Enzyme-modified tetrasulfide bond-doped mesoporous silica nanoparticles and their preparation method and application

Abstract

The present invention discloses enzyme-modified tetrasulfide-doped mesoporous silica nanoparticles, their preparation method, and application. The preparation method of the enzyme-modified tetrasulfide-doped mesoporous silica nanoparticles comprises the following steps: 1) preparing tetrasulfide-doped silica; 2) preparing tetrasulfide-doped mesoporous silica S4MSN; 3) preparing GOx-surface-modified S4MSN; and 4) preparing L-Arg-loaded GOx-S4MSN. The enzyme-modified tetrasulfide-doped mesoporous silica nanoparticles prepared by the present invention have an average particle size of 20 to 50 nm, and the loading rates of GOx and L-Arg are 10 to 20% and 8 to 16%, respectively. The preparation method of the present invention is simple to operate, has good reproducibility, and has a short preparation cycle and low cost, making it easy to promote for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial preparation, and in particular relates to enzyme-modified-tetrasulfide bond-doped mesoporous silica nanoparticles and a preparation method and application thereof. Background Art

[0002] Currently, the main treatments for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy, photothermal therapy, and starvation therapy, each with its own advantages and disadvantages. For example, chemotherapy is a common treatment option with good efficacy for most cancers, but it can also easily develop drug resistance, cause systemic toxicity, and hinder drug penetration into deep tumor tissues. Therefore, we are committed to developing novel therapeutic approaches to address this current dilemma. Studies have shown that glutathione (GSH) concentrations are higher in cancer cells than in normal cells, and downregulating GSH levels can disrupt tumor redox homeostasis, induce the accumulation of reactive oxygen species (ROS), and ultimately induce ferroptosis. Therefore, depleting GSH is considered a potential and compelling cancer treatment. However, during GSH depletion, a replenishment mechanism exists within the tumor that can mitigate the rapid decrease in GSH levels, restoring the internal environment to homeostasis and thus reducing the effectiveness of tumor treatment. Fortunately, GSH depletion can be synergistic with various therapeutic approaches, such as starvation therapy, to achieve the desired therapeutic effect.

[0003] Starvation therapy is one of the common methods in cancer treatment. It inhibits tumor growth and survival by blocking blood flow or depleting its essential nutrients / oxygen supply. Among them, consuming nutrients (such as glucose) in tumors is one of the most common starvation therapies and is widely used. The most typical example is glucose oxidase (GOx). Studies have shown that GOx can effectively consume glucose, thereby blocking the energy supply in the tumor and achieving a "starvation" effect, and the H2O2 in the byproduct is a highly toxic ROS that can further inhibit tumor growth. This shows that GOx, while serving as a starvation therapy supply agent, can also effectively promote the consumption of GSH, playing a synergistic therapeutic role.

[0004] In addition, in order to further improve the inhibition rate of tumor cells, we have introduced a "green" treatment method - gas therapy. At appropriate concentrations, gas molecules such as NO, CO, H2S, SO2, and H2 can significantly inhibit the survival of cancer cells without adversely affecting normal cells. Among them, NO has received increasing attention because of its anti-tumor effects through multiple pathways such as mitochondrial / DNA damage and the formation of highly cytotoxic peroxynitrite anions (ONOO-). In addition, a non-toxic and high-yield NO donor - L-arginine (L-Arg) has been proven by many experiments to be effective in cancer treatment. It is worth noting that the release of NO by L-Arg only has H + This is related to H2O2, which is in excess supply within tumor cells, and the byproduct of GOx consumption of glucose can also meet its release conditions.

[0005] The above shows that promoting GSH depletion and synergizing starvation therapy with NO gas therapy is a novel and feasible cancer therapy with great research significance in the field of tumor treatment and presents a promising paradigm. Therefore, it is feasible to develop a drug delivery system that can possess the above properties.

