Drug delivery system and preparation method and application thereof

Abstract

The invention provides a drug delivery system and a preparation method and application thereof. The drug delivery system comprises meso-porous silicon loaded with active components and hyaluronic acid wrapping the surface of the meso-porous silicon loaded with the active components; the active component comprises barium titanate and an anti-tumor drug. Mesoporous silicon is used as a drug delivery carrier, hyaluronic acid is used for mediating tumor specific targeting, and after barium titanate and anti-tumor drugs are loaded, the drugs are excited by ultrasound, so that the tumor killing effect is achieved. Cell experiments and animal studies show that the nano-drug delivery system can significantly inhibit tumor growth and improve a tumor hypoxic microenvironment; mechanism research shows that the nano-drug delivery system causes an ICD effect, the number of M1 type macrophages is up-regulated, and anti-tumor immune activation is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a drug delivery system and a preparation method and application thereof. Background Art

[0002] Colorectal cancer (CRC) is a heterogeneous tumor. In recent years, the incidence and mortality of colorectal cancer in my country have been increasing, and there is a trend of younger patients. Therefore, the diagnosis and treatment of CRC has received more and more attention from the country, and the research on CRC treatment has gradually shifted to individualization and refinement. At present, nano-drug therapy has the advantages of realizing multi-material loading and integrated diagnosis and treatment, and has become the main research direction of cancer treatment. However, single nano-drugs still have many defects, such as: (1) poor absorption effect, low solubility in water, low bioavailability, and difficulty in direct use; (2) the plasma half-life is too short, and continuous repeated administration is required; (3) poor targeting and toxic side effects on the body.

[0003] Therefore, finding a method to improve the targeting and bioavailability of nanomedicines is an urgent problem that needs to be solved. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a drug delivery system and its preparation method and application. The drug delivery system can achieve targeted and efficient delivery, overcome the shortcomings of poor water solubility and low bioavailability of barium titanate and anti-tumor drugs themselves, and improve anti-tumor performance.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a drug delivery system, comprising mesoporous silicon loaded with active components and hyaluronic acid wrapped on the surface of the mesoporous silicon loaded with active components;

[0007] The active components include barium titanate and anti-tumor drugs.

[0008] In the present invention, the mesoporous silicon has a rich pore structure and can physically embed barium titanate and anti-tumor drugs; the hyaluronic acid is wrapped on the surface of the mesoporous silicon through electrostatic interaction.

[0009] Preferably, the anti-tumor drug includes icariin.

[0010] Preferably, the molecular weight of the hyaluronic acid is 100,000 to 200,000 kDa.

[0011] Preferably, the mass ratio of the mesoporous silica, barium titanate, anti-tumor drug and hyaluronic acid is (1-2):(1-10):(1-6):(0.01-0.6).

[0012] Preferably, the drug delivery system is ultrasound responsive.

[0013] Preferably, the particle size of the drug delivery system is 100-500 nm, and the PDI is 0.08-0.09.

[0014] Preferably, the dosage form of the drug delivery system includes but is not limited to injection preparations.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned drug delivery system, comprising the following steps:

[0016] S1: Under alkaline conditions, barium titanate, a surfactant and a silicon source are reacted to obtain mesoporous silicon loaded with barium titanate;

[0017] S2: combining the mesoporous silicon loaded with barium titanate with the anti-tumor drug to obtain the mesoporous silicon loaded with barium titanate and the anti-tumor drug;

[0018] S3: Mesoporous silica loaded with barium titanate and anti-tumor drugs is combined with hyaluronic acid to obtain a drug delivery system.

[0019] Preferably, in step S2, the mesoporous silicon loaded with barium titanate is subjected to an amination treatment.

[0020] Preferably, the surfactant comprises cetyltrimethylammonium chloride and / or cetyltrimethylammonium bromide.

[0021] Preferably, the silicon source comprises tetraethyl silicate and / or 3-aminopropyltriethoxysilane.

[0022] Preferably, the barium titanate is obtained by reacting a barium source and a titanium source, the barium source includes barium nitrate and / or barium acetate, and the titanium source includes n-butyl titanate.

[0023] Preferably, the reaction temperature in step S1 is 50-80° C. and the reaction time is 5-24 h.

[0024] Preferably, the binding in steps S2 and S3 is performed at room temperature, and the binding time is independently 1 to 10 h.

[0025] In a third aspect, the present invention provides an application of the above-mentioned drug delivery system in the preparation of anti-tumor drugs.

[0026] Preferably, the tumor includes but is not limited to colorectal carcinoma.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a drug delivery system comprising mesoporous silicon loaded with active components and hyaluronic acid wrapped on the surface of the mesoporous silicon loaded with active components. The present invention can enhance the targeting of tumor tissues by using mesoporous silicon with a large specific surface area, a large number of pore structures and excellent biocompatibility as a carrier. After further using mesoporous silicon to carry active components (barium titanate and anti-tumor drugs), the mesoporous silicon loaded with active components is wrapped with hyaluronic acid, which can further increase the tumor targeting ability, not only can effectively achieve targeted and precise delivery of nanomedicines, but also can utilize the oxygen production and ROS production functions of barium titanate under ultrasonic conditions, which can improve hypoxia in tumors, enhance drug efficacy, and achieve precise treatment of tumors. In addition, the generated ROS can directly kill tumor cells, prompt them to release HMGB1, CRT, ATP, and be taken up by immune cells, thereby causing ICD effect, further enhancing the immunotherapy effect of tumors.

[0029] Taking the drug delivery system ICT-BTO@MSNs@H loaded with barium titanate and icariin as an example, in vivo anti-tumor studies have shown that ICT-BTO@MSNs@HA can inhibit tumor growth and reshape the tumor immune microenvironment under the action of ultrasound, and synergistically exert anti-tumor effects; mechanism studies have revealed that after ultrasonic excitation, ICT-BTO@MSNs@HA can cause tumor cells to release HMGB1, CRT, and ATP, induce ICD effect, promote the maturation of dendritic cells (DC cells), and upregulate the number of M1 macrophages to achieve anti-tumor immune activation; animal experiments showed that there was no significant change in body weight after ultrasonic excitation of ICT-BTO@MSNs@HA, indicating high biosafety.

