A composite nanoparticle, a preparation method and application thereof, and an antitumor drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kind of composite nanoparticles and its preparation method and application.The composite nanoparticles provided by the application include nano calcium peroxide, hyaluronic acid coated on the surface of the nano calcium peroxide and urease coated on the surface of the hyaluronic acid.In the application, the hyaluronic acid has tumor cell targeting property, which is conducive to accurately delivering the composite nanoparticles to tumor cells;at the same time, the hyaluronic acid can fix the urease on the surface of the nano calcium peroxide, which is conducive to improving the biological mineralization rate.In the application, when the composite nanoparticles enter the tumor microenvironment under the condition of providing urea outside, they are affected by the acidic microenvironment, the nano calcium peroxide decomposes to release calcium ions, and at the same time, the urease specifically decomposes urea in the microenvironment to release carbonate ions, the calcium ions and carbonate ions produce a mineral layer on the surface of the cell membrane through synergistic effect, thereby blocking the channel of cell material exchange and inducing cell apoptosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a composite nanoparticle, a preparation method and application thereof, and an antitumor drug. BACKGROUND

[0002] In recent years, the development of biomineralization in the biomedical field is particularly rapid. From being used as a physical shell for protecting cells to being an effective tool for killing cancer cells, tumor cell mineralization is also considered as a benign healing performance in the process of cancer treatment. Studies have shown that biomineralization can be artificially regulated by adjusting the medium homeostasis or nucleation site. Therefore, some researchers take biomineralization as a main treatment option, and scientists have proposed many biomineralization treatment schemes, including using biological molecules such as folic acid, chondroitin sulfate and polypeptides to induce mineralization.

[0003] However, in the existing biological molecule-induced mineralization process, the mineralization efficiency of the tumor site is low due to the influence of receptor circulation and local inhibiting factors, thereby limiting the treatment effect. SUMMARY

[0004] Therefore, the present application provides a composite nanoparticle, a preparation method and application thereof, and an antitumor drug. The composite nanoparticle provided by the present application has high mineralization efficiency for inducing mineralization of tumor cells.

[0005] In order to solve the above technical problems, the present application provides a composite nanoparticle, which comprises nano calcium peroxide, hyaluronic acid coated on the surface of the nano calcium peroxide, and urease coated on the surface of the hyaluronic acid.

[0006] Preferably, the average particle size of the nano calcium peroxide is 200-400 nm.

[0007] Preferably, the weight average molecular weight of the hyaluronic acid is 100000-200000.

[0008] Preferably, the mass ratio of the nano calcium peroxide and the hyaluronic acid is 1:1.156-2.

[0009] Preferably, the mass ratio of the calcium peroxide and the urease is 1:0.3-0.4.

[0010] The present application also provides a preparation method of the composite nanoparticle as described in the above technical solution, which comprises the following steps:

[0011] The nano calcium peroxide is dispersed in a hyaluronic acid solution for first coating to obtain hyaluronic acid-coated calcium peroxide nanoparticles;

[0012] Mixing the hyaluronic acid coated calcium peroxide nanoparticles, urease and phosphate buffer solution to perform a second coating to obtain the composite nanoparticles.

[0013] Preferably, the first coating is performed under stirring, and the time of the first coating is 6-12h.

[0014] Preferably, the molar concentration of the phosphate buffer solution is 0.01mol / L, and the pH value of the phosphate buffer solution is 7.2-7.6.

[0015] The mass of the urease and the volume of the phosphate buffer solution are in a ratio of 2-5mg:10mL.

[0016] The second coating is performed on a shaker, the rotation speed of the shaker is 40-50r / min, and the rotation time of the shaker is 22-26h.

[0017] The application further provides application of the composite nanoparticles in the above technical solution or the composite nanoparticles prepared by the preparation method in the above technical solution in preparation of an antitumor drug.

[0018] The application further provides an antitumor drug, which comprises composite nanoparticles and urea, and the composite nanoparticles are the composite nanoparticles in the above technical solution or the composite nanoparticles prepared by the preparation method in the above technical solution.

[0019] The application provides a composite nanoparticle, which comprises nano calcium peroxide, hyaluronic acid coated on the surface of the nano calcium peroxide and urease coated on the surface of the hyaluronic acid. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A preparation process diagram of the composite nanoparticles and a process diagram of inducing mineralization of tumor cells by the composite nanoparticles;

[0021] Figure 2TEM images of CaO2 nanoparticles and CaO2@HA@urease prepared for Example 1, wherein a is a TEM image of CaO2 nanoparticles, b is a TEM image of CaO2@HA@urease;

[0022] Figure 3 Columnar comparison chart of nanoparticle surface potential for Example 1 and Comparative Example 2;

[0023] Figure 4 Particle size distribution curve chart of nanoparticles for Example 1 and Comparative Example 2;

