CN113750233A

CN113750233A - Nano composite material and preparation method and application thereof

Info

Publication number: CN113750233A
Application number: CN202111159409.4A
Authority: CN
Inventors: 景香香; 张敏; 陈其青; 杨大艳; 冯桂英; 孙丹丹; 覃伶伶; 林凌
Original assignee: Individual
Current assignee: Hainan General Hospital
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-07
Anticipated expiration: 2041-09-30
Also published as: CN113750233B

Abstract

The invention belongs to the field of medicine, and particularly relates to a nano composite material as well as a preparation method and application thereof. The present invention provides a nanocomposite comprising: the BSA-based nano-material comprises an inorganic nano-material and BSA molecules which are modified on the surface of the inorganic nano-material; the inorganic nanomaterial includes: two-dimensional structure Ti₃C₂Nanosheet and supported on the two-dimensional structure Ti₃C₂CuO with positive charge on nanosheet surface₂And (4) nanodots. The invention is based on the positive and negative charge principle, and adopts a two-dimensional structure Ti₃C₂CuO is loaded on the surface of the nano sheet₂By using CuO₂Specific self-supply H in the tumor microenvironment₂O₂The triggered Fenton reaction and the exogenous ultrasonic stimulation realize the efficient ROS generation and synergistically improve the tumor treatment effect. The nano composite material provided by the invention is suitable for the synergistic and efficient treatment of the acoustic power and the chemical power of tumors, particularly glioma, and has important significance for the development of clinical tumor treatment and the progress of nano ultrasonic medicine.

Description

Nano composite material and preparation method and application thereof

Technical Field

The invention belongs to the field of medicine, and particularly relates to a nano composite material as well as a preparation method and application thereof.

Background

In 1989, Yumita et al used ultrasound to stimulate photosensitizers commonly used in photodynamic: hematoporphyrin and its derivatives, were also found to produce ROS that cause significant cytotoxicity, leading to apoptosis/necrosis of cancer cells. This novel approach to tumor treatment is known as sonodynamic therapy (SDT). Reactive Oxygen Species (ROS) are chemically reactive molecules including singlet oxygen (C¹O₂) Superoxide radical (O)₂·^-) Hydroxyl radical (. OH) and peroxide (O)₂ ^-) Plays an important role in the cell life cycle. At lower concentrations, ROS can act as key signaling molecules within the cell to regulate cell growth, proliferation, and differentiation. However, abnormal production of intracellular ROS will lead to oxidative damage of lipids, proteins or and DNA. Normally, cells are able to balance this oxidative damage to protect the cells from death. Excessive ROS can damage cell membranes, inhibit mitochondrial function, damage DNA, cause gene mutation, cause energy metabolism disorder, and cause calciumOverload, etc., causing irreversible oxidative damage to the cells and leading to apoptosis and programmed necrosis. Therefore, selective breakthrough of the threshold for ROS balance in tumor cells can effectively induce tumor cell death. SDT is a reactive oxygen-related tumor treatment that converts ambient oxygen into cytotoxic ROS by low frequency ultrasound (exogenous stimulus) irradiation-activated Sonosensitizers; the method is a representative method for treating cancer by using ROS, has great anti-tumor treatment potential, simultaneously has low toxic and side effects, and is not easy to generate drug resistance. Compared with traditional radiotherapy and chemotherapy, SDT has the advantages of non-invasiveness, no radiation, high selectivity and the like. Compared with optical therapy (photodynamic/photothermal therapy), the acoustodynamic therapy has excellent tissue penetrating power, the penetrating depth can reach tens of centimeters, and the acoustodynamic therapy can be used for treating deep tumors.

The organic sound-sensitive agents reported at present are porphyrin derivatives such as hematoporphyrin, protoporphyrin and phthalocyanine and fat-soluble small molecules. The organic sonosensitizer has small molecules and low water solubility, is easy to metabolize rapidly in vivo, has short blood circulation time, and cannot form effective enrichment at tumor parts, thereby failing to achieve ideal sonodynamic effect. Meanwhile, the photosensitizer is easy to generate phototoxicity and skin toxicity, and has low bioavailability. The emergence of nano biotechnology provides a new way for loading and transporting organic sound-sensitive agent molecules. By utilizing the novel carrier to deliver the sound-sensitive agent in a targeted manner, the enrichment amount and the accumulation amount of the sound-sensitive agent in the tumor can be effectively improved. Therefore, the development of more efficient, more stable and safer sonosensitizer for the efficient treatment of tumor SDT is urgently needed in clinic.

At present, the key point of research is to develop a novel and efficient sonosensitizer so as to improve the effect of sonodynamic therapy. Compared with organic sonosensitizer, inorganic sonosensitizer titanium oxide (TiO)₂) Nanoparticles are sound sensitizers of high stability and high biosafety developed in recent years. As a typical semiconductor nanomaterial, titanium oxide nanoparticles can realize electrons (e) under ultrasonic excitation^-) And a cavity (h)⁺) And generating ROS to kill the tumor cells. Research finds and usesTiO₂The effect of the nano-particles on the acoustic dynamic treatment of deep tissue tumor is less than that of the nano-particles added with TiO₂The treatment group of the nano-acoustic sensitivity agent is improved by 15 times. But TiO 2₂There are also disadvantages to be improved, such as a wide band gap, easy recombination of electrons and holes, etc., resulting in a decrease in the generation efficiency of ROS.