[0030] It can be seen that the drug delivery system provided by the present invention has a significant tumor-killing effect and provides a new idea for the transformation and application strategy of barium titanate and anti-tumor drugs (such as icariin). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the SEM image of BTO@MSN obtained in Preparation Example 1;

[0032] Figure 2 This is an image of mapping of ICT-BTO@MSNs@HA obtained in Preparation Example 1;

[0033] Figure 3 The particle size distribution image and particle size change image of ICT-BTO@MSNs@HA obtained in Preparation Example 1;

[0034] in, Figure 3 A is the particle size distribution image of ICT-BTO@MSNs@HA. Figure 3 B is the particle size change diagram of ICT-BTO@MSNs@HA during 7 days of storage;

[0035] Figure 4 This is the result diagram of oxygen production of BTO@MSNs@HA+US;

[0036] in, Figure 4 A is the result of oxygen production of barium titanate at different powers. Figure 4 B is the result diagram of oxygen production of barium titanate at different drug concentrations;

[0037] Figure 5 ESR images of BTO@MSNs@HA+US, BTO@MSNs@HA, US, and Control;

[0038] in, Figure 5 A is the singlet oxygen content image of different groups. Figure 5 B is the image of hydroxyl radical content in different groups;

[0039] Figure 6 Confocal microscopy images of Cy7-BTO@MSNs@HA after co-incubation with NCM460 and CT26 cells for 0 h, 0.5 h, 2 h, and 4 h, respectively;

[0040] in, Figure 6 A corresponds to the confocal microscopy image of Cy7-BTO@MSNs@HA co-incubated with NCM460 cells. Figure 6 B corresponds to the confocal microscopy image of Cy7-BTO@MSNs@HA co-incubated with CT26 cells;

[0041] Figure 7 Fluorescence images of CT26 tumor-bearing BALB / c mice, isolated organs and tumors after injection of different substances;

[0042] in, Figure 7 A is the fluorescence image of CT26 tumor-bearing BALB / c mice within 24 hours after ICG injection via tail vein. Figure 7 B is a fluorescence image of CT26 tumor-bearing BALB / c mice 24 hours after tail vein injection of ICG-BTO@MSNs@HA. Figure 7 C is the fluorescence image of isolated organs and tumors after ICG injection via tail vein. Figure 7 D is the fluorescence image of the isolated organs and tumors after injection of ICG-BTO@MSNs@HA via the tail vein;

[0043] Figure 8 The images show the killing effect on CT26 cells after treatment with different groups;

[0044] Fig. 9It is a summary of the relative growth curves of mouse tumors after treatment in different groups, a body weight analysis chart, and a chart showing changes in organ function indicators;

[0045] in, Fig. 9 A is a summary of the relative growth curves of mouse tumors after treatment in different groups. Fig. 9 B is the body weight analysis chart of different groups after treatment. Fig. 9 C is the graph showing the changes in organ function indicators after treatment in different groups;

[0046] Fig.10 The images of HMGB1 and CRT protein expression in CT26 cells after treatment with different groups;

[0047] Fig.11 The images are the flow cytometry analysis results of mature DC cells in the spleen tissue of mice, M1 macrophages, CD8+T cells, GZMB, and INF-γ in tumor tissue after treatment in different groups;

[0048] in, Fig.11 A is the flow cytometry analysis result of mature DC cells in mouse spleen tissue. Fig.11 B is the flow cytometry analysis result of M1 macrophages in mouse tumor tissue. Fig.11 C is the flow cytometry analysis result image of CD8+T cells. Fig.11 D is the flow analysis result image of GZMB. Fig.11 E is the image of flow cytometry analysis results of INF-γ;

[0049] in, Figure 8~Figure 11 The G1, G2, G3, G4, G5, G6, and G7 involved correspond as follows: G1 is Control (single PBS), G2 is US, G3 is BTO@MSNs, G4 is ICT@MSNs@HA, G5 is ICT-BTO@MSNs@HA, G6 is BTO@MSNs@HA+US, and G7 is ICT-BTO@MSNs@HA+US. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The present invention provides a drug delivery system, which is formed by mesoporous silicon loaded with active components and hyaluronic acid wrapped on the surface of the mesoporous silicon loaded with active components. Wherein, the mass ratio of the mesoporous silicon, barium titanate, anti-tumor drug and hyaluronic acid is (1-2): (1-10): (1-6): (0.01-0.6), preferably 1: (7-10): (4-6): (0.1-0.6), more preferably 1: 7.8: 4.4: 0.13.

[0052] In the present invention, the mesoporous silica as a carrier has a large specific surface area and a large number of pore structures, so that it can load hydrophobic drugs well. At the same time, the mesoporous silica also has good biocompatibility and can enhance the targeting of tumor tissues.

[0053] The present invention uses mesoporous silica (abbreviated as: MSNs) as a carrier to carry active components, and the active components include barium titanate and anti-tumor drugs.

[0054] Among them, the barium titanate, as a typical perovskite-type structure crystal, can be targeted and enriched in the tumor microenvironment due to its unique physical and chemical properties, and has the characteristics of high biocompatibility and low toxicity to normal cells. Under the action of ultrasound, the barium titanate in the present invention can decompose water molecules into oxygen and ROS (including superoxide anions, hydrogen peroxide, hydroxyl radicals, ozone, and singlet oxygen). Among them, oxygen can alleviate the hypoxic microenvironment of the tumor, and ROS can kill tumor tissue. Furthermore, ROS killing tumors can also induce ICD effect, stimulate the body's immune cells to kill tumors, and enhance the therapeutic effect.

[0055] It should be noted that there are many types of anti-tumor drugs. The present invention screens among many anti-tumor drugs and uses icariin as an anti-tumor drug, so that the prepared drug delivery system can have a more significant tumor-killing effect.

