Silicon-based nano material for targeted therapy of brucellosis as well as preparation method and application of silicon-based nano material

Abstract

The invention relates to a silicon-based nano material for targeted therapy of brucellosis as well as a preparation method and application of the silicon-based nano material. The invention relates to a preparation method of a silicon-based nano material for targeted therapy of brucellosis. The preparation method comprises the following steps: (1) preparing magnetic mesoporous silica; (2) aminating the magnetic mesoporous silica, and modifying the magnetic mesoporous silica with a responsive material and yeast beta-glucan to obtain modified magnetic mesoporous silica; and (3) carrying out antibiotic loading on the modified magnetic mesoporous silica. According to the silicon-based nano material for targeted therapy of brucellosis as well as the preparation method and the application of the silicon-based nano material, the surface of magnetic macroporous mesoporous silica is modified with yeast beta-glucan capable of targeting M cells and glutathione responsive molecules, and a drug delivery system sensitive to a brucellosis environment is synthesized; the nano-carrier has targeted targeting selectivity for delivering and releasing drugs, the activity, slow release and target cell uptake efficiency of antibiotics are ensured, and the brucellosis treatment is more accurate and efficient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocarrier materials, and particularly relates to a silicon-based nanomaterial for targeted treatment of brucellosis and a preparation method and application thereof. BACKGROUND

[0002] Brucellosis (brucellosis for short) is a bacterial infectious disease caused by Brucella. Brucella is a gram-negative coccobacillus, which is an intracellular parasitic bacteria with slow growth. The disease has characteristics of allergy, occupation and regionality. The strong pathogenicity of Brucella enables it to enter the body through inhalation, ingestion, exposure to mucous membranes or needle puncture wounds, and then produce endotoxins to cause bacteremia and toxemia in the body. The average clinical incubation period is 14-28 days. Brucellosis began to spread in China in the 1950s, and gradually reached a peak in the 1960s. The epidemic situation was more serious in various pastoral areas. Its main clinical manifestations include fever, fatigue, hearing loss, urinary or defecation dysfunction, limb paralysis and joint muscle, back pain and other various symptoms. Severe patients show severe lesions of multiple organs. One of the best ways to prevent and control brucellosis is to control infection and eradicate Brucella in livestock herds, which can be achieved by vaccination, elimination of infected animals, monitoring of livestock herds or a combination of the above, and treatment of brucellosis infection is also urgent.

[0003] At present, the treatment of brucellosis mainly relies on antibiotics. However, since Brucella enters the body in different stages and can evade immune system recognition, it faces the problems of low efficiency and side effects of liver and kidney toxicity. The premise of efficient treatment of intracellular infection is that the drug has sufficient concentration and antibacterial activity in the cells and sites where bacteria reside. However, most antibacterial drugs are difficult to penetrate cell membranes and are easily excluded or inactivated in cells. The study of nanodrug carriers is expected to solve such problems. It has designable physical, chemical and biological properties, thereby improving drug utilization, changing drug tissue and organ distribution, and improving drug biosafety. Therefore, designing an efficient nanocarrier that meets the requirements of long circulation, slow release, targeting, loading of drug molecules into intracellular, improving treatment efficiency, reducing side effects and shortening the course of disease is of great significance for the treatment of brucellosis.

[0004] Based on this, the present application provides a kind of silicon-based nanomaterial for targeted therapy brucellosis and its preparation method and application, which is a kind of silicon-based nanomaterial for targeted therapy brucellosis multiple lesion stage, the method combines magnetic mesoporous silica, glutathione responsive material and yeast β-glucan capable of targeting M cell to prepare drug delivery system, the prepared silicon-based nanomaterial has large aperture, large specific surface area, can efficiently load drug, and the responsive material makes it can be released slowly at brucella aggregation site specifically, prolongs drug release time, and the targeting molecule can also deliver material to immune cells, reduce the loss of drug in non-target site, significantly improve the proportion of target cells. SUMMARY

[0005] The application aims to provide a preparation method of a silicon-based nanomaterial for targeted therapy brucellosis, which combines silicon-based nanomaterial and active molecule yeast β-glucan capable of targeting M cell, and then modifies responsive material DTDP to prepare a targeted silicon-based nanodrug delivery system.

[0006] A preparation method of a silicon-based nanomaterial for targeted therapy brucellosis, comprising the following steps:

[0007] (1) preparing magnetic mesoporous silica;

[0008] (2) sequentially modifying the magnetic mesoporous silica with amino, responsive material and yeast β-glucan to obtain modified magnetic mesoporous silica;

[0009] (3) loading antibiotic drug on the modified magnetic mesoporous silica to obtain the silicon-based nanomaterial for targeted therapy brucellosis, which is a silicon-based nanodrug carrier capable of targeting M cell.

[0010] Further, in step (1), the mesoporous silica is spherical mesoporous silica.