Disclosure of Invention

In view of the above, the present invention aims to provide a nanocomposite material, and a preparation method and an application thereof, wherein the nanocomposite material provided by the present invention can realize efficient ROS generation when used as a sonosensitizer, and the material can perform sonodynamic therapy and chemical dynamic cooperative therapy on tumors, so that the treatment effect is more thorough, and tumor recurrence caused by residual tumors is effectively avoided.

The present invention provides a nanocomposite material comprising: the BSA-based nano-material comprises an inorganic nano-material and BSA molecules which are modified on the surface of the inorganic nano-material;

the inorganic nanomaterial includes: two-dimensional structure Ti₃C₂Nanosheet and supported on the two-dimensional structure Ti₃C₂CuO with positive charge on nanosheet surface₂And (4) nanodots.

The invention provides a preparation method of the nano composite material in the technical scheme, which comprises the following steps:

forming a two-dimensional structure Ti₃C₂Nanosheet, positively charged CuO₂And mixing the nanodots with bovine serum albumin, and performing solid-liquid separation to obtain the nano composite material.

Preferably, the two-dimensional structure Ti₃C₂The nano sheet is prepared according to the following steps:

mixing Ti₃AlC₂Sequentially carrying out hydrofluoric acid etching treatment and tetrapropyl ammonium hydroxide intercalation treatment on the powder to obtain Ti with a two-dimensional structure₃C₂Nanosheets.

Preferably, the Ti is₃AlC₂The powder is prepared according to the following steps:

mixing titanium powder, aluminum powder and graphite powder, and pressing to obtain Ti₃AlC₂Biscuit;

adding the Ti₃AlC₂Sintering the biscuit in protective gas atmosphere to obtain Ti₃AlC₂A bulk material;

to the Ti₃AlC₂Grinding the bulk material to obtain Ti₃AlC₂And (3) powder.

Preferably, the specific steps of sequentially performing the hydrofluoric acid etching treatment and the tetrapropylammonium hydroxide intercalation treatment include:

i) mixing Ti₃AlC₂Mixing the powder with HF aqueous solution, and carrying out solid-liquid separation to obtain Ti for completing hydrofluoric acid etching treatment₃C₂A material;

ii) Ti to be subjected to hydrofluoric acid etching treatment₃C₂Mixing the material with tetrapropylammonium hydroxide, and carrying out solid-liquid separation to obtain Ti with a two-dimensional structure₃C₂Nanosheets.

Preferably, in the step i), the concentration of the HF aqueous solution is 30-50 wt%; the Ti₃AlC₂The ratio of the powder to the aqueous HF solution was 10 g: (40-100) mL.

Preferably, in the step i), the mixing temperature is 15-35 ℃; the mixing speed is 400-1000 rpm; the mixing time is 1-5 days.

Preferably, in step ii), Ti is used₃AlC₂The Ti subjected to hydrofluoric acid etching treatment in the amount of powder used₃C₂The dosage ratio of the material to the tetrapropylammonium hydroxide is 10 g: (40-100) mL.

Preferably, in the step ii), the mixing temperature is 15-35 ℃; the mixing speed is 400-1000 rpm; the mixing time is 1-5 days.

Preferably, the positively charged CuO₂The nanodots are prepared according to the following steps:

mixing soluble salt of copper, polyvinylpyrrolidone and hydrogen peroxide in alkaline aqueous solution for reaction, and ultrafiltering to obtain CuO with positive charge₂And (4) nanodots.

Preferably, the two-dimensional structure Ti₃C₂Nanosheet, tapePositively charged CuO₂The molar ratio of the nanodots to the bovine serum albumin is 1: (1-5): (1-5).

The invention also provides application of the nano composite material in the technical scheme in preparation of a tumor sonodynamic and chemical dynamic cooperative therapy medicine.