[0056] The icariin is a monomer active ingredient extracted from the traditional Chinese herbal medicine Epimedium, and is a prenylated flavonoid derivative, which has the functions of blocking tumor cell cycle, promoting tumor cell apoptosis, inhibiting tumor cell metastasis and regulating body immunity. However, single icariin is generally used as an oral drug, resulting in low bioavailability. Therefore, the present invention uses it as one of the active components and carries it on a mesoporous silica carrier to improve its bioavailability.

[0057] It should be noted that if a single icariin or a single barium titanate (using ultrasonic excitation) is loaded on a carrier, the effect of the resulting drug delivery system in killing tumor cells will be significantly reduced. Therefore, the present invention uses icariin and barium titanate in combination, loads them together on a carrier and uses ultrasonic excitation, which can synergistically improve the effect of the drug delivery system in killing tumor cells.

[0058] It should be noted that barium titanate can rely on the high permeability and long retention effect (abbreviated as: EPR effect) to passively target and enrich in tumor tissues, but the efficiency of the EPR effect is low. The present invention preferably uses hyaluronic acid (HA) to wrap the above-mentioned mesoporous silicon nanoparticles carrying active components to enhance the specific targeting effect of the drug delivery system on tumor tissues.

[0059] In the present invention, the molecular weight of the hyaluronic acid is 100,000 to 200,000 kDa.

[0060] In summary, the present invention uses mesoporous silicon as a carrier to carry active components (including barium titanate and anti-tumor drugs), and uses hyaluronic acid to wrap the mesoporous silicon nanoparticles carrying the active components, which can increase the tumor targeting ability. The drug delivery system can be enriched in the tumor area after entering the body. The drug delivery system in the present invention has ultrasound responsiveness. By using ultrasound (parameters: 1 w / cm 2 , 1.0 MHz, 50% duty cycle) to stimulate the drug delivery system in the tumor area. Barium titanate can generate oxygen and ROS, which can not only alleviate the hypoxic microenvironment in the tumor and enhance the apoptosis-inducing effect of anti-tumor drugs, but also directly kill tumor cells through ROS, causing ICD effect and improving the body's immunotherapy function against tumors.

[0061] The present invention also provides a method for preparing the above-mentioned substance delivery system, comprising the following steps:

[0062] S1: Under alkaline conditions, barium titanate, a surfactant and a silicon source are reacted to obtain mesoporous silicon loaded with barium titanate;

[0063] S2: combining the mesoporous silicon loaded with barium titanate with the anti-tumor drug to obtain the mesoporous silicon loaded with barium titanate and the anti-tumor drug;

[0064] S3: Mesoporous silica loaded with barium titanate and anti-tumor drugs is combined with hyaluronic acid to obtain a drug delivery system.

[0065] In the present invention, barium titanate is first provided. The barium titanate is preferably obtained by reacting a barium source and a titanium source. The barium source includes barium nitrate and / or barium acetate, and the titanium source includes n-butyl titanate.

[0066] Exemplarily, the barium titanate is prepared according to the following method:

[0067] The barium source and alkaline substance are dissolved in water, preferably deionized water; the titanium source and dispersant are dissolved in 1-butanol. The alkaline substance is used to adjust the reaction environment, and the alkaline condition is conducive to the dissolution and subsequent reaction of the barium source and the titanium source; the dispersant is preferably oleic acid, which can be adsorbed on the surface of the nanoparticles to play a role in surface modification and improve the dispersibility of the particles.

[0068] Then, the above four parts of liquid are mixed, stirred, and reacted at high temperature. After cooling, the obtained liquid is resuspended and washed with anhydrous ethanol and deionized water, and after centrifugation, the precipitate is freeze-dried to obtain barium titanate (abbreviated as: BTO) solid powder.

[0069] The high temperature reaction is preferably carried out in a container lined with polytetrafluoroethylene. The temperature of the high temperature reaction is 130-145°C, preferably 135°C; the time is 16-20 h, preferably 18 h.

[0070] The number of resuspension, washing and centrifugation can be selected as needed, generally 2 to 5 times, preferably 3 times.

[0071] After obtaining barium titanate, according to the present invention, under alkaline conditions, barium titanate, a surfactant and a silicon source are reacted to obtain mesoporous silicon loaded with barium titanate.

[0072] In the present invention, the surfactant includes hexadecyltrimethylammonium chloride (abbreviated as CTAC) and / or hexadecyltrimethylammonium bromide; the silicon source includes tetraethyl silicate (abbreviated as TEOs) and / or 3-aminopropyltriethoxysilane. The reaction temperature is 50-80°C, preferably 70°C, and the reaction time is 5-24 h, preferably 12 h.

[0073] In some embodiments of the present invention, the mesoporous silicon loaded with barium titanate is prepared according to the following method:

[0074] Add chloroform dispersed with barium titanate to hexadecyltrimethylammonium chloride solution and stir for 0.5-5 h, preferably 1 h, to form an oil-in-water emulsion. Heat to 50-60 °C and continue stirring for 20-40 min to evaporate the chloroform. Raise the temperature to 70-80 °C, add water and alkaline solution (such as sodium hydroxide solution), and then add tetraethyl silicate. Reflux at 70-80 °C for 5-24 h. Stop heating after 5-24 h to terminate the reaction and obtain mesoporous silicon loaded with barium titanate (abbreviated as: BTO@MSNs).

[0075] In some preferred embodiments of the present invention, after the above-mentioned termination reaction, it is preferred to use anhydrous ethanol, centrifuge at a speed of 10000~3000 rpm for 5~30 min, and wash 2~3 times to remove residual raw materials and unreacted hexadecyltrimethylammonium chloride. Then, using a hydrochloric acid solution with a pH of 2~3, the centrifuged precipitate is dispersed in hydrochloric acid to remove hexadecyltrimethylammonium chloride. After stirring for 3~5 h, deionized water is used to centrifuge at a speed of 10000~3000 rpm for 5~30 min, and washed and centrifuged three times to remove residual hydrochloric acid and hexadecyltrimethylammonium chloride. The precipitate is collected and freeze-dried to obtain dry BTO@MSNs.

