

[0090] After incubating 10 μg of PDA@BTO nanoparticles with EDC and Sulfo-NHS solution (the molar ratio of EDC to Sulfo-NHS was 1:1) at pH 7 for 30 min, 100 μg of αCD56 antibody and αNK1.1 antibody were added to the mixture for incubation to obtain antibody αCD56 modified barium titanate nanoparticles (αCD56-PDA@BTO) and αNK1.1 antibody modified barium titanate nanoparticles (αNK1.1-PDA@BTO), respectively.

[0091] Example 6: In this example, the BTO nanoparticles and PDA@BTO nanoparticles prepared in Example 1 were subjected to scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), piezoelectric force microscopy (PFM) and oscilloscope-electrical signal characterization tests.

[0092] like Figure 1 As shown in Figure A, the scanning electron microscopy results show that the BTO nanoparticles have a cubic morphology, and the size of a single BTO nanoparticle is about 150 mm; Figure 2 As shown in A, the transmission electron microscopy results show that the morphology of BTO nanoparticles is consistent with the SEM results, and the lattice diffraction results show that the lattice of BTO nanoparticles is about 0.4 nm, with good crystallinity;

[0093] like Figure 1 As shown in Figure B, the SEM results show that the morphology of the PDA@BTO nanoparticles coated with polydopamine also presents a regular cubic morphology, and the size of a single PDA@BTO nanoparticle is also about 150 mm; Figure 2 As shown in B, the TEM results show that the morphology of PDA@BTO nanoparticles is consistent with the SEM results. Polydopamine forms a thin layer of polydopamine with a thickness of about 2.6 nm on the surface of BTO nanoparticles, without destroying the crystallinity of the BTO nanocrystals themselves.

[0094] like Figure 3As shown in Figure 2, the crystal structure information of BTO nanoparticles and PDA@BTO nanoparticles was detected by X-ray diffraction. The XRD results showed that a series of characteristic peaks appeared at 22.4°, 31.6°, 38.9°, 45.4°, 51.1°, 56.3°, 66.1° and 75.2°, which were consistent with the (100), (110), (111), (200), (210), (211), (220) and (310) crystal planes of the standard BTO nanoparticle tetragonal system.

[0095] like Figure 4 As shown in A and B, the hysteresis loop and butterfly curve results of PFM show that the synthesized BTO nanoparticles have good piezoelectric properties.

[0096] like Figure 5 As shown, the oscilloscope results show that PDA@BTO nanoparticles output electrical signals under ultrasound.

[0097] Example 7: This example tests the dispersibility of the BTO nanoparticles and PDA@BTO nanoparticles prepared in Example 1.

[0098] Experimental process: BTO nanoparticles and PDA@BTO nanoparticles were prepared into solutions of the same concentration and dispersed evenly at the same time, and a timer was used to record the sedimentation time of the BTO nanoparticle solution and the PDA@BTO nanoparticle solution.

[0099] like Figure 6 As shown in the figure, the sedimentation time of BTO nanoparticles is relatively fast. After 10 min, most of the BTO nanoparticles settle to the bottom of the test tube. However, the PDA@BTO nanoparticle solution coated with polydopamine has a high degree of dispersion and strong uniformity, and no obvious sedimentation phenomenon occurs, indicating that the presence of polydopamine is beneficial to improving the dispersion degree and stability of BTO nanoparticles in the solution system, that is, the PDA@BTO nanoparticles have good dispersibility and stability.

[0100] Example 8: This example tests the Fourier transform infrared spectra (FT-IR) of the αCD56-PDA@BTO nanoparticles prepared in Example 1.

[0101] like Figure 7 As shown in Ⅰ, Ⅱ and Ⅲ, the Fourier transform infrared spectra of BTO nanoparticles (Ⅰ), PDA@BTO nanoparticles (Ⅱ) and αCD56-PDA@BTO nanoparticles (Ⅲ) indicate that the antibody αCD56 is successfully modified. -1 The absorption peak corresponds to the amide group in αCD56-PDA@BTO nanoparticles, indicating that the coupling of PDA@BTO nanoparticles with αCD56 antibodies was successful.