[0011] Further, in step (1), the preparation method is as follows:

[0012] dissolve CTAT in water containing TEA, stir at 75-85°C for 1-2h, then add TEOS, react at 75-85°C for 1-2h, centrifuge and wash, vacuum dry, remove template, and obtain mesoporous silica nanospheres;

[0013] add the mesoporous silica nanospheres to a Gd2O3 aqueous solution of magnetic nanoparticles, ultrasonically stir, and obtain mesoporous silica coated with magnetic nanoparticles;

[0014] The mass ratio of the mesoporous silica nanospheres to the Gd2O3 magnetic nanoparticles is 1:1.5-2.5.

[0015] Further, in the step (2), the modification process is:

[0016] dispersing the magnetic mesoporous silica in a mixed solution of ethanol and 3- aminopropyl triethoxysilane, stirring at 75-85℃ for 20-28h, centrifugal washing, collecting the product, obtaining the amino-modified magnetic mesoporous silica;

[0017] dissolving the amino-modified magnetic mesoporous silica in PBS solution, mixing well, sequentially adding 3, 3-dithiodipropionic acid, EDC, NHS, stirring in the dark for 20-28h, centrifugal washing, collecting the product, vacuum drying, obtaining the magnetic mesoporous silica modified by the responsive material;

[0018] dissolving the magnetic mesoporous silica modified by the responsive material in DMSO, adding EDC, DMAP, stirring after activation, adding yeast β-glucan, stirring in the dark at 40-50℃ for 20-28h, centrifugal washing, freeze-drying, collecting the product.

[0019] Further, the volume of the ethanol and 3-aminopropyl triethoxysilane is 50-100:1;

[0020] The mass ratio of the amino-modified magnetic mesoporous silica to 3, 3-dithiodipropionic acid is 20:25-29, the mass ratio of EDC and NHS in PBS solution is 260-280mg:155-170mg, and the pH of PBS buffer is 6-8;

[0021] The mass ratio of the magnetic mesoporous silica modified by the responsive material to yeast β-glucan is 9-11:9, and the mass ratio of EDC and DMAP in DMSO buffer is 2-4:2.

[0022] Further, the volume of the ethanol and 3-aminopropyl triethoxysilane is 50:1;

[0023] The mass ratio of the amino-modified magnetic mesoporous silica to 3, 3-dithiodipropionic acid is 20:27, and the mass ratio of EDC and NHS in PBS solution is 270mg:163mg;

[0024] The mass ratio of the magnetic mesoporous silica modified by the responsive material to yeast β-glucan is 10:9, and the mass ratio of EDC and DMAP in DMSO buffer is 3:2.

[0025] Further, in the step (3), the antibiotic drug is doxycycline;

[0026] The mass ratio of the modified magnetic mesoporous silica to the antibiotic drug in the step (3) is 9-11:9.

[0027] Further, the mass ratio of the modified magnetic mesoporous silica to the antibiotic drug in the step (3) is 10:9.

[0028] Another object of the present application is to provide a silicon-based nanomaterial for targeted treatment of brucellosis, which is prepared by the above preparation method, and has the properties of targeting, magnetism, environmental responsiveness, large pore size and large specific surface area. The nanomaterial can directly reach the liver and spleen and other sites through magnetism and environmental responsiveness, and specifically responds to the survival microenvironment of brucella, and the yeast beta-glucan targets M cells, so as to be more accurately and quickly delivered to macrophages, enter cells and slowly release drugs, thereby providing an effective way for the treatment of brucellosis in the multiple lesion stage, i.e., the period when brucella reproduces in large quantities in the endothelial reticular system of the liver and spleen.

[0029] Another object of the present application is to provide the application of the above-mentioned silicon-based nanomaterial for targeted treatment of brucellosis in the treatment of brucellosis.

[0030] Compared with the prior art, the present application has the following advantages:

[0031] The silicon-based nanomaterial for targeted treatment of brucellosis and the preparation method and application thereof are a silicon-based nanomaterial for targeted treatment of brucellosis in the multiple lesion stage. Through the layer-by-layer structural modification of magnetism, environmental responsiveness and targeting molecules, a nanodrug carrier that can accurately and quickly reach the lesions of brucellosis is obtained. The antibiotic drug molecules are not easily rapidly degraded, the controlled slow release of the drug is realized, the utilization rate is improved, and the material contains magnetic molecules and can directly reach the place where brucella is located in the liver and spleen. The specific advantages are as follows:

[0032] 1. In the technical scheme of the present application, the prepared nanodelivery system is a large-pore spherical structure with large pore size and large specific surface area, can load magnetic molecules and drugs, reduces the waste of antibiotics, slowly releases the antibiotics, improves the utilization rate, and can be excreted out of the body through the kidney after a certain period of time, does not produce liver and kidney toxicity, can be synthesized in batches, and is easy to industrialize.