[0076] Then, according to the present invention, the mesoporous silicon loaded with barium titanate is combined with the anti-tumor drug to obtain the mesoporous silicon loaded with barium titanate and the anti-tumor drug.

[0077] In some embodiments of the present invention, the mesoporous silicon loaded with barium titanate is subjected to an amination treatment, preferably using 3-aminopropyltriethoxysilane (abbreviated as: APTEs) to aminate BTO@MSNs, and then mixed and reacted with anti-tumor drugs to allow the amino group and the anti-tumor drugs to produce electrostatic interaction, and the anti-tumor drugs are carried to BTO@MSNs by electrostatic interaction.

[0078] The mesoporous silica loaded with barium titanate can be combined with the anti-tumor drug at room temperature, and the combining time is 1 to 10 hours, preferably 5 to 6 hours.

[0079] For example, taking the anti-tumor drug icariin (abbreviated as ICT) as an example, in some embodiments of the present invention, the mesoporous silica loaded with barium titanate is combined with icariin according to the following method:

[0080] After BTO@MSNs are dispersed using an alcohol solvent such as isopropanol, 3-aminopropyltriethoxysilane is added. Stir at room temperature for 4 to 8 hours to achieve amination. Then, water, preferably deionized water, is centrifuged at 10,000 to 3,000 rpm for 5 to 30 minutes, washed 2 to 3 times to remove unreacted 3-aminopropyltriethoxysilane, and the precipitate is collected. Icariin and BTO@MSNs are configured in a mass ratio of 1:2, dissolved in dimethyl sulfoxide (DMSO), and stirred at room temperature for 1 to 10 hours to achieve BTO@MSNs carrying icariin to obtain ICT-BTO@MSNs.

[0081] In the present invention, after the above stirring for 6 to 10 hours, water, preferably deionized water, is used for centrifugation at a speed of 10,000 to 3,000 rpm for 5 to 30 minutes, and washed 2 to 3 times to remove the unloaded icariin, and then the precipitate is collected and freeze-dried to obtain dry ICT-BTO@MSNs.

[0082] Then, according to the present invention, mesoporous silica loaded with barium titanate and anti-tumor drugs is combined with hyaluronic acid to obtain a drug delivery system.

[0083] In the present invention, the reaction is carried out at room temperature for 1 to 10 h, preferably 5 to 6 h.

[0084] In the present invention, the room temperature is "10-30°C", preferably "15-25°C".

[0085] Exemplarily, in some embodiments of the present invention, the combination of mesoporous silica loaded with barium titanate and anti-tumor drugs and hyaluronic acid comprises the following steps:

[0086] Prepare a 1-3 wt% hyaluronic acid aqueous solution. Disperse mesoporous silica (e.g., ICT-BTO@MSNs) loaded with barium titanate and anti-tumor drugs with the hyaluronic acid solution, and stir the reaction at room temperature for 1-10 h to achieve surface hyaluronic acid modification of ICT-BTO@MSNs to obtain a drug delivery system.

[0087] In the present invention, after the stirring reaction is completed, deionized water is used again to centrifuge at a speed of 10000-3000 rpm for 5-30 min, washed 2-3 times to remove unreacted hyaluronic acid, and then the precipitate is collected. The collected precipitate is freeze-dried to obtain the final drug delivery system (such as ICT-BTO@MSNs@HA).

[0088] Taking ICT-BTO@MSNs@HA as an example, the drug delivery system provided by the present invention converts barium nitrate Ba(NO3) into 2 With n-butyl titanate C 16 H 36 O 4 Ti reacts to obtain barium titanate BaTiO 3 After that, it reacted with hexadecyltrimethylammonium chloride and tetraethyl silicate to obtain BTO@MSNs, and then 3-aminopropyltriethoxysilane was used to modify BTO@MSNs to make it amination. Finally, the amination BTO@MSNs was stirred with icariin and hyaluronic acid in turn to obtain ICT-BTO@MSNs@HA.

[0089] After testing and analysis, the particle size range of ICT-BTO@MSNs@HA prepared by the present invention is 100-500 nm, the average particle size is 291.5 nm, and the PDI is 0.08-0.09.

[0090] It can be seen that the preparation method of the drug delivery system provided by the present invention is simple, convenient, easy to implement, and is conducive to industrial production or large-scale production.

[0091] In summary, the present invention provides a drug delivery system, which uses mesoporous silicon with rich pore structure as an active component carrier, carries barium titanate through physical embedding, and then carries anti-tumor drugs through electrostatic interaction and physical embedding, and then the hyaluronic acid is wrapped on the surface of mesoporous silicon through electrostatic interaction, which plays a role in mediating targeting. Among them, barium titanate, as one of the active components, produces oxygen and reactive oxygen (ie, ROS) under the action of ultrasound, alleviates the hypoxic microenvironment of the tumor, enhances the efficacy of anti-tumor drugs (such as icariin), and reactive oxygen directly kills tumors and induces ICD effect. At the same time, anti-tumor drugs (such as icariin) can induce apoptosis of cancer cells. In addition, hyaluronic acid and barium titanate can synergistically improve the targeting effect of the drug delivery system.

[0092] Based on this, the present invention also provides an application of the above drug delivery system in the preparation of anti-tumor drugs. The tumor includes but is not limited to colorectal cell carcinoma.

[0093] In the present invention, the dosage form of the drug delivery system includes but is not limited to injection preparations.

[0094] In order to further illustrate the present invention, the following examples are provided for detailed description.

[0095] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0096] Drug: Barium nitrate (Ba(NO 3 ) 2 ) was purchased from Chengdu Jinshan Chemical, n-butyl titanate (C 16 H 36 O 4 Ti) and icariin (ICT) were purchased from Aladdin, cetyltrimethylammonium chloride (CTAC) and hyaluronic acid (HA) were purchased from Maclean, and tetraethyl orthosilicate (TEOs) and 3-aminopropyltriethoxysilane (APTEs) were purchased from Adamas.