Compared with the prior art, the invention provides a nano composite material and a preparation method and application thereof. The present invention provides a nanocomposite comprising: the BSA-based nano-material comprises an inorganic nano-material and BSA molecules which are modified on the surface of the inorganic nano-material; the inorganic nanomaterial includes: two-dimensional structure Ti₃C₂Nanosheet and supported on the two-dimensional structure Ti₃C₂CuO with positive charge on nanosheet surface₂And (4) nanodots. The invention is based on the positive and negative charge principle, and adopts a two-dimensional structure Ti₃C₂CuO is loaded on the surface of the nano sheet₂By using CuO₂Specific self-supply H in Tumor Microenvironment (TME)₂O₂The fenton reaction and the exogenous ultrasonic stimulation that trigger have realized efficient ROS and have produced, promote tumour treatment effect in coordination, more specifically: 1) the invention adopts a two-dimensional structure Ti₃C₂The nanosheet replaces the traditional TiO₂Sonosensitizers, two-dimensional structures Ti₃C₂The nano sheet has large specific surface area, can provide storage and anchoring sites for drugs or functional nano materials through Van der Waals force, and has high stability, good biological safety and clinical application potential; and, Ti₃C₂H capable of being affected by the tumor area when participating in the treatment₂O₂Oxidation in situ generation of Ti₃C₂/TiO₂The presence of carbon (C) being advantageous for TiO₂The separation of the ultrasound-excited electrons and holes improves the ROS generation efficiency, thereby enhancing the effect of SDT. 2) Using CuO₂Has the characteristics of tumor microenvironment response, CuO₂Can generate Cu in the slightly acidic environment of tumor focus area when participating in treatment²⁺And H₂O₂Thereby in-situ lifting H in the tumor₂O₂In an amount of H₂O₂And Cu²⁺For the reaction mass to generateThe chemical kinetic treatment (CDT) function of generating hydroxyl free radicals by the Fenton reaction causes the apoptosis of tumor cells, thereby improving the treatment effect of the tumor. In addition, the size, the shape and the performance of the material are further adjusted by modifying BSA molecules on the surface of the material, so that the material has good dispersibility and high stability in aqueous solution and physiological environment. The invention provides a nano composite material (Ti)₃C₂/CuO₂BSA) is suitable for the synergistic and efficient treatment of the acoustic power and the chemical power of tumors, particularly glioma, and has important significance for the development of clinical tumor treatment and the progress of nano ultrasonic medicine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 shows a multilayer Ti film provided in example 1 of the present invention₃C₂SEM electron micrograph of (1);

FIG. 2 shows Ti provided in example 1 of the present invention₃C₂TEM micrograph of the nanosheets;

FIG. 3 shows Ti provided in example 1 of the present invention₃C₂/CuO₂TEM micrograph of nanoparticles prepared with a/BSA molar ratio of 1:2: 2;

FIG. 4 shows Ti provided in example 1 of the present invention₃C₂/CuO₂TEM micrograph of nanoparticles prepared with a/BSA molar ratio of 1:1: 2;

FIG. 5 shows Ti provided in example 1 of the present invention₃C₂/CuO₂TEM micrograph of nanoparticles prepared with a/BSA molar ratio of 1:3: 2;

FIG. 6 shows Ti provided in example 1 of the present invention₃C₂/CuO₂-a HADDF-STEM map of BSA nanoparticles;

FIG. 7 shows Ti provided in example 1 of the present invention₃C₂/CuO₂-an energy dispersive mapping plot of BSA nanoparticles;

FIG. 8 shows Ti provided in example 1 of the present invention₃C₂/CuO₂-atomic force microscopy of BSA nanoparticles;

FIG. 9 is an atomic force microscope measurement of Ti as provided in example 1 of the present invention₃C₂/CuO₂-thickness profile of BSA nanoparticles;

FIG. 10 is a DLS map provided in example 1 of the present invention;

FIG. 11 is a bar graph of the cytotoxicity of U87 glioma cells versus different material concentrations provided by an embodiment of the present invention;

FIG. 12 is a graph of body weight for mice in groups of materials of varying concentrations provided by an embodiment of the present invention;

FIG. 13 is a graph of HE staining of major organs of mice in different concentration groups according to the present invention;

FIG. 14 is a tumor image taken in different groups according to an embodiment of the present invention;

FIG. 15 is a graph of relative tumor volume versus time for different groups provided by an embodiment of the present invention;

FIG. 16 is a graph of HE and immunofluorescent staining of various groups of tumor tissue provided by an embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention also provides a preparation method of the nano composite material in the technical scheme, which comprises the following steps:

forming a two-dimensional structure Ti₃C₂Nanosheet, positively charged CuO₂And mixing the nanodots with Bovine Serum Albumin (BSA), and performing solid-liquid separation to obtain the nano composite material.

In the preparation method provided by the invention, the two-dimensional structure Ti₃C₂The nanosheet is preferably prepared according to the following steps:

mixing Ti₃AlC₂Sequentially carrying out hydrofluoric acid etching treatment and tetrapropyl ammonium hydroxide intercalation treatment on the powder to obtain Ti with a two-dimensional structure₃C₂Nanosheets; wherein the etching treatment is for removing Ti₃AlC₂The Al layer in the nano-film is subjected to intercalation treatment so as to convert the material into a nano-film with an ultrathin two-dimensional structure.

In the present invention, the above-mentioned two-dimensional structure Ti is provided₃C₂In the step of preparing nanosheet, the Ti₃AlC₂The particle size of the powder is preferably 50-800 nm, specifically 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm or 800 nm; the Ti₃AlC₂The powder is preferably prepared according to the following steps:

The above-mentioned Ti is provided in the present invention₃AlC₂In the powder preparation step, the particle size of the titanium powder is preferably 200-400 meshes, and more preferably 325 meshes; the particle size of the aluminum powder is preferably 200-400 meshes, and more preferably 200-400 meshes325 mesh; the particle size of the graphite powder is preferably 200-400 meshes, and more preferably 300 meshes; the molar ratio of the titanium powder in terms of Ti atoms, the aluminum powder in terms of Al atoms, and the graphite powder in terms of C atoms is preferably 2:1: 1; the mixing mode is preferably ball milling; the mixing time is preferably 5-20 h, and more preferably 10 h; the pressing pressure is preferably 5-20 MPa, and more preferably 10 MPa; the shielding gas includes, but is not limited to, argon; the sintering temperature is preferably 1200-1800 ℃, and more preferably 1500 ℃; the sintering time is preferably 1-3 h, and more preferably 2 h.