[0033] 2. In the technical scheme of the present application, Dectin-1 on M cells has specific recognition ability for yeast beta-glucan, can transport it from the mucosal cavity to the lymph tissue on the basal side, and deliver it to antigen-presenting cells such as macrophages and dendritic cells. When the APC recognizes the signal of beta-glucan, the recognition mechanism can quickly start the defense mechanism of the body.

[0034] 3. In the technical solution of the present invention, by modifying the magnetic material and the responsive material DTDP, the nanomaterial can directly reach the Brucella aggregation sites such as the liver and spleen to release drugs in response.

[0035] 4. In the technical solution of the present invention, the modification of Gd2O3, DTDP and yeast β-glucan has high universality. During the modification process, the linkage is carried out by amidation reaction of carboxyl and amino groups or esterification reaction of carboxyl and hydroxyl groups. The final synthesized drug carrier has the characteristics of strong synergistic targeting, low toxicity and side effects and high biocompatibility. Attached Figure Description

[0036] Figure 1 These are TEM images of the magnetic silicon-based nanomedicine carrier before and after modification in Example 1;

[0037] Figure 2 The images show the BET and BJH diagrams of the silicon-based nanomedicine carrier before and after modification in Example 1.

[0038] Figure 3 The surface potential changes of the silicon-based nanomedicine carrier during the modification process in Example 1 are shown.

[0039] Figure 4 The thermogravimetric changes of the silicon-based nanomedicine carrier during the modification process in Example 1 are shown.

[0040] Figure 5 The infrared spectral analysis of the silicon-based nanomedicine carrier during the modification process in Example 1;

[0041] Figure 6 The ultraviolet spectral analysis of the silicon-based nanomedicine carrier during the modification process in Example 1;

[0042] Figure 7 This is a graph showing the evaluation results of the blood compatibility of the silicon-based nanomedicine carrier during the modification process in Example 1;

[0043] Figure 8 This is a graph showing the toxicity of different carrier concentrations of the silicon-based nanomedicine carrier before and after modification to cells in Example 1. Detailed Implementation

[0044] To further illustrate the silicon-based nanomaterial for targeted treatment of brucellosis, its preparation method, and its application, and to achieve the intended purpose of the invention, the following detailed description, in conjunction with preferred embodiments, details the specific implementation methods, structures, features, and effects of the silicon-based nanomaterial for targeted treatment of brucellosis proposed in this invention. In the following description, different "embodiments" or "embodiments" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable manner.

[0045] A kind of silicon-based nanometer material for targeted treatment brucellosis and its preparation method and application will be further described in detail below in conjunction with specific examples:

[0046] The silicon-based nanometer material for targeted treatment brucellosis and its preparation method and application of the application, mesoporous silica spheres are prepared by soft template method, then magnetic nanoparticles are prepared, and the two are synthesized by ultrasonic stirring to form magnetic mesoporous silica, then the magnetic mesoporous silica is subjected to amidation reaction to connect responsive material DTDP and esterification reaction to connect active molecule yeast β-glucan, and finally antibiotic drug doxycycline is loaded to obtain a silicon-based nanometer drug carrier that can target M cells.

[0047] The technical scheme of the application is as follows:

[0048] A preparation method of a silicon-based nanometer material for targeted treatment brucellosis includes the following steps:

[0049] (1) preparing magnetic mesoporous silica;

[0050] (2) sequentially modifying the magnetic mesoporous silica by amination, responsive material modification and yeast β-glucan modification to obtain modified magnetic mesoporous silica;

[0051] (3) loading antibiotic drug on the modified magnetic mesoporous silica to obtain the silicon-based nanometer material for targeted treatment brucellosis.

[0052] Preferably, in step (1), the mesoporous silica is spherical mesoporous silica.

[0053] Further preferably, in step (1), the preparation method is as follows:

[0054] Dissolve CTAT in water containing TEA, stir at 75-85℃ for 1-2h, then add TEOS, react at 75-85℃ for 1-2h, centrifuge and wash, vacuum dry, remove the template, and obtain mesoporous silica nanospheres;

[0055] Add the mesoporous silica nanospheres to a Gd2O3 aqueous solution of magnetic nanoparticles, and after ultrasonic stirring, obtain mesoporous silica coated with magnetic nanoparticles;

[0056] The mass ratio of the mesoporous silica nanospheres to the Gd2O3 magnetic nanoparticles is 1:1.5-2.5.

[0057] Preferably, in step (2), the modification process is as follows:

[0058] The magnetic mesoporous silica is dispersed in a mixed solution of ethanol and 3-aminopropyl triethoxysilane, stirred at 75-85℃ for 20-28h, centrifuged, washed, and the product is collected to obtain the amino-modified magnetic mesoporous silica;

[0059] The amino-modified magnetic mesoporous silica is dissolved in PBS solution, mixed well, and then 3,3-dithiodipropionic acid, EDC, and NHS are added in sequence, stirred and reacted in the dark for 20-28h, centrifuged, washed, and the product is collected, vacuum dried to obtain the magnetic mesoporous silica modified by the responsive material;

[0060] The magnetic mesoporous silica modified by the responsive material is dissolved in DMSO, EDC and DMAP are added, stirred and activated, and then yeast β-glucan is added, stirred and reacted in the dark at 40-50℃ for 20-28h, centrifuged, freeze-dried, and the product is collected.