[0097] Experimental cell lines: Mouse colorectal cancer cell line CT26, mouse embryonic fibroblast cell line NIH / 3T3, and human normal colon epithelial cell line NCM460 were purchased from ATCC American Cell Bank.

[0098] Experimental animals: BALB / c mice (4 weeks old, female) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. All animals were housed in the SPF environment of the animal laboratory of Guangxi Medical University.

[0099] All animal experiments followed relevant ethical standards and were approved by the Ethics Committee of the Affiliated Cancer Hospital of Guangxi Medical University.

[0100] Preparation Example 1- Preparation of ICT-BTO@MSNs@HA

[0101] (1) Synthesis of barium titanate

[0102] Dissolve 261.34 mg of barium nitrate and 500 mg of sodium hydroxide in 5 mL of deionized water, and dissolve 340.22 mg of tetrabutyl titanate and 2.5 mL of oleic acid in 5 mL of 1-butanol. Mix the above 4 liquids, stir, and pour into a container lined with polytetrafluoroethylene for high-temperature reaction at 135°C for 18 h. After the reaction is completed and cooled, centrifuge the obtained liquid at a speed of 8000 rpm for 5 min, and use anhydrous ethanol and deionized water to repeatedly resuspend, wash, and centrifuge for 3 times. Finally, freeze-dry the obtained precipitate to obtain barium titanate solid powder, referred to as BTO.

[0103] (2) Synthesis of BTO@MSNs

[0104] Weigh 50 mg of barium titanate and disperse it with 1 mL of chloroform. Weigh 0.2 g of hexadecyltrimethylammonium chloride and add it to 12.5 mL of deionized water. In a 100 mL round-bottom flask, add 10 mL of hexadecyltrimethylammonium chloride solution, and drop 1 mL of the dispersed barium titanate in chloroform into the hexadecyltrimethylammonium chloride solution and stir for 1 h to form an oil-in-water emulsion. Then place the flask in a water bath and heat it to 60°C, and continue stirring for 20 min to evaporate the chloroform. Raise the water bath temperature to 70°C, add 25 mL of deionized water and 1.8 mL of 2 mol / L sodium hydroxide solution, and then add 0.36 mL of tetraethyl silicate. Reflux at 70°C for 12 h, and stop heating after 12 h to terminate the reaction. Use anhydrous ethanol and centrifuge at 10,000 rpm for 5 min, and wash three times to remove residual raw materials and unreacted hexadecyltrimethylammonium chloride. Use 20 mL of hydrochloric acid solution with pH 2-3 to disperse the precipitate after centrifugation in hydrochloric acid to remove hexadecyltrimethylammonium chloride. After stirring for 3 h, use deionized water to centrifuge at 10000 rpm for 5 min, wash and centrifuge three times to remove residual hydrochloric acid and hexadecyltrimethylammonium chloride. Collect the precipitate and freeze-dry it to obtain BTO@MSNs.

[0105] (3) Preparation of ICT-BTO@MSNs@HA

[0106] In a 50 mL round-bottom flask, 20 mL of isopropanol was used to disperse BTO@MSNs, and 0.5 mL of 3-aminopropyltriethoxysilane was added. Stir at room temperature for 4 h to achieve amination. Deionized water was centrifuged at 10,000 rpm for 5 min, washed three times to remove unreacted 3-aminopropyltriethoxysilane, and the precipitate was collected. Icariin and BTO@MSNs were configured in a mass ratio of 1:2, dissolved in DMSO, stirred at room temperature for 6 h, and BTO@MSNs were loaded with icariin. Deionized water was centrifuged at 10,000 rpm for 5 min, washed three times to remove unloaded icariin, and then the precipitate was collected and freeze-dried. A 1 wt% hyaluronic acid aqueous solution was prepared. The collected precipitate was dispersed with hyaluronic acid solution and stirred at room temperature for 6 h to achieve surface hyaluronic acid modification of ICT-BTO@MSNs. After the reaction, the mixture was centrifuged at 10,000 rpm for 5 min with deionized water and washed three times to remove the unreacted hyaluronic acid, and then the precipitate was collected and freeze-dried to obtain the final ICT-BTO@MSNs@HA.

[0107] The present invention performs SEM characterization on BTO@MSNs, and the obtained SEM images are as follows: Figure 1 shown.

[0108] The present invention performs a mapping test on ICT-BTO@MSNs@HA, and the results are as follows: Figure 2 shown.

[0109] The present invention performs particle size distribution analysis on ICT-BTO@MSNs@HA, and the obtained particle size distribution diagram is as follows: Figure 3 As shown in A, the change in particle size during the 7-day period is shown in Figure 3 As shown in B.

[0110] The present invention uses a dissolved oxygen meter to test the oxygen production capacity of BTO@MSNs@HA+US (US refers to ultrasound), and the results are as follows: Figure 4 As shown. Among them, Figure 4 A is the oxygen production of barium titanate at different powers. Figure 4 B is the oxygen production of barium titanate at different drug concentrations.

[0111] The present invention uses ESR to test the ROS generation of BTO@MSNs@HA+US, BTO@MSNs@HA, US, and Control groups, wherein the Control group refers to a single PBS solution; the US group refers to the use of ultrasound on the basis of the Control group, that is, the PBS solution is ultrasounded; the BTO@MSNs@HA+US group refers to the use of ultrasound on the basis of the BTO@MSNs@HA group, that is, the BTO@MSNs@HA is ultrasounded, and the results are as follows Figure 5 As shown. (Among them, Figure 5 A is the singlet oxygen content image of different groups. Figure 5 B is the image of the content of hydroxyl radicals in different groups.