[0024] Figure 5 Columnar comparison chart of cytotoxicity of nanoparticles prepared for Example 1 and Comparative Examples 1-3 at different concentrations;

[0025] Figure 6 Dot-line chart of tumor volume after different treatment times for the test group, control 1 group and control 2 group

[0026] Figure 7 In vivo tumor imaging chart of mice after different treatment times for the test group, control 1 group and control 2 group;

[0027] Figure 8 Micro-CT chart of tumor mineralization of mice in the test group. DETAILED DESCRIPTION

[0028] The present application provides a composite nanoparticle, which comprises nano calcium peroxide, hyaluronic acid coated on the surface of the nano calcium peroxide, and urease coated on the surface of the hyaluronic acid. In the present application, all raw materials are conventional commercially available products if not otherwise specified.

[0029] In the present application, the average particle size of the nano calcium peroxide is preferably 200-400 nm, and more preferably 200-300 nm.

[0030] In the present application, the preparation method of the nano calcium peroxide comprises the following steps:

[0031] The hyaluronic acid solution, calcium chloride solution, ammonia water and hydrogen peroxide solution are mixed to obtain a mixed solution;

[0032] After adding sodium hydroxide solution to the mixed solution, solid-liquid separation is performed to obtain the nano calcium peroxide.

[0033] The present application mixes a hyaluronic acid solution, a calcium chloride solution, ammonia water and a hydrogen peroxide solution to obtain a mixed solution. In the present application, the weight average molecular weight of the hyaluronic acid in the hyaluronic acid solution is preferably 100000-200000, more preferably 150000-200000. In the present application, the mass concentration of the hyaluronic acid solution is preferably 10-21 mg / mL, more preferably 14-15 mg / mL. The present application preferably dissolves hyaluronic acid in water to obtain a hyaluronic acid solution. In the present application, the water is preferably ultrapure water. In the present application, the dissolution is preferably carried out under stirring. The present application has no special requirements for the stirring, as long as it can be completely dissolved. Because hyaluronic acid and calcium ions have high affinity, calcium peroxide is easily synthesized in hyaluronic acid.

[0034] In the present application, the mass concentration of the calcium chloride solution is preferably 0.08-0.12 g / mL, more preferably 0.1 g / mL. In the present application, the volume ratio of the hyaluronic acid solution and the calcium chloride solution is preferably 7-10:1.5-2, more preferably 7:1.5.

[0035] In the present application, the molar concentration of the ammonia water is preferably 0.8-1.2 mol / L, more preferably 1 mol / L. In the present application, the volume ratio of the hyaluronic acid solution and the ammonia water is preferably 7-10:1, more preferably 7:1.

[0036] In the present application, the mass concentration of the hydrogen peroxide solution is preferably 28-32%, more preferably 30%. In the present application, the volume ratio of the hyaluronic acid solution and the hydrogen peroxide solution is preferably 7-10:1.5-2, more preferably 7:1.5.

[0037] In the present application, the mixing is preferably carried out under stirring. In the present application, the stirring is preferably electromagnetic stirring; the stirring time is preferably 2.8-3.2 h, more preferably 3 h. The present application has no special requirements for the stirring speed, as long as it can be uniformly mixed.

[0038] After obtaining the mixed solution, the present application adds a sodium hydroxide solution to the mixed solution, and then separates the solid and liquid to obtain the nano calcium peroxide. In the present application, the molar concentration of the sodium hydroxide solution is preferably 0.8-1.2 mol / L, more preferably 1 mol / L. In the present application, the volume ratio of the hyaluronic acid solution and the sodium hydroxide solution is preferably 7-10:1.5-2, more preferably 7:2. The present application preferably accompanies ultrasonic when adding the sodium hydroxide solution to the mixed solution. The present application has no special requirements for the ultrasonic, as long as it can be uniformly mixed.

[0039] In the present application, the solid-liquid separation is preferably centrifugation; the rotation speed of the centrifugation is preferably 10000-13000 r / min, more preferably 11000-12000 r / min; the centrifugation time is preferably 5-10 min, more preferably 6-8 min.

[0040] The present application preferably washes the solid obtained by solid-liquid separation. In the present application, the washing includes sodium hydroxide solution washing, ultrapure water washing and anhydrous ethanol washing in sequence. In the present application, the molar concentration of the sodium hydroxide solution used in the sodium hydroxide solution washing is preferably 0.1 mol / L. In the present application, the number of times of the sodium hydroxide solution washing is preferably 1; the number of times of the ultrapure water washing is preferably 1; and the number of times of the anhydrous ethanol washing is preferably 1.