[0061] Further preferably, the volume ratio of the ethanol and 3-aminopropyl triethoxysilane is 50-100:1;

[0062] The mass ratio of the amino-modified magnetic mesoporous silica to 3,3-dithiodipropionic acid is 20:25-29, the mass ratio of EDC to NHS in PBS solution is 260-280mg:155-170mg, and the pH of the PBS buffer is 6-8;

[0063] The mass ratio of the magnetic mesoporous silica modified by the responsive material to yeast β-glucan is 9-11:9, and the mass ratio of EDC to DMAP in the DMSO buffer is 2-4:2.

[0064] Further preferably, the volume ratio of the ethanol and 3-aminopropyl triethoxysilane is 50:1;

[0065] The mass ratio of the amino-modified magnetic mesoporous silica to 3,3-dithiodipropionic acid is 20:27, and the mass ratio of EDC to NHS in PBS solution is 270mg:163mg;

[0066] The mass ratio of the magnetic mesoporous silica modified by the responsive material to yeast β-glucan is 10:9, and the mass ratio of EDC to DMAP in the DMSO buffer is 3:2.

[0067] Preferably, in step (3), the antibiotic drug is doxycycline;

[0068] In step (3), the mass ratio of the modified magnetic mesoporous silica to the antibiotic drug is 9-11:9.

[0069] Further preferably, in the step (3), the mass ratio of the modified magnetic mesoporous silica to the antibiotic drug is 10:9.

[0070] Example 1.

[0071] The specific operation steps are as follows:

[0072] (1) Preparation of magnetic mesoporous silica:

[0073] Preparation of mesoporous silica nanospheres: 0.96 g of CTAT was dissolved in 50 ml of water containing 120 μl of TEA, and after stirring at 80°C for 1.5 h, 7.8 ml of TEOS was quickly added. After reacting at 80°C for 2 h, the product was washed with water and ethanol for three times, vacuum dried, and calcined at high temperature in a muffle furnace to remove the template to obtain mesoporous silica nanospheres.

[0074] The preparation method of the magnetic nanoparticle Gd2O3 is as follows: 2.4 g of GdCl3·6H2O was dissolved in 40 ml of DEG solution, and after stirring at 80°C and 750 rpm for 1 h, 4.5 ml of 1 mM / L NaOH was added to adjust the pH to 11, and the mixture was fully mixed. After stirring at 140°C and 750 rpm for 1 h, the reaction conditions were changed to stirring at 180°C and 750 rpm for 4 h, and then the reaction was stopped. The product was naturally cooled, 400 ml of ultrapure water was added, the mixture was fully mixed, and the product was stored at 4°C.

[0075] The preparation method of the magnetic mesoporous silica is as follows: 587 mg of mesoporous silica was dissolved in the above-mentioned 440 ml of magnetic Gd2O3 aqueous solution (the mass ratio of mesoporous silica to magnetic nanoparticle Gd2O3 was 1:2), and after ultrasonic stirring for 24 h, the mesoporous silica coated with magnetic nanoparticles, i.e., the magnetic mesoporous silica Gd2O3@MSN, was obtained.

[0076] (2) Modification of the magnetic mesoporous silica

[0077] 0.5 g of the magnetic mesoporous silica was dispersed in a mixed solution of 50 ml of ethanol and 1 ml of 3-aminopropyltriethoxysilane, and after stirring at 80°C for 24 h, the product was collected by centrifugal washing to obtain Gd2O3@MSN-NH2.

[0078] 200 mg of Gd2O3@MSN-NH2 was dissolved in 40 ml of PBS solution with a pH of 7.4, and after fully stirring to mix, 270 mg of 3,3-dithiodipropionic acid, 270 mg of EDC, and 163 mg of NHS were sequentially added. After avoiding light and stirring for 24 h, the product was collected by centrifugal washing, water washing for three times, and vacuum drying at 60°C to obtain Gd2O3@MSN-DTDP.

[0079] Gd2O3@MSN-DTDP-β-Glucan was prepared by dissolving 50 mg Gd2O3@MSN-DTDP in 20 ml DMSO solution, adding 90 mg EDC, 60 mg DMAP, stirring and activating, then adding 45 mg yeast β-glucan dissolved in 10 ml DMSO, stirring at 45°C in the dark for 24 h, centrifuging and washing, water washing, freeze-drying, and collecting the product.