[0112] Preparation Example 2-Preparation of ICT@MSNs@HA

[0113] (1) Synthetic MSNs

[0114] Weigh 0.2 g of hexadecyltrimethylammonium chloride and add it to 12.5 mL of deionized water. In a 100 mL round-bottom flask, add 10 mL of hexadecyltrimethylammonium chloride solution, then place the flask in a water bath and heat it to 70°C, add 25 mL of deionized water and 1.8 mL of 2 mol / L sodium hydroxide solution, and then add 0.36 mL of tetraethyl silicate. Reflux at 70°C for 12 h, stop heating after 12 h, and terminate the reaction. Use anhydrous ethanol, centrifuge at 10000 rpm for 5 min, and wash three times to remove residual raw materials and unreacted hexadecyltrimethylammonium chloride. Use 20 mL of hydrochloric acid solution with pH 2-3 to disperse the precipitate after centrifugation in hydrochloric acid to remove hexadecyltrimethylammonium chloride. After stirring for 3 h, use deionized water to centrifuge at 10000 rpm for 5 min, wash and centrifuge three times to remove residual hydrochloric acid and hexadecyltrimethylammonium chloride. Collect the precipitate and freeze-dry it to obtain MSNs.

[0115] (3) Preparation of ICT@MSNs@HA

[0116] In a 50 mL round-bottom flask, 20 mL of isopropanol was used to disperse the MSNs, and 0.5 mL of 3-aminopropyltriethoxysilane was added. Stir at room temperature for 4 h to achieve amination. Deionized water was centrifuged at 10,000 rpm for 5 min, washed three times to remove unreacted 3-aminopropyltriethoxysilane, and the precipitate was collected. Icariin and MSNs were configured in a mass ratio of 1:2, dissolved in DMSO, stirred at room temperature for 6 h, and MSNs were loaded with icariin. Deionized water was centrifuged at 10,000 rpm for 5 min, washed three times to remove unloaded icariin, and then the precipitate was collected and freeze-dried. A 1 wt% hyaluronic acid aqueous solution was prepared. The collected precipitate was dispersed with hyaluronic acid solution and stirred at room temperature for 6 h to achieve surface hyaluronic acid modification of ICT@MSNs. After the reaction, the mixture was centrifuged at 10,000 rpm for 5 min with deionized water and washed three times to remove unreacted hyaluronic acid, and then the precipitate was collected and freeze-dried to obtain the final ICT@MSNs@HA.

[0117] Preparation Example 3-Preparation of BTO@MSNs@HA

[0118] (1) Synthesis of barium titanate

[0119] The steps are the same as those in Preparation Example 1.

[0120] (2) Synthesis of BTO@MSNs

[0121] The steps are the same as those in Preparation Example 1.

[0122] (3) Preparation of BTO@MSNs@HA

[0123] In a 50 mL round-bottom flask, 20 mL of isopropanol was used to disperse BTO@MSNs, and 0.5 mL of 3-aminopropyltriethoxysilane was added. Stir at room temperature for 4 h to achieve amination. Deionized water was used to centrifuge at 10,000 rpm for 5 min, washed three times to remove unreacted 3-aminopropyltriethoxysilane, and the precipitate was collected. A 1 wt% hyaluronic acid aqueous solution was prepared. The collected precipitate was dispersed with hyaluronic acid solution and stirred at room temperature for 6 h to achieve surface hyaluronic acid modification of BTO@MSNs. After the reaction was completed, deionized water was used again to centrifuge at 10,000 rpm for 5 min, washed three times to remove unreacted hyaluronic acid, and then the precipitate was collected. The collected precipitate was freeze-dried to obtain the final BTO@MSNs@HA.

[0124] Example 1

[0125] This example explores the targeted delivery effect of ICT-BTO@MSNs@HA on tumor cells in an in vitro experiment, using CT26 cells for the study and NCM460 cells as a control group. The method is as follows:

[0126] Fluorescence confocal microscopy was used to evaluate the in vitro targeted delivery function of ICT-BTO@MSNs@HA. CT26 and NCM460 cells were cultured at 5.0×10 4 The cells were seeded at a density of 100 cells / dish in a confocal dish, and an appropriate amount of RPMI-1640 medium was added to each dish. The cells were placed in a 5% CO 2 Content, cultured in a 37℃ constant temperature and humidity incubator with a relative humidity of 95% for 12 h. BTO@MSNs@HA loaded with Cy7 was co-incubated with CT26 and NCM460 cells according to a time gradient. After fixation with paraformaldehyde at room temperature, the membrane was broken with Triton X-100 at room temperature, blocked with 5% BSA at room temperature in the dark, and then a 0.5% actin solution dissolved in BSA was added. The cells were incubated in a light-proof shaking table, and an anti-fluorescence quencher (including DAPI) was added. The cells were stored in a light-proof environment at 4℃ and observed using a confocal microscope. The results are as follows Figure 6 As shown in the figure, after CT26 cells were co-cultured with Cy7-BTO@MSNs@HA, the increase in intracellular Cy7 fluorescence intensity was positively correlated with time, while the increase in NCM460 fluorescence intensity was relatively insignificant. The results showed that CT26 cells can effectively take up nanomedicines, proving that the latter has good tumor cell targeting.

[0127] Example 2

[0128] This example explores the targeting effect of ICT-BTO@MSNs@HA on tumors in vivo, and uses a BTO@MSNs@HA nano-delivery system loaded with ICG to simulate the distribution of ICT-BTO@MSNs@HA in animals, with ICG as the control group. The method is as follows:

[0129] Six female BALB / c mice were inoculated with 1×10 6 One week later, when the tumor volume was about 200 mm 3 The mice were randomly divided into ICG-BTO@MSNs@HA group and ICG group (3 mice in each group) and injected with 10 mg / kg ICG-BTO@MSNs@HA or ICG via the tail vein. In vivo fluorescence images were taken at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug injection. The mice were killed by cervical dislocation 24 h after injection, and the main organs and tumor tissues including heart, liver, spleen, lung, and kidney were removed and obtained. Fluorescence images of ex vivo organs and tumor tissues were taken. The results are shown in Figure 7 As shown, the nano-delivery system provided by the present invention soon showed obvious tumor enrichment after intravenous injection, and had a good retention effect, and among the major organs, only the liver had a slightly higher fluorescence intensity, which indicated that the nano-drug had a good tumor targeting effect and was metabolized by the liver. The long-term retention of the drug could effectively ensure the effect of ultrasound-induced drug excitation and the sustained apoptosis-promoting ability of icariin.