[0041] In the present application, the weight average molecular weight of the hyaluronic acid (HA) is preferably 100000-200000, more preferably 150000-200000. In the present application, the mass ratio of the nano calcium peroxide and the hyaluronic acid is preferably 1:1.156-2, more preferably 1:1.156. In the present application, the hyaluronic acid is rich in carboxyl groups, which can fix urease on the surface of the nanoparticles through electrostatic interaction, thereby improving the mineralization rate of the composite nanoparticles.

[0042] In the present application, the mass ratio of the calcium peroxide and urease is preferably 1:0.3-0.4, more preferably 1:0.308.

[0043] The composite nanoparticles provided by the present application have good biocompatibility.

[0044] The composite nanoparticles provided by the present application can be used in a tumor microenvironment-responsive enzyme-induced biological mineralization treatment system. With the assistance of urea, the composite nanoparticles autonomously decompose urea and calcium peroxide in the tumor microenvironment, generate a large amount of calcium ions, ammonia and carbonate ions, and ultimately form a large amount of calcified substances, which can effectively solve the problems of insufficient mineralization efficiency in the existing biological mineralization anti-tumor treatment.

[0045] The present application also provides a preparation method of the composite nanoparticles described in the above technical solution, which comprises the following steps:

[0046] The nano calcium peroxide is dispersed in a hyaluronic acid solution for first coating to obtain hyaluronic acid-coated calcium peroxide nanoparticles;

[0047] The hyaluronic acid-coated calcium peroxide nanoparticles, urease and phosphate buffer solution are mixed for second coating to obtain the composite nanoparticles.

[0048] The present application disperses nano calcium peroxide in a hyaluronic acid solution to perform a first coating, and obtains hyaluronic acid-coated calcium peroxide nanoparticles. In the present application, the weight average molecular weight of the hyaluronic acid in the hyaluronic acid solution is preferably 100000-200000, and more preferably 150000-200000. In the present application, the mass concentration of the hyaluronic acid solution is preferably 8-10 mg / mL, and more preferably 8-9 mg / mL. In the present application, the mass ratio of the nano calcium peroxide to the volume of the hyaluronic acid solution is preferably 150-200 mg:10-15 mL, and more preferably 150 mg:10 mL.

[0049] The present application does not have special requirements for the dispersion, as long as it can be uniformly dispersed.

[0050] In the present application, the first coating is preferably performed under stirring; the time of the first coating is preferably 6-12 h, and more preferably 8-10 h; the temperature of the first coating is preferably room temperature, and the temperature of the room temperature is preferably 20-35℃, and more preferably 25-30℃. The present application does not have special requirements for the stirring speed, as long as it can be uniformly mixed.

[0051] In the present application, after the completion of the first coating, preferably further comprising: performing solid-liquid separation on the system after the first coating; and the solid-liquid separation is preferably centrifugation. In the present application, the speed of the centrifugation is preferably 5000-8000 r / min, and more preferably 6000-7000 r / min; and the time of the centrifugation is preferably 2-5 min, and more preferably 3-4 min.

[0052] The present application preferably washes the solid obtained by the solid-liquid separation. In the present application, the washing preferably comprises sequentially performing water washing and phosphate buffer washing. In the present application, the water used for the water washing is preferably ultrapure water; and the number of times of the water washing is preferably 2-3 times. In the present application, the pH value of the phosphate buffer used for the phosphate buffer washing is preferably 7.4; and the number of times of the phosphate buffer washing is preferably 1 time.

[0053] After obtaining the hyaluronic acid-coated calcium peroxide nanoparticles, the present application mixes the hyaluronic acid-coated calcium peroxide nanoparticles, urease and a phosphate buffer solution to perform a second coating, and obtains the composite nanoparticles. In the present application, the pH value of the phosphate buffer solution (PBS) is preferably 7.2-7.6, and more preferably 7.4.

[0054] In the present application, the mass ratio of the nano calcium peroxide to the urease is preferably 150-200:2-5, and more preferably 150-180:2-3. In the present application, the mass ratio of the urease to the volume of the phosphate buffer solution is preferably 2-5 mg:10 mL, and more preferably 3-5 mg:10 mL.

[0055] The mixing has no special requirements in the application, as long as it can be uniformly mixed.

[0056] In the application, the second coating is preferably carried out on a shaker, and the rotation speed of the shaker is preferably 40-50 r / min, more preferably 42.8 r / min; and the rotation time of the shaker is preferably 22-26 h, more preferably 24 h.

[0057] In the application, after the completion of the second coating, the system after the second coating is preferably subjected to solid-liquid separation; and the solid-liquid separation is preferably centrifugation. In the application, the rotation speed of the centrifugation is preferably 5000-8000 r / min, more preferably 6000-7000 r / min; and the centrifugation time is preferably 2-5 min, more preferably 3-4 min.