[0080] (3) Modification of the magnetic mesoporous silica loaded antibiotic drug prepared in step (2)

[0081] Gd2O3@MSN-DTDP-β-Glucan was prepared by dissolving 50 mg Gd2O3@MSN-DTDP in 20 ml DMSO solution, adding 90 mg EDC, 60 mg DMAP, stirring and activating, then adding 45 mg yeast β-glucan dissolved in 10 ml DMSO, stirring at 45°C in the dark for 24 h, centrifuging and washing, water washing, freeze-drying, and collecting the product.

[0082] Example 2.

[0083] TEM test method: A small amount of Gd2O3, Gd2O3@MSN and Gd2O3@MSN-DTDP-β-Glucan prepared in Example 1 was dissolved and ultrasonically dispersed with anhydrous ethanol, 20 μl of the sample was added dropwise on a copper mesh, and TEM transmission electron microscope was used for scanning observation.

[0084] Results: As shown in Figure 1 . Figure 1 a is the TEM picture of Gd2O320 nm, the size is about 3-5 nm; 1b is the TEM picture of Gd2O3@MSN 100 nm, the mesoporous silica dendrite channel can be seen filled with Gd2O3; 1c is the TEM picture of Gd2O3@MSN-DTDP-β-Glucan 200 nm, the dendritic channel is covered, indicating that the surface is modified with organic matter.

[0085] Example 3.

[0086] BET, BJH test was performed on Gd2O3, Gd2O3@MSN-DTDP-β-Glucan prepared in Example 1.

[0087] Method: Nitrogen adsorption-desorption isotherm was measured by A500 instrument (Mike, USA), and specific surface area was calculated from adsorption data in low pressure range using Brunauer-Emmett-Teller (BET) model, and pore size was determined according to Barrett Joyner-Halenda (BJH) method.

[0088] Result: As Figure 2 As shown, MSN exhibits typical Langmuir type IV hysteresis curves before and after modification, indicating that the material has a mesoporous structure. The changes in pore size, specific surface area, and pore volume before and after modification also indicate that Gd2O3@MSN-DTDP-β-Glucan was successfully prepared.

[0089] Example 4.

[0090] The surface charge changes of the nanoparticles in Example 1 were characterized.

[0091] Methods: The nanoparticles Gd2O3@MSN, Gd2O3@MSN-NH2, Gd2O3@MSN-DTDP, and Gd2O3@MSN-DTDP-β-Glucan prepared in Example 1 were dispersed in 5 mL of water, and after sonication for 15 min, the surface charge changes of the mixed solutions were characterized.

[0092] Result: As Figure 3 As shown, the introduction of DTDP makes it negatively charged due to the presence of carboxyl groups after being linked to amino groups. After modifying yeast β-glucan, it is still negatively charged but the negative charge is reduced. The continuous change of Zeta potential further proves the successful modification and alteration of mesoporous silica.

[0093] Example 5.

[0094] Thermogravimetric analysis was performed on the nanoparticles in Example 1.

[0095] Method: The nanoparticles Gd2O3@MSN, Gd2O3@MSN-DTDP, and Gd2O3@MSN-DTDP-β-Glucan prepared in Example 1 were subjected to thermogravimetric analysis at 10 K / min in an N2 atmosphere between 25 and 800 °C.

[0096] Result: As Figure 4 As shown, Gd2O3@MSN exhibits a thermogravimetric loss of 9.45% between 25℃ and 800℃. However, after modification with DTDP and yeast β-glucan, the thermogravimetric loss rates reach 13.09% and 23.96%, respectively. This is because both DTDP and yeast β-glucan are organic compounds, and the increase in the content of organic components leads to an increase in the carbon content lost, thus resulting in a continuous increase in the weight loss rate. This also demonstrates the successful modification with DTDP and yeast β-glucan.

[0097] Example 6.

[0098] The nanoparticles in Example 1 were subjected to infrared functional group testing and analysis.

[0099] Method: The nanoparticles Gd2O3@MSN, Gd2O3@MSN-DTDP, Gd2O3@MSN-DTDP-β-Glucan, β-Glucan powder in the preparation process of Example 1 were mixed and ground with potassium bromide powder, the mass ratio was about 1:100, then tabletting was carried out for infrared test.

[0100] Results: As shown in Figure 5 , compared with Gd2O3@MSN, Gd2O3@MSN-DTDP appeared amide bond 1550cm -1 of amide reaction at the specific position marked, Gd2O3@MSN-DTDP-β-Glucan appeared ester bond 1740cm -1 of esterification reaction.

[0101] Example 7.

[0102] The nanoparticles Gd2O3@MSN, Gd2O3@MSN-DTDP, Gd2O3@MSN-DTDP-β-Glucan, Gd2O3@MSN-DTDP-β-Glucan@DH, DH in Example 1 were tested and analyzed by ultraviolet spectrum.

[0103] Method: UV-Vis spectrum was used to confirm each modification process.

[0104] Results: As shown in Figure 6 , the curve change after modification of DTDP and β-Glucan proved the successful modification of mesoporous silica, in addition, the appearance of two peaks same as doxycycline DH also proved the effective loading of Gd2O3@MSN-DTDP-β-Glucan to DH.