[0130] Example 3

[0131] This example explores the effects of various components of the nano-delivery system and ultrasound on CT26 cells, and uses the CCK-8 method to evaluate the killing effects of various components of the nano-delivery system and ultrasound on CT26 cells. The method is as follows:

[0132] Cells in logarithmic growth phase were cultured in 96-well plates with a cell density of 10,000 cells / well and 200 μL RPMI-1640 medium per well. The cells were placed in a 5% CO 2 Content, cultured in a constant temperature and humidity incubator at 37℃ with a relative humidity of 95% for 24 h. After 24 h, the cells were divided into groups according to Control, US, BTO@MSNs, ICT@MSNs@HA, ICT-BTO@MSNs@HA, BTO@MSNs@HA+US, and ICT-BTO@MSNs@HA+US, and the original culture medium was replaced with RPMI-1640 culture medium dissolved with different groups of drugs. The drug concentration was 100 μg / mL, and the total liquid volume was 200 μL. The Control group was replaced with 200 μL RPMI-1640 culture medium. After the cells were cultured for another 6 h, ultrasonic excitation was used according to the group, and the ultrasonic parameter was 1 w / cm 2 , 1.0 MHz, 50% duty cycle. 10 μL CCK8 solution was added to each well and the relative survival rate of cells was measured using an ELISA reader after 1 h of incubation. The test results are shown in Figure 8As shown in the figure (where G1, G2, G3, G4, G5, G6, and G7 correspond to Control (i.e., single PBS), US (ultrasound on the basis of Control), BTO@MSNs, ICT@MSNs@HA, ICT-BTO@MSNs@HA, BTO@MSNs@HA+US, and ICT-BTO@MSNs@HA+US), nanodelivery has a significant inhibitory ability on CT26 cells after ultrasound excitation, proving that the nanodelivery system has the potential to treat tumors. At the same time, ICT@MSNs@HA, ICT-BTO@MSNs@HA, BTO@MSNs From the comparison between @HA+US and ICT-BTO@MSNs@HA+US, it can be seen that whether it is single icariin loaded on a mesoporous silicon carrier, single barium titanate loaded on a mesoporous silicon carrier (using ultrasonic excitation at the same time), or icariin and barium titanate loaded on a mesoporous silicon carrier (without ultrasonic excitation), compared with icariin and barium titanate loaded on a mesoporous silicon carrier (using ultrasonic excitation at the same time), the inhibitory ability of the former three on tumor cells is poor. It can be seen that icariin and barium titanate using ultrasonic excitation have the ability to synergistically inhibit tumor cells, that is, the drug delivery system provided by the present invention needs to be under the action of ultrasound to achieve excellent inhibitory ability on tumor cells.

[0133] Example 4

[0134] This example investigates the tumor inhibition effect of ICT-BTO@MSNs@HA on CT26 colorectal tumors under ultrasound and the biosafety evaluation on mice. The method is as follows:

[0135] Thirty-five female BALB / c mice were subcutaneously inoculated with 1×10 6 CT26 mouse colorectal cancer cells / 100 μL, and wait until the average volume reaches 200 mm 3 Afterwards, they were randomly divided into Control, US, BTO@MSNs, ICT@MSNs@HA, ICT-BTO@MSNs@HA, BTO@MSNs@HA+US, and ICT-BTO@MSNs@HA+US groups, with 5 mice in each group. Drug administration began at the same time, with an injection every 3 days, for a total of 3 times. Ultrasound excitation was used according to the group 6 hours after drug administration. The body weight and tumor volume of the mice were recorded before each administration. After the treatment, the mouse eye blood was extracted to evaluate the impact of different group interventions on the biosafety of mice. Fig. 9As shown, after three treatments, the Control, US, and BTO@MSNs groups showed the highest tumor growth rates, followed by the ICT@MSNs@HA, ICT-BTO@MSNs@HA, and BTO@MSNs@HA+US groups. The growth rate of the tumor in the final group ICT-BTO@MSNs@HA+US was significantly inhibited after treatment, indicating that under ultrasound excitation, the nano delivery system can inhibit tumor growth and serve as a drug for tumor treatment. The weight of mice in the seven groups did not change significantly during the treatment period, and there were no significant differences in various serum functional indicators, indicating that the nano drug had no significant toxicity to mice and had good biosafety.

[0136] Example 5

[0137] This example explores the mechanism of ultrasound-mediated immunogenic cell death (ICD) effect of ICT-BTO@MSNs@HA on CT26 cells. Protein expression was analyzed by Western Blot technology to evaluate the ability of ICT-BTO@MSNs@HA to induce ICD effect in CT26 cells under in vitro ultrasound. The method is as follows:

[0138] Cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 200,000 cells per well, and 2 mL of RPMI-1640 medium was added. 2 , and cultured in a 37°C constant temperature and humidity incubator with a relative humidity of 95% for 12 h. After 24 h, according to the grouping of Control, US, BTO@MSNs, ICT@MSNs@HA, ICT-BTO@MSNs@HA, BTO@MSNs@HA+US, and ICT-BTO@MSNs@HA+US, the original culture medium was replaced with RPMI-1640 culture medium containing the corresponding drugs. The drug concentration was 100 μg / mL and the total volume was 2 mL. The Control group was replaced with only 2 mL of RPMI-1640 culture medium. After the cells were cultured for another 6 h, ultrasonic excitation was used according to the grouping, and the ultrasonic parameters were set to 1 w / cm 2 , frequency 1.0 MHz, duty cycle 50%. Cells were lysed using RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) to extract total cell protein. Protein concentration was determined by BCA method and the loading amount was calculated; loading buffer was added to the protein solution and boiled at 100°C for 10 min, the calculated protein solution and loading buffer were added to the electrophoresis gel for electrophoresis and transfer; then blocked with 5% milk for 2 h; incubated with the corresponding primary antibody at 4°C overnight, and incubated with the corresponding secondary antibody at room temperature for 2 h. After washing, color development was performed to observe the target band. The results are shown in Fig.10As shown in the figure, after ultrasonic excitation of ICT-BTO@MSNs@HA, the expression of intracellular HMGB1 was significantly decreased and the expression of CRT was significantly increased, confirming that ultrasonic excitation of ICT-BTO@MSNs@HA can effectively induce the ICD effect of CT26 cells and promote subsequent immune response.