[0058] The solid obtained by the solid-liquid separation is preferably washed in the application. In the application, the washing is preferably phosphate buffer washing. In the application, the pH value of the phosphate buffer used for the phosphate buffer washing is preferably 7.4; and the phosphate buffer washing is preferably carried out 2-3 times.

[0059] The preparation method provided by the application has the advantages of simple process, convenient operation, energy saving and environmental protection, low raw material cost and easy availability.

[0060] The obtained composite nanoparticles are preferably dispersed in a phosphate buffer solution with a pH value of 7.4 for storage.

[0061] The application also provides the use of the composite nanoparticles in the above technical solution or the composite nanoparticles prepared by the preparation method in the above technical solution in the preparation of an antitumor drug. Figure 1 Figure 1 is a schematic diagram of the structure of the composite nanoparticles, and Figure 2 is a schematic diagram of the process of inducing mineralization of tumor cells by the composite nanoparticles provided by the application. During the process of inducing mineralization, calcium peroxide in the composite nanoparticles decomposes to form calcium ions, urease decomposes urea provided from the outside to form carbonate ions, and the calcium ions and the carbonate ions combine to form calcium that coats the surface of the tumor cells.

[0062] The application also provides an antitumor drug, which comprises composite nanoparticles and urea, and the composite nanoparticles are the composite nanoparticles in the above technical solution or the composite nanoparticles prepared by the preparation method in the above technical solution. The application has no special requirements for the dosage form of the antitumor drug, and a conventional dosage form in the art can be used.

[0063] The present application utilizes the characteristic of calcium peroxide hydrolysis to release calcium ions under the acidity of tumor microenvironment, prepares calcium peroxide nanoparticles as endogenous calcium supply, uses hyaluronic acid to fix urease on the surface of nanoparticles as urea hydrolysis catalyst, and uses ammonia and carbonate ions generated by urea hydrolysis to improve the efficiency of biomineralization. The present application has the advantages of easy synthesis, high stability, significant improvement of mineralization efficiency, and effective inhibition of tumor cell proliferation, and is a great innovation in tumor-targeted biomineralization.

[0064] The composite nanoparticles provided by the present application can be used as a good biomineralization drug for treating tumors, do not need external calcium ion supply, are simple to synthesize, and have low cost, and are a great innovation in the field of biomineralization treatment. Compared with the prior art, the present application has the following outstanding beneficial technical results: the composite nanoparticles provided by the present application have good dispersibility, good stability in a physiological environment, easy-to-meet preparation conditions, abundant raw material sources, and very low cost; the composite nanoparticles provided by the present application as a biomineralization treatment system do not need additional calcium ion supply as a mineral source, can release sufficient calcium ions through self-decomposition, can maintain stable structure before reaching the tumor microenvironment, and can only respond and play a role in the tumor microenvironment; the composite nanoparticles provided by the present application as a biomineralization treatment drug have extremely small toxicity, good dispersibility, good biocompatibility, high enzyme activity, can realize efficient cancer cell mineralization according to the acidic conditions of the tumor microenvironment, and can play a very significant anti-tumor activity.

[0065] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application. Examples

[0066] (1) Preparation of CaO2 nanoparticles: 100 mg of hyaluronic acid with a weight average molecular weight of 100,000 was dissolved in 7 mL of ultrapure water at 25°C under stirring to obtain a hyaluronic acid solution; the hyaluronic acid solution, 1.5 mL of calcium chloride solution with a mass concentration of 0.1 g / mL, 1 mL of ammonia water with a molar concentration of 1 mol / L, and 1.5 mL of hydrogen peroxide solution with a mass concentration of 30% were electromagnetically stirred at 25°C for 3 h to obtain a mixed solution; 2 mL of sodium hydroxide solution with a molar concentration of 1 mol / L was added to the mixed solution under ultrasonic conditions, and then centrifuged at a speed of 10,000 r / min for 10 min; the supernatant was discarded, and the obtained solid was washed with 0.1 mol / L sodium hydroxide solution, ultrapure water, and anhydrous ethanol in sequence to obtain CaO2 nanoparticles;

[0067] (2) 150 mg of CaO2 nanoparticles were dispersed in 10 mL of hyaluronic acid solution with a mass concentration of 8 mg / mL and a weight average molecular weight of 200,000. The mixture was stirred at 25 °C for 6 h to perform the first coating. After centrifugation at 5000 r / min for 5 min, the supernatant was discarded. The solid obtained by centrifugation was washed 3 times with ultrapure water and then washed once with PBS with a molar concentration of 0.01 mol / L and a pH of 7.4 to obtain hyaluronic acid-coated calcium peroxide nanoparticles, denoted as CaO2@HA.