[0105] Example 8: Drug loading performance analysis

[0106] Method: The following different concentration gradient of doxycycline solution was configured, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL, 1mg / mL, then 1mg / mL of mesoporous silica microsphere carrier was prepared and added into the above solution, avoiding light stirring for 24h, 10000rpm centrifugation for 10min, to get the precipitate. The precipitate was washed with ultrapure water to remove free drugs and surface adsorbed drugs, to get drug-loaded nanoparticles Gd2O3@MSN-DTDP-β-Glucan@DH, the supernatant was collected to determine the drug absorbance at 346nm. Then the drug loading rate was calculated according to the formula:

[0107] DLC(%)=(Wz -W s ) / W t ×100%

[0108] wherein, W z is the total mass of DH added; W s is the mass of DH in the supernatant; W t is the total mass of the carrier and drug after drug loading.

[0109] It was tested that the drug loading rate was the highest at 0.9 mg / mL of DH concentration, which was 12.24%.

[0110] Example 9.

[0111] The blood compatibility of nanoparticles Gd2O3@MSN, Gd2O3@MSN-DTDP-β-Glucan, Gd2O3@MSN-DTDP-β-Glucan@DH in Example 1 was evaluated.

[0112] Method: Fresh sterile sheep blood was centrifuged at 4℃ for 10 min at a speed of 2000 rpm, and washed with PBS solution for 5 times to obtain red blood cells (RBCs), and then diluted with PBS solution to obtain a 5% RBCs suspension. Then 0.5 mL of nanoparticle sample solution (2 mg / mL) was added to 0.5 mL of 5% RBCs suspension, and the same volume of PBS and 0.1% Triton X-100 mixed RBCs were used as negative and positive controls, respectively, and the mixed solution was vortexed and incubated in a constant temperature shaking incubator at 37℃ for 2 h. Subsequently, all samples were centrifuged at 4℃ at a speed of 2000 rpm for 10 min, and the absorbance value of the supernatant at 540 nm was measured to calculate the hemolysis rate.

[0113] Results: As shown in Table 1, according to the standard of the International Organization for Standardization, when the hemolysis rate exceeds 5%, the nanomaterial is determined to have a risk of hemolysis, although the hemolysis rate of Gd2O3@MSN is 29.69%, but the hemolysis rate of mesoporous silica after final modification is all less than 5%, which has good biocompatibility. Figure 7

[0114] Example 10.

[0115] The cytotoxicity analysis of different carrier concentrations of nanoparticles Gd2O3@MSN-DTDP-β-Glucan, Gd2O3@MSN-DTDP-β-Glucan@DH in Example 1 was performed.

[0116] ​Method: Macrophages were seeded in 96-well plates at 5000 cells per well and incubated with different concentrations of the above materials (12.5, 25, 50, 100, 200 μg / mL) for 24 h, then the cell viability was detected by CCK-8 method, the toxicity of different pure carrier treatments on cells was determined by comparing the cell viability, and the cell survival rate was calculated according to the formula:

[0117] Cell survival rate (%) = [(OD1-OD0) / (OD2-OD0)]x100

[0118] In the formula: OD1 is the absorbance of the experimental group; OD2 is the absorbance of the control group; OD0 is the absorbance of the blank group.

[0119] Dosing group: cells + nanoparticles + medium + CCK-8

[0120] Control group: cells + medium + CCK-8

[0121] Blank group: medium + CCK-8

[0122] Results: As shown in Figure 8 Gd2O3@MSN-DTDP-β-Glucan showed good biocompatibility with macrophages, and although Gd2O3@MSN-DTDP-β-Glucan@DH had a slightly greater effect on cells than Gd2O3@MSN-DTDP-β-Glucan, the cell survival rate was still greater than 85%, indicating that the final modified nanomedicine carrier had low cytotoxicity and would not produce toxic side effects on normal macrophages not infected with Brucella.

[0123] Example 11.

[0124] The specific operation steps are as follows:

[0125] (1) Preparation of magnetic nanoparticle-coated mesoporous silica

[0126] Preparation of mesoporous silica nanospheres: 0.96 g of CTAT was dissolved in 50 ml of water containing 120 μl of TEA, stirred at 75°C for 2 h, then 7.8 ml of TEOS was quickly added, and reacted at 75°C for 1.5 h, then washed with water and ethanol for three times each, vacuum dried, and calcined at high temperature in a muffle furnace to remove the template to obtain mesoporous silica nanospheres.

[0127] Preparation of magnetic nanoparticles Gd2O3: 2.4 g of GdCl3·6H2O was dissolved in 40 ml of DEG solution, stirred at 80°C and 750 rpm for 1 h, 4.5 ml of 1 mM / L NaOH was added to adjust the pH to 11.5, fully mixed, stirred at 140°C and 750 rpm for 1 h, then the reaction conditions were changed to 180°C and 750 rpm for 4 h, the reaction was stopped, and the product was naturally cooled, 400 ml of ultrapure water was added, fully mixed, and stored at 4°C.