[0139] Example 6

[0140] This example explores the mechanism by which ICT-BTO@MSNs@HA inhibits CT26 colorectal tumors under ultrasound. The ability of ICT-BTO@MSNs@HA to induce ICD effect on mouse CT26 subcutaneous tumor model under ultrasound was evaluated by extracting mature DC cells from mouse spleen, M1 macrophages and CD8+T cells from mouse tumors for flow cytometry analysis. The method is as follows:

[0141] According to the animal model construction method and grouping method described in Example 4, 35 female BALB / c mice were selected to construct a CT26 cell model. After the treatment, the mice were killed by cervical dislocation, and the spleen and tumor tissue of the mice were extracted by dissection and stored in serum-free RPMI-1640 at 4°C. For the extraction of mature DC cells in the spleen, the spleen was washed, ground, and filtered, and then resuspended with red blood cell lysis buffer. After the lysis, serum-free RPMI-1640 was added, and the supernatant was removed by centrifugation and resuspended with PBS. Flow cytometry antibodies were added for incubation. After the incubation was completed, PBS was added to stop the incubation, and the supernatant was removed by centrifugation and resuspended with PBS. Flow cytometry data was collected and analyzed. For the extraction of M1 macrophages and CD8+T cells in tumor tissues, the tumor tissues were washed, ground, and filtered, and then digested with collagenase and DNase to prepare single-cell suspensions. Flow cytometry antibodies were added for staining. After the incubation was completed, PBS was added to stop the incubation, and the supernatant was removed by centrifugation and resuspended with PBS. Flow cytometry data was collected and analyzed. The results are as follows. Fig.11 As shown in the figure, after being treated with different conditions, the immune cells extracted from the spleen and tumor tissue of mice were subjected to flow cytometry grouping and analysis. The results showed that after ultrasonic stimulation of ICT-BTO@MSNs@HA, the mature DC cells in the spleen tissue increased significantly, the number of M1 macrophages in the tumor tissue increased, and the content of CD8+T cells, GZMB and INF-γ increased, indicating that ultrasonic stimulation of ICT-BTO@MSNs@HA can stimulate the body's ICD effect, activate immune cells, promote DC cell maturation, upregulate the number of M1 macrophages, recruit CD8 + T cells gather in tumor tissues and secrete GZMB and INF-γ to kill tumor tissues.

[0142] In summary, the present invention provides a nano drug delivery system comprising mesoporous silicon loaded with active components and hyaluronic acid wrapped on the surface of the mesoporous silicon loaded with active components. The present invention uses mesoporous silicon as a drug delivery carrier, hyaluronic acid mediates tumor-specific targeting, and after loading barium titanate and anti-tumor drugs, ultrasound excites the drugs to achieve a tumor killing effect. Cell experiments and animal studies have shown that the nano drug delivery system can significantly inhibit tumor growth and improve the hypoxic microenvironment of tumors; mechanism studies have revealed that the nano drug delivery system causes an ICD effect, upregulates the number of M1 macrophages, and achieves anti-tumor immune activation.

[0143] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A drug delivery system, characterized in that It comprises mesoporous silicon loaded with active components and hyaluronic acid wrapped on the surface of the mesoporous silicon loaded with active components; The active components include barium titanate and anti-tumor drugs.

2. The drug delivery system according to claim 1, characterized in that The anti-tumor drug includes icariin.

3. The drug delivery system according to claim 1 or 2, characterized in that The molecular weight of the hyaluronic acid is 100,000 to 200,000 kDa.

4. The drug delivery system according to any one of claims 1 to 3, characterized in that The mass ratio of the mesoporous silica, barium titanate, anti-tumor drug and hyaluronic acid is (1-2):(1-10):(1-6):(0.01-0.6).

5. The drug delivery system according to any one of claims 1 to 4, characterized in that The drug delivery system has ultrasound responsiveness; The particle size of the drug delivery system is 100-500 nm, and the PDI is 0.08-0.09; The dosage form of the drug delivery system includes, but is not limited to, injection preparations.

6. A method for preparing a drug delivery system as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: S1: Under alkaline conditions, barium titanate, a surfactant and a silicon source are reacted to obtain mesoporous silicon loaded with barium titanate; S2: combining the mesoporous silicon loaded with barium titanate with the anti-tumor drug to obtain the mesoporous silicon loaded with barium titanate and the anti-tumor drug; S3: Mesoporous silica loaded with barium titanate and anti-tumor drugs is combined with hyaluronic acid to obtain a drug delivery system.

7. The preparation method according to claim 6, characterized in that: In step S2, the mesoporous silicon loaded with barium titanate is subjected to an amination treatment.

8. The preparation method according to claim 6 or 7, characterized in that: The surfactant includes hexadecyltrimethylammonium chloride and / or hexadecyltrimethylammonium bromide; The silicon source includes tetraethyl silicate and / or 3-aminopropyltriethoxysilane; The barium titanate is obtained by reacting a barium source and a titanium source, wherein the barium source includes barium nitrate and / or barium acetate, and the titanium source includes n-butyl titanate; The reaction temperature in step S1 is 50-80°C and the reaction time is 5-24 h; The binding in steps S2 and S3 is performed at room temperature, and the binding time is independently 1 to 10 h.

9. Use of the drug delivery system according to any one of claims 1 to 5 or the drug delivery system prepared by the preparation method according to any one of claims 6 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor includes, but is not limited to, colorectal cancer.

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