[0068] (3) After mixing CaO2@HA, 10 mL of 0.01 mol / L PBS with a pH of 7.4 and 2 mg of urease, place the mixture on a shaker and rotate it for 24 h at a temperature of 25 °C and a speed of 42.5 r / min. Then, centrifuge it at a speed of 5000 r / min for 5 min, discard the supernatant, and wash the solid obtained by centrifugation twice with 0.01 mol / L PBS with a pH of 7.4 to obtain composite nanoparticles, which are denoted as CaO2@HA@urease.

[0069] Transmission electron microscopy (TEM) was performed on the CaO2 nanoparticles and CaO2@HA@urease prepared in Example 1, and the TEM images are shown below. Figure 2 As shown, a is a TEM image of CaO2 nanoparticles, and b is a TEM image of CaO2@HA@urease. (From...) Figure 2 It can be seen that the morphology of the calcium peroxide nanoparticles coated with hyaluronic acid and urease did not change significantly compared with that before coating, and a layer of hyaluronic acid can be seen on the surface of CaO2@HA@urease. Example

[0070] (1) Preparation of nano-CaO2: 100 mg of hyaluronic acid with a weight average molecular weight of 150,000 was dissolved in 10 mL of ultrapure water at 25 °C to obtain a hyaluronic acid solution; the hyaluronic acid solution, 1.5 mL of calcium chloride solution with a mass concentration of 0.1 g / mL, 1 mL of ammonia water with a molar concentration of 0.8 mol / L and 1.5 mL of hydrogen peroxide solution with a mass concentration of 28% were electromagnetically stirred at 25 °C for 2.8 h to obtain a mixed solution; 2 mL of sodium hydroxide solution with a molar concentration of 0.8 mol / L was added to the mixed solution under ultrasonic conditions and centrifuged at 11000 r / min for 9 min. The supernatant was discarded and the solid obtained by centrifugation was washed once with sodium hydroxide solution with a molar concentration of 0.1 mol / L, ultrapure water and anhydrous ethanol, respectively, to obtain CaO2 nanoparticles;

[0071] (2) CaO2 nanoparticles 150 mg were dispersed in 10 mL of a hyaluronic acid solution with a mass concentration of 9 mg / mL and a weight average molecular weight of 150,000, and first coating was performed by stirring at 25°C for 7 h; centrifugation was performed at a speed of 6000 r / min for 4 min, the supernatant was discarded, the solid obtained by centrifugation was washed with ultrapure water for 3 times, and then washed with PBS with a molar concentration of 0.01 mol / L and a pH value of 7.2 for 1 time, to obtain hyaluronic acid-coated calcium peroxide nanoparticles, denoted as CaO2@HA;

[0072] (3) After mixing CaO2@HA, 10 mL of PBS with a molar concentration of 0.01 mol / L and a pH value of 7.2, and 3 mg of urease, the mixture was rotated on a shaker at a temperature of 25°C and a speed of 40 r / min for 24 h, and then centrifuged at a speed of 6000 r / min for 4 min, the supernatant was discarded, and the solid obtained by centrifugation was washed with PBS with a molar concentration of 0.01 mol / L and a pH value of 7.2 for 2 times, to obtain composite nanoparticles, denoted as CaO2@HA@urease. Example

[0073] (1) The preparation of CaO2 nanoparticles: 100 mg of hyaluronic acid with a weight average molecular weight of 180,000 was dissolved in 8 mL of ultrapure water by stirring at 25°C, to obtain a hyaluronic acid solution; the hyaluronic acid solution, 1.5 mL of a calcium chloride solution with a mass concentration of 0.12 g / mL, 1 mL of an ammonia water solution with a molar concentration of 1.2 mol / L, and 1.5 mL of a hydrogen peroxide solution with a mass concentration of 32% were subjected to electromagnetic stirring at 25°C for 3.2 h, to obtain a mixed solution; 2 mL of a sodium hydroxide solution with a molar concentration of 1.2 mol / L was added to the mixed solution under ultrasonic conditions, and then centrifugation was performed at a speed of 12000 r / min for 8 min, the supernatant was discarded, and the solid obtained by centrifugation was washed with a sodium hydroxide solution with a molar concentration of 0.1 mol / L, ultrapure water, and anhydrous ethanol in sequence for 1 time, to obtain CaO2 nanoparticles;

[0074] (2) CaO2 nanoparticles 150 mg were dispersed in 10 mL of a hyaluronic acid solution with a mass concentration of 10 mg / mL and a weight average molecular weight of 180,000, and first coating was performed by stirring at 25°C for 10 h; centrifugation was performed at a speed of 7000 r / min for 3 min, the supernatant was discarded, the solid obtained by centrifugation was washed with ultrapure water for 3 times, and then washed with PBS with a molar concentration of 0.01 mol / L and a pH value of 7.3 for 1 time, to obtain hyaluronic acid-coated calcium peroxide nanoparticles, denoted as CaO2@HA;

[0075] (3) CaO2@HA, 10 mL of 0.01 mol / L PBS with pH value of 7.3, 5 mg of urease were mixed, and then placed on a shaker to rotate at a temperature of 25°C and a speed of 42.8 r / min for 26 h. After centrifugation at a speed of 7000 r / min for 4 min, the supernatant was discarded, and the obtained solid was washed twice with 0.01 mol / L PBS with pH value of 7.3 to obtain the composite nanoparticles, which were recorded as CaO2@HA@urease.