[0128] Preparation of magnetic mesoporous silica: 587 mg of mesoporous silica was dissolved in the magnetic Gd2O3 aqueous solution prepared by the above method (the mass ratio of mesoporous silica to magnetic nanoparticles Gd2O3 was 1:1.5), and after ultrasonic stirring for 24 h, magnetic mesoporous silica coated with magnetic nanoparticles was obtained, which was referred to as magnetic mesoporous silica.

[0129] (2) Modification

[0130] The magnetic mesoporous silica was modified by amination, responsive material and yeast β-glucan. Specifically:

[0131] 0.5 g of magnetic mesoporous silica was dispersed in a mixture of 80 ml of ethanol and 1 ml of 3-aminopropyltriethoxysilane, stirred at 75°C for 28 h, centrifuged and washed, and the product was collected to obtain aminated modified mesoporous silica Gd2O3@MSN-NH2.

[0132] 200 mg of Gd2O3@MSN-NH2 was dissolved in 40 ml of PBS solution with pH 6.0, fully stirred and mixed, 250 mg of 3,3-dithiodipropionic acid, 260 mg of EDC and 155 mg of NHS were added, and the reaction was stirred in the dark for 20 h, then centrifuged and washed with water three times, the product was collected and dried at 60°C under vacuum to obtain responsive material modified mesoporous silica Gd2O3@MSN-DTDP.

[0133] 45 mg of Gd2O3@MSN-DTDP was dissolved in 20 ml of DMSO solution, 60 mg of EDC and 60 mg of DMAP were added, stirred and activated, then 45 mg of yeast β-glucan dissolved in 10 ml of DMSO was added, stirred in the dark at 40°C for 28 h, centrifuged and washed with water, and then freeze-dried to collect the product to obtain Gd2O3@MSN-DTDP-β-Glucan.

[0134] (3) Loading of antibiotic drugs on the magnetic mesoporous silica modified in step (2)

[0135] The final modified Gd2O3@MSN-DTDP-β-Glucan was dispersed in a 900 mg / L doxycycline solution (mass ratio of Gd2O3@MSN-DTDP-β-Glucan to doxycycline was 9:9), stirred in the dark for 24 h, centrifuged at 10,000 rpm, and then washed with deionized water three times. After freeze-drying, Gd2O3@MSN-DTDP-β-Glucan@DH was obtained.

[0136] Example 12.

[0137] The specific operation steps are as follows:

[0138] (1) Preparation of magnetic nanoparticle-coated mesoporous silica

[0139] Preparation of mesoporous silica nanospheres: 0.96 g of CTAT was dissolved in 50 ml of water containing 120 μl of TEA, stirred at 85°C for 1 h, then 7.8 ml of TEOS was quickly added, and reacted at 85°C for 1 h. After washing with water and ethanol three times each, vacuum drying, and high-temperature calcination in a muffle furnace to remove the template, mesoporous silica nanospheres were obtained.

[0140] Preparation of magnetic nanoparticles Gd2O3: 2.4 g of GdCl3·6H2O was dissolved in 40 ml of DEG solution, stirred at 80°C and 750 rpm for 1 h, 4.5 ml of 1 mM / L NaOH was added to adjust the pH to 12, and then fully mixed. Stir at 140°C and 750 rpm for 1 h, then change the reaction conditions to 180°C and 750 rpm for 4 h. Stop the reaction and cool naturally. Move the product to a container, add 400 ml of ultrapure water, mix thoroughly, and store at 4°C.

[0141] Preparation of magnetic mesoporous silica: 500 mg of mesoporous silica was dissolved in the magnetic Gd2O3 aqueous solution prepared by the above method (mass ratio of mesoporous silica to magnetic nanoparticles Gd2O3 was 1:2.5), and ultrasonic stirring was performed for 24 h to obtain magnetic nanoparticle-coated mesoporous silica, which was referred to as magnetic mesoporous silica.

[0142] (2) Modification

[0143] The magnetic mesoporous silica was modified by amination, a responsive material, and yeast β-glucan. Specifically:

[0144] 0.5 g of magnetic mesoporous silica was dispersed in a mixture of 100 ml of ethanol and 1 ml of 3-aminopropyltriethoxysilane, stirred at 85°C for 20 h, centrifuged and washed, and the product was collected to obtain aminated modified mesoporous silica Gd2O3@MSN-NH2.

[0145] The 200mg Gd2O3@MSN-NH2 is dissolved in 50ml PBS solution with pH of 8.0, mixed well by stirring, 290mg 3,3-dithiodipropionic acid, 280mg EDC, 170mg NHS are added, and the reaction is stirred for 28h in dark, centrifuged and washed, washed with water for three times, the product is collected, dried in vacuum at 60℃, and the mesoporous silica Gd2O3@MSN-DTDP after modification of the response material is obtained.