In the present invention, the above-mentioned two-dimensional structure Ti is provided₃C₂In the step of preparing the nanosheet, the specific steps of sequentially performing the hydrofluoric acid etching treatment and the tetrapropyl ammonium hydroxide intercalation treatment preferably include:

ii) Ti to be subjected to hydrofluoric acid etching treatment₃C₂Mixing the material with tetrapropylammonium hydroxide (TPAOH), and carrying out solid-liquid separation to obtain Ti with a two-dimensional structure₃C₂Nanosheets.

In the treatment step provided by the invention, in the step i), the concentration of the HF aqueous solution is preferably 30-50 wt%, and more preferably 40 wt%; the Ti₃AlC₂The ratio of powder to aqueous HF solution is preferably 10 g: (40-100) mL, more preferably 10 g: 60 mL; the mixing temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the mixing rotating speed is preferably 400-1000 rpm, and more preferably 600 rpm; the mixing time is preferably 1 to 5 days, and more preferably 3 days.

In the above processing step provided by the present invention, in step i), the solid-liquid separation is preferably performed by centrifugal separation, and the rotation speed of the centrifugal separation is preferably 10000-15000 rpm, and more preferably 13000 rpm; after solid-liquid separation, the obtained Ti which is etched by hydrofluoric acid is treated₃C₂The material is washed, preferably by water and ethanol.

In the above treatment step provided by the present invention, in step ii), Ti is used₃AlC₂The Ti subjected to hydrofluoric acid etching treatment in the amount of powder used₃C₂The ratio of the amount of material to tetrapropylammonium hydroxide used is preferably 10 g: (40-100) mL, more preferably 10 g: 60 mL; the mixing temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the mixing rotating speed is preferably 400-1000 rpm, and more preferably 600 rpm; the mixing time is preferably 1 to 5 days, and more preferably 3 days.

In the above treatment step provided by the present invention, in step ii), the solid-liquid separation is preferably performed by centrifugal separation, and the rotation speed of the centrifugal separation is preferably 10000 to 15000rpm, and more preferably 13000 rpm; after solid-liquid separation, the obtained two-dimensional structure Ti is subjected to₃C₂Washing the nanosheets to remove residual tetrapropylammonium hydroxide, wherein the washing mode is preferably water washing and ethanol washing, and the washing is preferably repeated for 3 times.

In the preparation method provided by the invention, the CuO with positive charge₂The nanodots are preferably prepared according to the following steps:

mixing soluble salt of copper, polyvinylpyrrolidone (PVP) and hydrogen peroxide in alkaline aqueous solution, reacting, ultrafiltering to obtain CuO with positive charge₂And (4) nanodots.

The above-mentioned positively charged CuO according to the present invention₂In the nanodot preparation step, the soluble salt of copper is preferably copper chloride (CuCl)₂) (ii) a The viscosity-average molecular weight (Mv) of the polyvinylpyrrolidone is preferably 5000-20000, and specifically can be 5000, 10000, 15000 or 20000; the alkalinity is preferably provided by an alkali metal hydroxide, preferably sodium hydroxide. In the present invention, the specific process of the mixing reaction is preferably: mixing copper salt aqueous solution, polyvinylpyrrolidone, alkali metal hydroxide aqueous solution and hydrogen peroxide for reaction; wherein the concentration of the copper salt aqueous solution is preferably 0.005-0.02 mol/L, and more preferably 0.01 mol/L; the concentration of the alkali metal hydroxide aqueous solution is preferably 0.01-0.05 mol/L, and more preferably 0.02 mol/L; the dioxygenThe concentration of the water is preferably 10-50 wt%, and more preferably 30 wt%; the dosage ratio of the copper salt aqueous solution, the polyvinylpyrrolidone, the alkali metal hydroxide aqueous solution and the hydrogen peroxide is preferably (1-10) mL: (0.1-1) g: (1-10) mL: 100 μ L, more preferably 5 mL: 0.5 g: 5mL of: 100 μ L. In the invention, the temperature of the mixing reaction is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the mixing rotating speed is preferably 400-1000 rpm, and more preferably 600 rpm; the mixing time is preferably 10-60 min, and more preferably 30 min.

The above-mentioned positively charged CuO according to the present invention₂In the nano-dot preparation step, the cut-off molecular weight of an ultrafiltration tube used for ultrafiltration is preferably 10000-50000, and more preferably 30000; the rotating speed of the ultrafiltration is preferably 3000-6000 rpm, and more preferably 4500 rpm; the number of times of ultrafiltration is preferably 1-5, more preferably 3; the total time consumption of the ultrafiltration is preferably 20-60 min, and more preferably 40 min. After completion of ultrafiltration, the resulting positively charged CuO is preferably used₂The nanodots were redispersed in water and dialyzed to remove residual polyvinylpyrrolidone.

In the preparation method provided by the invention, the molecular weight of the bovine serum albumin is preferably 50-100 kDa, and more preferably 66 kDa; the bovine serum albumin is preferably supplied by Macklin (Macklin).