[0102] Example 9: Targeting experiment of αCD56-PDA@BTO nanoparticles and αNK1.1-PDA@BTO nanoparticles

[0103] The αCD56-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with NK92 cells and K562 cells, and the αNK1.1-PDA@BTO nanoparticles were co-incubated with mouse spleen NK cells and Yac-1 cells, and then washed with PBS and centrifuged. The binding of αCD56-PDA@BTO nanoparticles to NK92 cells, K562 cells, and αNK1.1-PDA@BTO nanoparticles to mouse spleen NK cells and Yac-1 cells was observed using confocal microscopy.

[0104] like Figure 8 As shown in middle A, the confocal results show that αCD56-PDA@BTO nanoparticles bind well to NK92 cells and are evenly distributed; however, αCD56-PDA@BTO nanoparticles hardly bind to the surface of K562 cells;

[0105] like Figure 8 As shown in B, the confocal results show that αNK1.1-PDA@BTO nanoparticles also bind to the surface of mouse spleen NK cells, but there is no obvious binding of nanoparticles on the surface of Yac-1 cells. The above results indicate that αCD56-PDA@BTO nanoparticles and αNK1.1-PDA@BTO nanoparticles have certain targeting properties.

[0106] Example 10: Cytotoxicity test

[0107] The αCD56-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with NK92 cells, and the αNK1.1-PDA@BTO nanoparticles were co-incubated with mouse spleen NK cells, and then treated with ultrasound (ultrasonic power 80 W, ultrasound time 2 min), and the viability of NK92 cells and mouse spleen NK cells was detected using a cell live / dead staining kit.

[0108] like Fig. 9As shown in A and B, the live / dead staining of NK92 cells and mouse spleen NK cells was analyzed by confocal observation. The confocal results showed that compared with the blank control group, the addition of αCD56-PDA@BTO nanoparticles and αNK1.1-PDA@BTO nanoparticles did not affect the viability of NK92 cells and mouse spleen NK cells; and under the condition of ultrasonic stimulation, the cell viability of NK cells loaded with nanoparticles was roughly the same as that of the blank control group. After co-incubation of αCD56-PDA@BTO nanoparticles with NK92 cells and αNK1.1-PDA@BTO nanoparticles with mouse spleen NK cells, ultrasonic treatment (ultrasonic power 80 W, ultrasonic time 2 min) was used, and the viability of NK92 cells was calculated using trypan blue staining.

[0109] like Fig.10 As shown in A and B, the trypan blue staining results show that compared with the blank control group NK cells, the presence of αCD56-PDA@BTO nanoparticles and the use of ultrasound to stimulate NK92 cells do not cause the death of NK92 cells; αNK1.1-PDA@BTO nanoparticles also have little effect on the viability of mouse spleen NK cells. The above results show that αCD56-PDA@BTO nanoparticles, αNK1.1-PDA@BTO nanoparticles and ultrasound systems have no cytotoxicity to NK cells.

[0110] Example 11: In vitro experiment of NK cells killing tumor cells

[0111] After the αCD56-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with NK92 cells, they were co-cultured with K562-Luc cells, HepG2-Luc cells, and U251-Luc cells at an effector-target ratio of 0.5:1 and 1:1, respectively, and the cells were treated with ultrasound (ultrasonic power 80 W, ultrasound time 2 min), and the ability of NK92 cells to kill tumor cells was detected using a Luciferase kit after 5 hours;

[0112] like Fig.11 As shown in Figures AD, under the effector-target ratio system of 0.5:1 and 1:1, the ability of NK92 cells treated with ultrasound to kill different tumor cells was enhanced compared with that of NK92 cells in the blank control group, indicating that the mechanical force signals and electrical signals generated by αCD56-PDA@BTO nanoparticles under ultrasound stimulation improved the anti-tumor ability of NK92 cells.