[0076] The CaO2 nanoparticles prepared in Example 1 were used as Comparative Example 1.

[0077] The composite nanoparticles were prepared according to the method of Example 1, except that step (3) was omitted, and the obtained composite nanoparticles were recorded as CaO2@HA.

[0078] The performance of the composite nanoparticles was detected according to the following method:

[0079] Determination of urease activity:

[0080] The urease activity of the composite nanoparticles prepared in Example 1 was 3 U / ml, which indicated that the composite nanoparticles provided by the present application had urease activity and could catalyze the hydrolysis of urea.

[0081] Determination of particle size and surface potential:

[0082] The Malvern nanoparticle size potential instrument was used to determine Examples 1-3 and Comparative Examples 1 and 2. The conditions for detecting particle size were: the refractive index was set to 1.590, the absorption coefficient was set to 0.010, the temperature was set to 25°C, and the measurement mode was set to automatic, with Z average statistical value as the determination result. The test object was measured three times, and the average value of the three measurement values was taken as the measurement result, which was listed in Table 1. The conditions for detecting surface potential were: the dielectric constant was set to 79, the viscosity coefficient was set to 0.8872, the temperature was set to 25°C, and the measurement mode was set to automatic. The test object was measured three times, and the average value of the three measurement values was taken as the measurement result, which was listed in Table 1. The measured results were particle size of 200-400 nm and potential of -10 to -20 mV.

[0083] The application effect of the composite nanoparticles as a biological mineralization therapeutic agent in the preparation of a tumor treatment drug was divided into in vitro and in vivo applications:

[0084] Detection of anti-tumor activity in vitro: 4T1 mouse breast cancer cells were used as the cancer cells to be investigated. The 4T1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin mixture, and the incubator conditions were 37°C, 5% CO2, and the cells were passaged every 2 days. When the cells grew to 70%-80%, the related experiments were performed. Different amounts of composite nanoparticles were dispersed in RPMI1640 culture solution to obtain culture solutions with composite nanoparticle mass concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 50 μg / mL, and 60 μg / mL, respectively; the CCK-8 method was used to detect the toxic effect of the dispersion on 4T1 cells. The logarithmic phase cells were collected, and the cell suspension concentration was adjusted, 200 μL was added to each well of a 96-well plate, and the cells were plated to a density of 1×10 4 per well (the edge wells were filled with PBS). After incubation at 37°C for 24 h in a 5% CO2 incubator until the cell monolayer covered the bottom of the well (96-well flat-bottom plate), culture solutions with concentration gradients (0 μg / mL, 20 μg / mL, 40 μg / mL, 50 μg / mL, and 60 μg / mL) were added, the culture solution with a mass concentration of 0 was used as a blank control, and duplicate wells were set up. After 12 h, the drug-containing culture medium was removed and replaced with fresh culture medium containing 8 mol / L urea (urea was provided) for continued culture for 24 h. A control group experiment was set up, the composite nanoparticles were prepared according to Example 1, and the detection was performed according to the above method, except that after 12 h, the drug-containing culture medium was removed and replaced with fresh culture medium without urea (the control group did not provide urea compared to Example 1) for continued culture for 24 h, which was recorded as the control group. 10 μL of CCK-8 was added to each well, and the culture plate was placed back in the incubator at 37°C for 1 h, and then the OD value was measured at 450 nm. The cell survival rate was calculated according to Formula 1: cell survival rate = 1 - experimental group value OD value / control group value OD value, Formula 1, wherein the values of the experimental group and the control group were both deducted from the blank control, and the results are shown in Table 1.

[0085] Table 1 Performance of nanoparticles of Examples 1-3, Comparative Examples 1-2, and the control group

[0086]

[0087] A bar chart comparing the surface potentials of Example 1 and Comparative Example 2 according to the data in Table 1 is shown in Figure 3 A particle size distribution curve graph is plotted according to the particle size distribution data of Example 1 and Comparative Example 2 in Table 1, as shown in Figure 4 According to Table 1 and Figure 3 , Figure 4 it can be seen that hyaluronic acid and urease have been successfully modified on the surface of the nanoparticles.

[0088] A bar contrast chart was drawn according to the data of cell survival rate of Example 1 and Comparative Examples 1-3 in Table 1, as shown in Figure 5 Fig. 1. In combination with Table 1 and Figure 5 It can be seen that the composite nanoparticles provided by the present application can produce a dense calcified mineral layer on the surface of cancer cell membrane when used as a biomimetic mineralization drug in the presence of urea, effectively leading to the death of cancer cells.