In the preparation method provided by the invention, the two-dimensional structure Ti₃C₂Nanosheet and CuO with positive charge₂The molar ratio of nanodots is preferably 1: (1-5), specifically 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1: 2; the two-dimensional structure Ti₃C₂The molar ratio of the nanosheets to bovine serum albumin is preferably 1: (1-5), specifically 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1:2.

In the preparation method provided by the invention, the two-dimensional structure Ti₃C₂Nanosheet, positively charged CuO₂The rotation speed for mixing the nanodots and the bovine serum albumin is preferably 300-1000 rpm, and more preferably 600 rpm; the mixing time is preferably 2-6 h, and is more preferablyIs selected as 4 h.

In the preparation method provided by the invention, the two-dimensional structure Ti₃C₂Nanosheet, positively charged CuO₂After the nanodots and the bovine serum albumin are mixed, solid-liquid separation is preferably performed, the rotation speed of the centrifugal separation is preferably 10000-15000 rpm, more preferably 13000rpm, and the time of the centrifugal separation is preferably 5-30 min, more preferably 10 min; after solid-liquid separation, the obtained nanocomposite (Ti)₃C₂/CuO₂BSA), the wash preferably being Phosphate Buffered Saline (PBS).

In the preparation method provided by the invention, after the nano composite material is prepared, the nano composite material is preferably dispersed into a physiological saline solution and refrigerated for storage.

The technical scheme provided by the invention is based on the positive and negative charge principle, and adopts a two-dimensional structure Ti₃C₂CuO is loaded on the surface of the nano sheet₂By using CuO₂Specific self-supply H in Tumor Microenvironment (TME)₂O₂The fenton reaction and the exogenous ultrasonic stimulation that trigger have realized efficient ROS and have produced, promote tumour treatment effect in coordination, more specifically: 1) the invention adopts a two-dimensional structure Ti₃C₂The nanosheet replaces the traditional TiO₂Sonosensitizers, two-dimensional structures Ti₃C₂The nano sheet has large specific surface area, can provide storage and anchoring sites for drugs or functional nano materials through Van der Waals force, and has high stability, good biological safety and clinical application potential; and, Ti₃C₂H capable of being affected by the tumor area when participating in the treatment₂O₂Oxidation in situ generation of Ti₃C₂/TiO₂The presence of carbon (C) being advantageous for TiO₂The separation of the ultrasound-excited electrons and holes improves the ROS generation efficiency, thereby enhancing the effect of SDT. 2) Using CuO₂Has the characteristics of tumor microenvironment response, CuO₂Can generate Cu in the slightly acidic environment of tumor focus area when participating in treatment²⁺And H₂O₂Thereby in-situ lifting H in the tumor₂O₂In an amount of H₂O₂And Cu²⁺The chemical kinetic therapy (CDT) function of generating hydroxyl free radicals for the Fenton reaction of reactants causes the apoptosis of tumor cells, thereby improving the tumor treatment effect. In addition, the size, the shape and the performance of the material are further adjusted by modifying BSA molecules on the surface of the material, so that the material has good dispersibility and high stability in aqueous solution and physiological environment. The invention provides a nano composite material (Ti)₃C₂/CuO₂BSA) is suitable for the synergistic and efficient treatment of the acoustic power and the chemical power of tumors, particularly glioma, and has important significance for the development of clinical tumor treatment and the progress of nano ultrasonic medicine.

For the sake of clarity, the following examples are given in detail.

Example 1

1) Ultra-thin two-dimensional structure Ti₃C₂Preparing a nano sheet:

preparing ultrathin two-dimensional structure Ti by adopting hydrofluoric acid (HF) etching and cation intercalator (TPAOH) intercalation liquid phase stripping method₃C₂The nano sheet comprises the following specific processes: mixing titanium powder (Ti, 325 meshes), aluminum powder (Al, 325 meshes) and graphite powder (graphite, 300 meshes) according to the molar ratio of 2:1:1, ball-milling for 10h, and pressing into cylindrical Ti under the pressure of 10MPa₃AlC₂Biscuit; under the protection of argon (Ar), the biscuit is heated to 1500 ℃ in a high-temperature sintering furnace for sintering, the furnace is cooled to room temperature (25 ℃) after two hours of heat preservation and sintering, and the MAX phase ceramic block material (Ti) is obtained₃AlC₂) (ii) a Crushing Ti with mortar and pestle₃AlC₂The ceramic block is used for obtaining powder with the particle size of 300-500 nm, and 10g of the powder is magnetically stirred in 60mL of HF aqueous solution (the concentration is 40 wt%) at the room temperature at 600rpm for 3 days; then, the precipitate was collected by centrifugation (13000rpm), washed 3 times with water and ethanol; subsequently, the precipitate was further dispersed in 60mL TPAOH (tetrapropylammonium hydroxide) and magnetically stirred at room temperature for 3 days; finally, the product Ti₃C₂The nano-sheets are collected by centrifugation (the centrifugal speed is 13000rpm), and are mixed with ethanolWater rinse three times to remove residual TPAOH.