[0113] The αNK1.1-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with mouse spleen NK cells, and then co-cultured with B16-Luc cells at an effector-target ratio of 5:1. The cells were treated with ultrasound (ultrasonic power 80 W, ultrasound time 2 min), and the ability of mouse spleen NK cells to kill tumor cells was detected using a Luciferase kit after 5 h.

[0114] like Fig.12 As shown in the figure, under the effector-target ratio system of 5:1, compared with the spleen NK cells of mice in the blank control group, the ability of the mouse spleen NK cells treated with ultrasound to kill B16-luc cells was enhanced, indicating that the mechanical force signals and electrical signals generated by αNK1.1-PDA@BTO nanoparticles under ultrasound stimulation improved the anti-tumor ability of mouse spleen NK cells.

[0115] Example 12: NK cell effector function experiment

[0116] The αCD56-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with NK92 cells and then co-cultured with HepG2 cells for 24 h. During the co-culture period, the cells were treated with ultrasound twice (ultrasonic power 80 W, ultrasound time 2 min), and the cytokine secretion level of NK92 cells in the cell supernatant was detected by ELISA;

[0117] like Fig.13 As shown in A and B, the ELISA results showed that in the co-culture system of NK92 cells and HepG2 cells, compared with the NK92 cells in the blank control group, the NK92 cells loaded with αCD56-PDA@BTO nanoparticles had a higher ability to secrete TNF-α and IFN-γ under ultrasound stimulation, indicating that under the action of ultrasound, the NK92 cells loaded with αCD56-PDA@BTO nanoparticles had a higher effector function.

[0118] The αNK1.1-PDA@BTO nanoparticles prepared in Example 1 were co-incubated with mouse spleen NK cells and then co-cultured with B16 / F10 cells for 24 h. During the co-culture period, the cells were treated with ultrasound twice (ultrasonic power 80 W, ultrasound time 2 min). The cytokine secretion level of mouse spleen NK cells in the cell supernatant was detected by ELISA.

[0119] like Fig.13As shown in C and D, the ELISA results showed that in the co-culture system of mouse spleen NK cells and B16 / F10 cells, compared with the mouse spleen NK cells in the blank control group, the mouse spleen NK cells loaded with αNK1.1-PDA@BTO nanoparticles had a higher ability to secrete TNF-α and IFN-γ under ultrasound stimulation, indicating that under the action of ultrasound, the mouse spleen NK cells loaded with αNK1.1-PDA@BTO nanoparticles have higher effector function.

[0120] Example 13: Tumor inhibition experiment

[0121] Since mouse NK cells do not express CD56, CD56 in human NK cells can be used as a marker. The marker of mouse NK cells is NK1.1. When conducting mouse tumor inhibition experiments, mouse-derived cells need to be infused back. Only anti-NK1.1 antibodies can bind to mouse NK cells. Therefore, this embodiment provides a tumor inhibition experiment in which αNK1.1-PDA@BTO nanoparticles are co-incubated with mouse spleen NK cells.

[0122] Mouse spleen was ground into tissue suspension, centrifuged and then red blood cell lysis solution was added, and spleen mononuclear cells were obtained after centrifugation. After NK cells were sorted using a magnetic bead purification kit, αNK1.1-PDA@BTO nanoparticles were co-incubated with mouse spleen NK cells for later use.

[0123] Each mouse was inoculated subcutaneously in the left groin with 5 × 10 5 The mice were randomly divided into four groups and treated with NK cells transfused into the adjacent part of the tumor 12 days later (day 0): blank group 1 was transfused with PBS in a volume of 100 μL; blank group 2 was transfused with PBS in a volume of 100 μL, accompanied by ultrasound treatment (ultrasound power 80 W, ultrasound duration 5 min, duty cycle 50%, treatment once every other day); the control group was transfused with 1×10 6 The experimental group received 1×10 6 Mouse spleen NK cells loaded with αNK1.1-PDA@BTO nanoparticles were injected into the NK cells in a system volume of 100 μL and accompanied by ultrasound treatment. The treatment was terminated on day 6 and the tumor volume and survival of the mice were recorded every day.