[0146] The 55mg Gd2O3@MSN-DTDP is dissolved in 30ml DMSO solution, 120mg EDC, 60mg DMAP are added, stirred and activated, 45mg yeast β-glucan dissolved in 10ml DMSO is added, stirred for 20h in dark at 50℃, centrifuged and washed, washed with water, freeze-dried, the product is collected, and the Gd2O3@MSN-DTDP-β-Glucan is obtained.

[0147] (3) The magnetic mesoporous silica loaded antibiotic drug after modification in step (2)

[0148] The Gd2O3@MSN-DTDP-β-Glucan after final modification is dispersed in 900mg / L doxycycline solution (the mass ratio of Gd2O3@MSN-DTDP-β-Glucan to doxycycline is 11:9), stirred for 24h in dark, centrifuged at 10000rpm, the supernatant is removed, washed with deionized water for three times, freeze-dried, and the Gd2O3@MSN-DTDP-β-Glucan@DH is obtained.

[0149] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A method for preparing a silicon-based nanomaterial for targeted treatment of brucellosis, characterized in that, The method comprises the following steps: (1) preparing magnetic mesoporous silica; (2) sequentially modifying the magnetic mesoporous silica with amino, responsive material and yeast beta-glucan to obtain modified magnetic mesoporous silica; (3) loading antibiotic drugs on the modified magnetic mesoporous silica to obtain the silica-based nanomaterial for targeted treatment of Buruli ulcer.

2. The preparation method according to claim 1, wherein in the step (1), the mesoporous silica is spherical mesoporous silica.

3. The preparation method according to claim 2, wherein in the step (1), the preparation method is as follows: dissolving CTAT in water containing TEA, stirring at 75-85 DEG C for 1-2 h, then adding TEOS, reacting at 75-85 DEG C for 1-2 h, centrifuging and washing, vacuum drying, removing the template to obtain mesoporous silica nanospheres; adding the mesoporous silica nanospheres into a Gd2O3 aqueous solution of magnetic nanoparticles, ultrasonically stirring to obtain mesoporous silica coated with magnetic nanoparticles; the mass ratio of the mesoporous silica nanospheres to the Gd2O3 is 1:1.5-2.

5.

4. The preparation method according to claim 1, wherein in the step (2), the modification process is as follows: dispersing the magnetic mesoporous silica in a mixed solution of ethanol and 3-aminopropyl triethoxysilane, stirring at 75-85 DEG C for 20-28 h, centrifuging and washing to collect the product to obtain amino-modified magnetic mesoporous silica; dissolving the amino-modified magnetic mesoporous silica in a PBS solution, thoroughly mixing, sequentially adding 3,3-dithiodipropionic acid, EDC and NHS, stirring in the dark for 20-28 h, centrifuging and washing to collect the product, vacuum drying to obtain responsive material-modified magnetic mesoporous silica; dissolving the responsive material-modified magnetic mesoporous silica in DMSO, adding EDC and DMAP, stirring and activating, then adding yeast beta-glucan, stirring in the dark at 40-50 DEG C for 20-28 h, centrifuging and freeze-drying to collect the product.

5. The preparation method according to claim 4, wherein the volume ratio of the ethanol to 3-aminopropyl triethoxysilane is 50-100:1; the mass ratio of the amino-modified magnetic mesoporous silica to 3,3-dithiodipropionic acid is 20:25-29, the mass ratio of EDC to NHS in the PBS solution is 260-280 mg:155-170 mg, and the pH of the PBS buffer is 6-8; the mass ratio of the responsive material-modified magnetic mesoporous silica to yeast beta-glucan is 9-11:9, and the mass ratio of EDC to DMAP in the DMSO buffer is 2-4:

2.

6. The preparation method according to claim 5, wherein the volume ratio of the ethanol to 3-aminopropyl triethoxysilane is 50:

1. ​ ​ ​ ​ ​ The mass ratio of the amino-modified magnetic mesoporous silica and 3,3-dithiodipropionic acid is 20:27, and the mass ratio of EDC and NHS in PBS solution is 270mg:163mg; The mass ratio of the responsive material-modified magnetic mesoporous silica and yeast β-glucan is 10:9, and the mass ratio of EDC and DMAP in DMSO buffer is 3:

2.

7. The preparation method of claim 1, wherein, In the step (3), the antibiotic drug is doxycycline. In the step (3), the mass ratio of the final delivery system / modified magnetic mesoporous silica and the antibiotic drug is 9-11:

9.

8. The preparation method of claim 1, wherein, In the step (3), the mass ratio of the modified magnetic mesoporous silica and the antibiotic drug is 10:

9.

9. A silicon-based nanomaterial for targeted treatment of brucellosis, characterized in that, The preparation method of any one of claims 1-8.

10. The silicon-based nanomaterial for targeted treatment of brucellosis prepared by the preparation method of claim 9.

Images