In this example, a multilayer structure Ti obtained after hydrofluoric acid (HF) etching₃C₂SEM micrograph of the following is shown in FIG. 1, which shows Ti obtained after intercalation of cationic intercalator (TPAOH)₃C₂TEM micrograph of the nanoplatelets is shown in FIG. 2.

2) CuO having positive charge₂Preparing nano dots:

5mL of 0.01mol/L CuCl₂Aqueous solution, 0.5g PVP (MV 10000), 5mL of 0.02mol/L NaOH aqueous solution and 100. mu.L of 30 wt% H₂O₂Magnetically stirring the aqueous solution at the room temperature at 600rpm for 30 minutes; then the solution was ultrafiltered 3 times with an ultrafiltration tube with molecular weight cut-off of 30000 at 4500rpm for 40 min; finally, the product obtained by ultrafiltration, namely CuO with positive charges₂The nanodots were redispersed in aqueous solution and dialyzed against dialysis bags for 24 hours to remove residual PVP.

3) Nanocomposite (Ti)₃C₂/CuO₂-BSA) preparation:

ti prepared as described above₃C₂Nanosheet, CuO₂The nanodots and BSA (66kDa, Meclin) were stirred at room temperature (600rpm) for 4 hours, centrifuged (13000rpm) for 10 minutes and washed 3 times with PBS to obtain Ti in a molar ratio of 1:1:2, 1:2:2, and 1:3:2, respectively₃C₂/CuO₂-BSA nanoparticles; the resulting nanoparticles were dispersed in physiological saline solution and stored in a 4 ℃ refrigerator for subsequent experiments.

Ti prepared in different molar ratios for the examples₃C₂/CuO₂TEM observation of BSA nanoparticles is shown in FIGS. 3-5, FIG. 3 is Ti provided in example 1 of the present invention₃C₂/CuO₂Nanoparticles prepared with a molar ratio of 1:2: 2/BSA, FIG. 4 is Ti provided in example 1 of the present invention₃C₂/CuO₂Nanoparticles prepared with a molar ratio of 1:1: 2/BSA, FIG. 5 is Ti provided in example 1 of the present invention₃C₂/CuO₂Nanoparticles prepared with a/BSA molar ratio of 1:3: 2. Tong (Chinese character of 'tong')As can be seen from FIGS. 3 to 5, the nanoparticles prepared at a molar ratio of 1:2:2 are uniform in size, free from agglomeration and stacking, and CuO₂The nano-dots successfully pass through self-assembly and Ti₃C₂The optimal molar ratio is obtained.

For this example, Ti₃C₂/CuO₂Ti prepared under the condition of 1:2: 2/BSA molar ratio₃C₂/CuO₂HADDF-STEM detection of BSA nanoparticles, the results are shown in FIG. 6, FIG. 6 is Ti provided in example 1 of the present invention₃C₂/CuO₂HADDF-STEM map of BSA nanoparticles. As can be seen from FIG. 6, the nanoparticles prepared at a molar ratio of 1:2:2 were uniform in size, free from agglomeration and stacking, and CuO₂The nano-dots successfully pass through self-assembly and Ti₃C₂The optimal molar ratio is obtained.

For this example, Ti₃C₂/CuO₂Ti prepared under the condition of 1:2: 2/BSA molar ratio₃C₂/CuO₂The result of energy dispersion spectroscopy detection of BSA nanoparticles is shown in FIG. 7, and FIG. 7 shows Ti provided in example 1 of the present invention₃C₂/CuO₂-energy dispersive spectroscopy mapping plot of BSA nanoparticles. As can be seen from FIG. 7, Ti, Cu, O and C elements were successfully supported on the nano-sheet, confirming that Ti was present₃C₂/CuO₂And (4) successfully synthesizing.

For this example, Ti₃C₂/CuO₂Ti prepared under the condition of 1:2: 2/BSA molar ratio₃C₂/CuO₂The results of atomic force microscope observation of the BSA nanoparticles are shown in FIGS. 8-9, and FIG. 8 shows Ti provided in example 1 of the present invention₃C₂/CuO₂FIG. 9 is an AFM (atomic force microscope) measurement of Ti by the use of AFM provided in example 1 of the present invention₃C₂/CuO₂-layer thickness profile of BSA nanoparticles. From this, it was found that the thickness of the nanoparticles was 1.2 nm.

Separately for Ti prepared in step 1) of this example₃C₂Nanosheet and CuO prepared in step 2)₂Nanodots, Ti prepared in step 3)₃C₂/CuO₂-BSA nanoparticles (Ti)₃C₂/CuO₂a/BSA molar ratio of 1:2:2), and Ti prepared according to step 3) without addition of BSA₃C₂/CuO₂Nanoparticles (Ti)₃C₂/CuO₂Molar ratio of 1:2) was measured, and the results are shown in fig. 10, which is a DLS graph provided in example 1 of the present invention. As can be seen from FIG. 10, Ti produced in this example₃C₂/CuO₂-the hydrated particle size of the BSA nanoparticles is about 189 nm.