[0089] Determination of in vivo anti-tumor activity: The composite nanoparticles prepared in Example 1 were dispersed in physiological saline to obtain a dispersion with a mass concentration of 120 μg / mL. 4T1 cells were subcutaneously inoculated in the right anterior axillary fossa of BALB / c nude mice at a concentration of 3×10 6 cells per mouse. After 7 days of tumor inoculation, 15 nude mice with a tumor volume of ≥100 mm 3 The nude mice were randomly divided into three groups, namely control group 1, control group 2 and test group, with 5 mice in each group. The control group 1 was injected with physiological saline, the control group 2 was injected with a urea solution with a molar concentration of 8 mol / L, and the test group was injected with 50 μL of the dispersion with a mass concentration of 120 μg / mL, and then 50 μL of a urea solution with a molar concentration of 8 mol / L was injected intratumorally 20 minutes later (urea was provided). The three groups were all given drugs intratumorally, once every two days, with 200 μL of physiological saline or the dispersion with a mass concentration of 120 μg / mL each time, for a total of 6 times. During the entire experiment, the mice were observed daily, their body weight was measured every two days, and the long diameter (A) and short diameter (B) of the sarcoma were measured using a vernier caliper. The tumor volume V was calculated according to the formula 2: V = 1 / 2 (A×B), and the test results are shown in Table 2. We performed in vivo tumor imaging on any two mice in each group on the first day, the third day and the tenth day of drug administration, and obtained Figure 7 ; we dissected the test group mice on the sixth day of treatment and obtained tumor tissues, and scanned the calcification of the tumor tissues using micro-CT, obtaining Figure 8 , Figure 8 The circles in the middle are labeled mineralization signals scanned.

[0090] Table 2 Tumor volumes after different treatment times in the test group, control group 1 and control group 2

[0091]

[0092] A dot-line chart of tumor volumes after different treatment times in the test group, control group 1 and control group 2 was drawn according to the data in Table 2, as shown in Figure 6 Fig. 2. In combination with Table 2 and Figure 6 It can be seen that the increase in tumor volume of the nude mice treated with the composite nanoparticles provided by the present application as a biomimetic mineralization drug was significantly inhibited.

[0093] From the aboveFigure 7 and Figure 8 It can be seen that the tumor growth of the mice treated with nanoparticles is inhibited, and the white mineralization signal appears in the tumor tissue of the test mice, proving that the biological mineralization induced by nanoparticles occurs inside the tumor.

[0094] The biological mineralization efficiency of Examples 1-3 was characterized by a quartz crystal microbalance. First, mouse breast cancer cells 4T1 were fixed on a quartz crystal microbalance sensor, and then the composite nanoparticles obtained in Examples 1-3 were added to the open sample pool of the microbalance, respectively. After co-incubation, the free nanoparticles were carefully washed away with fresh culture medium, and fresh culture medium containing 8 mol / L urea was added. The decrease in the resonance frequency signal of the quartz crystal microbalance sensor was detected, and the results are shown in Table 3. After 16 h of reaction, the resonance frequency tended to be stable, and the decrease in the resonance frequency represented the amount of mineral produced by biological mineralization.

[0095] Table 3 Quartz crystal microbalance resonance frequency decrease after biological mineralization of tumor cells in Examples 1-3

[0096] Example Example 1 Example 2 Example 3 Resonance frequency decrease (Hz) 4289.77 4422.7 4146.2

[0097] As can be seen from Table 3, the CaO2@HA@urease nanoparticles obtained from Examples 1-3 can all cause biological mineralization of tumor cells, and the mineralization can be detected by the quartz crystal microbalance sensor, and the decrease in the resonance frequency is close, indicating that the nanoparticles obtained from Examples 1-3 have stable performance.

[0098] The mineralization efficiency of Example 1, CaO2@HA particles without modified urease (Comparative Example 2), folic acid (a biological mineralization inducer), and chondroitin sulfate (a biological mineralization inducer) was characterized by a quartz crystal microbalance, and 4T1 tumor cells were used as a blank control. The resonance frequency was converted to mass change using the Sauerbrey equation, and the results are shown in Table 4.

[0099] Specific operation: Example 1: First, fix the sensor wafer with the fixed cells on the quartz crystal microbalance open module, set the temperature to 37°C, and add 1 mL of serum-free DMEM medium. After the signal is stable, slowly add the CaO2@HA@urease nanoparticle solution (150 μg / mL) to the wall, and then add 1 mL of serum-free DMEM medium to the wall. After 16 h of reaction, the resonance frequency of the quartz crystal microbalance sensor was measured, and the results are shown in Table 3. -1, 500 μL), and incubated for 2 h. The supernatant was removed and the untargeted bound nanoparticles were washed away by gently rinsing twice with serum-free DMEM medium, and 1 mL of 8 mol / L urea in serum-free medium was added. The frequency change of the microbalance was recorded for 16 h, and the data were processed by Qsoft401, Qtools and OriginLab software to quantify the mineralization efficiency of CaO2@HA@urease, and the results are listed in Table 4.