[0124] like Fig.14As shown in Figures AC, the mouse tumor volume (A) on day 6, the mouse tumor growth curve (B) and the mouse survival curve (C) showed that under ultrasonic treatment, the mouse spleen NK group that was re-infused with αNK1.1-PDA@BTO nanoparticles had the smallest tumor volume, the slowest tumor growth rate and the highest mouse survival rate.

[0125] The tumor was removed on day 6 and ground. Fig.15 As shown in Figures AD, under ultrasound treatment, the proportion of infiltrating NK cells in the tumor of the mouse spleen NK group loaded with αNK1.1-PDA@BTO nanoparticles was the highest (A), and the expression levels of IFN-γ (B), CD107a (C) and TNF-α (D) of NK cells in the tumor were higher, indicating that under ultrasound stimulation, the mechanical force signals and electrical signals generated by the αNK1.1-PDA@BTO nanoparticles loaded on the surface of NK cells can improve the effector function of NK cells in vivo. The improvement of the above indicators shows that under ultrasound, αNK1.1-PDA@BTO nanoparticles can improve the function of NK cells and inhibit tumor growth in vivo.

[0126] 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. An antibody-modified barium titanate nanoparticle, characterized in that: The nanoparticles include polydopamine-barium titanate nanoparticles and antibodies modified on the surface of the nanoparticles, and the antibodies are selected from one of αCD56 and αNK1.

1.

2. A method for preparing antibody-modified barium titanate nanoparticles according to claim 1, characterized in that: The following steps are involved: S1, Ba(OH)2 . 8H2O and TiO2 are mixed and reacted to obtain barium titanate nanoparticles; S2, dispersing dopamine hydrochloride into the solution and incubating the solution with barium titanate nanoparticles to obtain polydopamine-modified barium titanate nanoparticles; S3. Dispersing the polydopamine-modified barium titanate nanoparticles into a solution for incubation, and then adding antibodies for co-incubation to obtain antibody-modified barium titanate nanoparticles.

3. The preparation method according to claim 2, characterized in that: In step S1, the Ba(OH)2 . The mass of 8H2O is 9.0~10.0 g, and the mass of TiO2 is 0.5~1.0 g.

4. The preparation method according to claim 2, characterized in that: In step S1, the heating temperature of the reaction is 200-220° C., and the heating time is 36-60 h.

5. The preparation method according to claim 2, characterized in that: In step S1, the reaction mixture needs to be cooled, washed and dried in sequence; wherein the washing refers to washing and centrifuging with HCl and ultrapure water; the concentration of HCl is 0.1-0.2 M.

6. The preparation method according to claim 2, characterized in that: In step S2, the mass of the barium titanate nanoparticles is 0.1-0.5 g; the concentration of the dopamine hydrochloride is 0.01-0.02 M.

7. The preparation method according to claim 6, characterized in that: In step S2, the dopamine hydrochloride is dispersed in a Tris-HCl solution; the concentration of the Tris-HCl solution is 0.01-0.05 M, and the volume of the Tris-HCl solution is 100-500 mL; the co-incubation temperature is room temperature, and the co-incubation time is 4-8 h.

8. The preparation method according to claim 2, characterized in that: In step S3, the mass of the polydopamine-modified barium titanate nanoparticles is 10-50 μg; the mass of the antibody is 10-200 μg.

9. The preparation method according to claim 2, characterized in that: In step S3, the polydopamine-modified barium titanate nanoparticles are dispersed in a mixed solution of EDC and Sulfo-NHS, and then the pH value of the mixed solution is adjusted to be neutral and incubated, and then the antibody is added to the mixture and incubated to obtain antibody-modified polydopamine-barium titanate nanoparticles; the molar ratio of EDC and Sulfo-NHS is 0.5~1.5:0.5~1.

5.

10. Use of the antibody-modified barium titanate nanoparticles according to claim 1 in the preparation of drugs for solid tumor targeting and treatment; The drug is a drug that enhances the activation level of NK cells or a drug that enhances the anti-tumor function of NK cells; The tumors include lung cancer, melanoma, glioma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, and blood tumors.

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