Example 2

Ti prepared in example 1₃C₂/CuO₂-BSA nanoparticles (Ti)₃C₂/CuO₂Biosafety evaluation with a/BSA molar ratio of 1:2: 2):

1) in vitro culture of U87 glioma cells and HUVEC human venous endothelial cells, evaluation of Ti₃C₂/CuO₂-the toxicity of BSA nanoparticles on tumor cells, comprising the following steps:

arranging 2 96-hole culture plates, wherein each plate is divided into 7 groups, and each group is provided with 5 multiple holes; a plate with 100uL of 1X 10 density added per well⁴Glioma cells, b plates added 100uL per well at a density of 5X 10³Culturing human vein endothelial cells for 24h, adding Ti with different concentrations diluted in equal proportion after the cells adhere to the wall₃C₂/CuO₂And (3) continuously culturing the BSA nanoparticles for 24h, adding CCK-8, incubating for 1h, detecting the cell survival rate in each hole, and measuring the absorbance at 450nm by using an enzyme-labeling instrument.

Ti₃C₂/CuO₂Results of evaluating tumor cytotoxicity of BSA nanoparticles are shown in fig. 11, and fig. 11 is a bar graph of U87 glioma cytotoxicity versus different material concentrations provided by the examples of the present invention. As can be seen from FIG. 11, the cell activity gradually decreased with increasing Ti concentration, and was 40% when the Ti concentration reached 50. mu.g/ml.

2)30 ICR mice were randomly divided into 4 groups of 5 mice each, and Ti was injected via tail vein at different doses₃C₂/CuO₂-BSA nanoparticles, (control)l, 5mg/kg, 10mg/kg, 15mg/kg) was sacrificed after 30 days, specific observations included:

2.1 general state: the mental state, diet, body temperature, etc. were observed daily. Growth curve: the mouse body weight was measured by an electronic scale every two days.

The body weight curve of the mice with different concentration material groups is shown in fig. 12, and fig. 12 is a body weight curve graph of the mice with different concentration material groups provided by the embodiment of the invention. As can be seen from fig. 12, there was no significant difference in body weight of mice between the groups.

2.2) changes in major organs and hematological indices: after 30 days, blood is taken from veins behind the eyeball of the mouse, and hematology index detection is carried out; after blood sampling, mice were sacrificed and major organs (heart, liver, spleen, lung, kidney) were collected, fixed with 4% paraformaldehyde, and gross tissue specimens were sectioned and stained to observe major organ histological structures.

The results of HE staining of the main organs of the mice in the material groups with different concentrations are shown in fig. 13, and fig. 13 is a graph of HE staining of the main organs of the mice in the material groups with different concentrations according to the embodiment of the present invention. As can be seen from fig. 13, there was no significant damage to the major organs in each group of mice.

Example 3

Ti prepared in example 1₃C₂/CuO₂-BSA nanoparticles (Ti)₃C₂/CuO₂The BSA molar ratio is 1:2:2) and the effect of the acoustic power/chemical power synergetic treatment on the tumors is evaluated:

firstly, detecting Tetramethylbenzidine (TMB) color reaction and 1, 3-diphenyl isobenzofuran (DPBF) color fading reaction with different reaction time by using an ultraviolet spectrophotometer and an enzyme-labeling instrument; 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO), BMPO (CAS:387334-31-8) in combination with electron spin resonance spectroscopy (ESR) analysis of the singlet oxygen (1O)₂) And a hydroxyl radical (. OH). Next, the cell level was observed for the efficiency of ROS production using the ROS fluorescent probe DCFH-DA (2, 7-dichlorfluorogenic Cindilacete).

The 96-well culture plate is divided into 10 areas, each area has 5 multiple wells, and 3 culture plates are arranged in total. Adding 100uL of glioma cells with the density of 1 × 104 into each hole, culturing for 24h, grouping according to different powers and ultrasonic time after the cells adhere to the wall, continuously culturing for 24h, adding CCK-8, incubating for 1h, detecting the survival rate of the cells in each hole, and measuring the absorbance at 450nm by using an enzyme-labeling instrument.

Experimental grouping and respective corresponding processing methods: control-cells were not treated; pure low intensity ultrasound group-therapeutic ultrasound irradiation of the cells to be treated through the bottom of the culture plate; ti₃C₂Group-addition of Ti of the same Ti concentration₃C₂A nanoparticle; CuO (copper oxide)₂group-CuO added at the same Cu concentration₂A nanoparticle; ti₃C₂/CuO₂Group- -addition of Ti at the same Ti and Cu concentrations₃C₂/CuO₂A nanoparticle; low intensity ultrasound + Ti₃C₂/CuO₂Combined with a chemical-adding method like Ti₃C₂/CuO₂The group is characterized in that therapeutic ultrasonic irradiation is carried out on cells to be treated through the bottom of a culture plate, meanwhile, in order to further analyze the killing effect of a synergistic treatment mode on breast cancer cells, the cells of each group are treated differently and then are put into an incubator to be cultured for 1h, live cells and dead cells are respectively dyed through Calcein-AM and propidium iodide, and then the survival condition of the cells is observed under a fluorescence microscope. In order to further quantitatively analyze the apoptosis state of the cells, the cells are digested and collected after different treatments, and the apoptosis state of the cells is quantitatively analyzed by the fluorescence intensity of the dye through a flow cytometer after dyeing. Ultrasonic irradiation conditions: frequency 1MHz, duty cycle 50%, sound intensity: 1.5W/cm²And ultrasonically irradiating for 5 s.