[0100] Control group: First, the sensor chip with fixed cells was fixed on the quartz crystal microbalance open module, the temperature was set to 37 ℃, 1 mL of serum-free DMEM medium was added, and after the signal was stable, 500 μL of folic acid solution (containing 3 mg mL -1 folic acid and 500 μL of DMEM), chondroitin sulfate solution (containing 3 mg mL -1 chondroitin sulfate and 500 μL of DMEM containing 7.5 mM Na2CO3) and CaO2@HA nanoparticle solution (containing 150 μg mL -1 nanoparticles and 500 μL of DMEM) were added to each group, respectively. The data were processed by Qsoft401, Qtools and OriginLab software to quantify the mineralization efficiency of different materials, and the results are listed in Table 4.

[0101] Table 4 Resonance frequency drop of quartz crystal microbalance after biomineralization of tumor cells in different ways

[0102] Example Blank control Example 1 Comparative Example 2 Folic acid Chondroitin sulfate Resonance frequency decrease (Hz) 2 4420 126 271 64 Mineralized mass produced (ng) 0.7 1606.8 45.8 98.5 23.3

[0103] As can be seen from Table 4, the resonance frequency signal drop of Example 1 is significantly greater than that of other control groups, which proves that the mass produced by the biomineralization of Example 1 is the most, i.e., the mineralization efficiency of Example 1 is the highest in the same time.

[0104] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A composite nanoparticle, characterized in that, The composite nanoparticles include nano-calcium peroxide, hyaluronic acid coated on the surface of the nano-calcium peroxide, and urease coated on the surface of the hyaluronic acid. The preparation method of the composite nanoparticles includes the following steps: Nano-sized calcium peroxide is dispersed in a hyaluronic acid solution for a first coating to obtain hyaluronic acid-coated calcium peroxide nanoparticles. The weight-average molecular weight of the hyaluronic acid in the hyaluronic acid solution is 100,000-20,000, the mass concentration of the hyaluronic acid solution is 8-10 mg / mL, and the mass-to-volume ratio of the nano-sized calcium peroxide to the hyaluronic acid solution is 150-200 mg:10-15 mL. The first coating is carried out under stirring conditions for 6-12 hours. The hyaluronic acid-coated calcium peroxide nanoparticles, urease, and phosphate buffer solution are mixed for a second coating to obtain the composite nanoparticles. The mass ratio of the nano-calcium peroxide to urease is 150-200:2-5, and the mass ratio of the urease to the volume ratio of the phosphate buffer solution is 2-5 mg:10 mL. The molar concentration of the phosphate buffer solution is 0.01 mol / L, and the pH value of the phosphate buffer solution is 7.2-7.

6. The second coating is carried out on a shaker at a speed of 40-50 r / min for 22-26 h.

2. The method for preparing the composite nanoparticles according to claim 1, comprising the following steps: Nano-calcium peroxide was dispersed in a hyaluronic acid solution for the first coating, resulting in hyaluronic acid-coated calcium peroxide nanoparticles. The hyaluronic acid solution has a weight-average molecular weight of 100,000 to 20,000 and a mass concentration of 8 to 10 mg / mL. The mass ratio of the nano-calcium peroxide to the volume of the hyaluronic acid solution is 150 to 200 mg: 10 to 15 mL. The first coating is carried out under stirring conditions for 6 to 12 hours. The hyaluronic acid-coated calcium peroxide nanoparticles, urease, and phosphate buffer solution are mixed for a second coating to obtain the composite nanoparticles. The mass ratio of the nano-calcium peroxide to urease is 150-200:2-5, and the mass ratio of the urease to the volume ratio of the phosphate buffer solution is 2-5 mg:10 mL. The molar concentration of the phosphate buffer solution is 0.01 mol / L, and the pH value of the phosphate buffer solution is 7.2-7.

6. The second coating is carried out on a shaker at a speed of 40-50 r / min for 22-26 h.

3. The application of the composite nanoparticles of claim 1 or the composite nanoparticles prepared by the preparation method of claim 2 and urea in the preparation of antitumor drugs; The antitumor drug is used to treat 4T1 breast cancer cells.

4. An antitumor drug, characterized in that, It includes composite nanoparticles and urea, wherein the composite nanoparticles are the composite nanoparticles of claim 1 or the composite nanoparticles prepared by the preparation method of claim 2.

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