By subcutaneous injection of nude mice at a density of 1X 10⁷And (3) after the glioma cells establish a nude mouse glioma transplantation tumor model, measuring the maximum diameter and the minimum diameter of the mouse tumor to calculate the tumor volume. All tumor-bearing mice 6 groups were divided into control group, US group and Ti group according to the random principle₃C₂Group, CuO₂Group Ti₃C₂/CuO ₂5 mice bearing tumors per group. In the experiment, the injection dosage of the nanoparticles is 10mg/kg, the nanoparticles are respectively injected through tail veins on the 1 st day and the 3 rd day of treatment, and the ultrasonic-related treatment groups are respectively 24 days after the nanoparticle injectionUltrasonic irradiation is carried out for hours, namely day 2 and day 4; ultrasonic irradiation conditions: frequency 1MHz, duty cycle 50%, sound intensity: 1.5W/cm²And carrying out ultrasonic irradiation for 5 min. Experimental grouping and respective corresponding processing methods: the control group, tumor-bearing mice, was not treated at all; the simple low-intensity ultrasonic group-tumor-bearing mice only carry out therapeutic ultrasonic irradiation; ti₃C₂Group-tumor-bearing mice injected with Ti through tail vein₃C₂Nanoparticles; CuO (copper oxide)₂Group-tumor-bearing mice injected with CuO through tail vein₂Nanoparticles; ti₃C₂/CuO₂Group-tumor-bearing mice by tail intravenous injection of Ti₃C₂/CuO₂Nanoparticles; low intensity ultrasound + Ti₃C₂/CuO₂Group-injection method of nanoparticles as same as Ti₃C₂/CuO₂Group, while therapeutic ultrasound irradiation is performed.

The observation of therapeutic effects includes the following aspects:

1) tumor growth curves: measuring longest diameter (L) and shortest diameter (W) of tumor by vernier caliper every two days, respectively, and obtaining the value of L.W by formula V²And/2, calculating the tumor volume until the whole treatment course is finished, and drawing a tumor growth curve. Tumor Inhibition Rate (IR) ═ tumor mass of control group-tumor mass of treatment group)/tumor mass of control group × 100%.

2) Post-treatment neoplastic pathological changes: after finishing the 15-day treatment course, killing A group of tumor-bearing mice, taking tumor specimens, and respectively weighing and recording the volume and the weight of tumor tissues; 4% paraformaldehyde was fixed for further pathological examination of gross tumor histological sections.

The experimental results are shown in fig. 14-16, wherein fig. 14 is a tumor live image of different groups provided by the embodiment of the present invention, fig. 15 is a relative tumor volume-time curve of different groups provided by the embodiment of the present invention, and fig. 16 is an HE and immunofluorescent staining image of tumor tissues of different groups provided by the embodiment of the present invention. As can be seen in FIG. 14, Ti was present in 6 different treatment groups₃C₂/CuO₂+ US group tumors were minimal; as can be seen in FIG. 15, Ti was present in 6 different treatment groups₃C₂/CuO₂+ the best tumor treatment in US group; tong (Chinese character of 'tong')As can be seen in FIG. 16, Ti was present in 6 different treatment groups₃C₂/CuO₂The + US group had the most area of tumor hemorrhage and necrosis, while the proliferation activity of tumor tissue was the lowest.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A nanocomposite material comprising: the BSA-based nano-material comprises an inorganic nano-material and BSA molecules which are modified on the surface of the inorganic nano-material;

2. A method of preparing the nanocomposite of claim 1, comprising the steps of:

3. The production method according to claim 2, wherein the two-dimensional structure Ti₃C₂The nano sheet is prepared according to the following steps:

4. The method according to claim 3, wherein the Ti is₃AlC₂The powder is prepared according to the following steps:

5. The method according to claim 3, wherein the steps of sequentially performing the hydrofluoric acid etching treatment and the tetrapropylammonium hydroxide intercalation treatment comprise:

6. The preparation method according to claim 5, wherein in step i), the concentration of the HF aqueous solution is 30 to 50 wt%; the Ti₃AlC₂The ratio of the powder to the aqueous HF solution was 10 g: (40-100) mL;

in the step i), the mixing temperature is 15-35 ℃; the mixing speed is 400-1000 rpm; the mixing time is 1-5 days.

7. The method according to claim 5, wherein in step ii), Ti is used as the material for the catalyst layer₃AlC₂The Ti subjected to hydrofluoric acid etching treatment in the amount of powder used₃C₂The dosage ratio of the material to the tetrapropylammonium hydroxide is 10 g: (40-100) mL;

in the step ii), the mixing temperature is 15-35 ℃; the mixing speed is 400-1000 rpm; the mixing time is 1-5 days.

8. The method of claim 2Method, characterized in that said positively charged CuO₂The nanodots are prepared according to the following steps:

9. The production method according to claim 2, wherein the two-dimensional structure Ti₃C₂Nanosheet, positively charged CuO₂The molar ratio of the nanodots to the bovine serum albumin is 1: (1-5): (1-5).

10. The use of the nanocomposite material of claim 1 in the preparation of a medicament for the sonodynamic and chemodynamic co-therapy of tumors.