[0006] Nowadays, nano drug carriers have great application prospects in cancer treatment. They can effectively solve problems such as low drug bioavailability and solve many difficult problems in cancer treatment. Among them, silica has high stability, chemical versatility and biocompatibility, and can store and gradually release therapeutic drugs. Therefore, it is widely used in the field of drug sustained release. In addition, silica carriers can also be combined with different new cancer treatment methods to achieve synergistic treatment, improve treatment effects, and reduce toxic side effects. Therefore, designing a nano drug delivery system with silica as the main body to achieve synergistic treatment effects is of practical significance. Summary of the Invention

[0007] The purpose of the present invention is to provide an enzyme-modified tetrasulfide bond-doped mesoporous silica nanoparticle that can achieve GSH consumption to promote cell ferroptosis, synergize with starvation therapy and gas therapy, and can be quickly cleared in the body, as well as a preparation method and application thereof.

[0008] This invention leverages the GSH-depleting function of tetrasulfide bonds. Tetrasulfide-doped mesoporous silica (S4MSN) is used as the main component, with glucose oxidase (GOx) modified on the surface to achieve starvation therapy. L-arginine (L-Arg) is also loaded as a NO donor. The resulting nanoparticles, GOx-S4MSN@L-Arg, have GOx and L-Arg loading ratios of 10-20% and 8-16%, respectively.

[0009] The enzyme-modified tetrasulfide-doped mesoporous silica nanoparticles GOx-S4MSN@L-Arg of the present invention are composed of tetrasulfide-doped mesoporous silica S4MSN nanomaterials, GOx and L-Arg. The GOx is loaded on the surface of the S4MSN nanomaterial, and the L-Arg is loaded in the pores of the S4MSN nanomaterial.

[0010] Preferably, the specific surface area of ​​the S4MSN nanomaterial is 200 to 500 m 2 / g, pore volume is 0.5~0.8cm 3 / g, pore size is 2~5nm.

[0011] Preferably, the average particle size of the GOx-S4MSN@L-Arg is 20 to 50 nm.

[0012] The preparation method of the enzyme-modified and tetrasulfide-doped mesoporous silica nanoparticles GOx-S4MSN@L-Arg of the present invention comprises the following steps:

[0013] 1) Preparation of tetrasulfide-doped silica: CTAB and bis-[γ-(triethoxysilyl)propyl]-tetrasulfide (TESPTS) were added to solvent 1 and stirred until the CTAB was dissolved and the TESPTS was evenly dispersed. The dissolved CTAB solution and the evenly dispersed TESPTS were then added to solvent 2 and the reaction was continued with stirring. After the reaction was completed, tetrasulfide-doped silica was obtained.

[0014] 2) Preparation of tetrasulfide bond-doped mesoporous silica S4MSN: The tetrasulfide bond-doped silica material was dispersed in solvent 3, and the CTAB was removed by stirring to obtain S4MSN nanomaterials;

[0015] 3) Preparation of GOx-surface-modified S4MSN: S4MSN nanomaterials were dispersed in a toluene solution, and 3-aminopropyltriethoxysilane (APTES) was added. The mixture was heated and stirred to react, yielding amino-modified S4MSN (NH2-S4MSN). The carboxyl groups on GOx were activated and added to deionized water containing the NH2-S4MSN. The mixture was stirred to react, yielding GOx-surface-modified S4MSN (GOx-S4MSN).

[0016] 4) Preparation of L-Arg-loaded GOx-S4MSN: GOx-S4MSN was dispersed in deionized water containing L-Arg and stirred overnight at room temperature. After stirring, the mixture was washed and vacuum-dried to obtain L-Arg-loaded GOx-S4MSN (GOx-S4MSN@L-Arg).

[0017] Preferably, in step 1), solvent 1 is a mixed solvent consisting of deionized water and ethanol, wherein the volume ratio of deionized water to ethanol is (20-40):(10-20); the stirring temperature is 25-80°C, and the stirring time is 20-50 min; solvent 2 is a mixed solvent consisting of deionized water, ethanol and ammonia water, wherein the volume ratio of deionized water, ethanol and ammonia water is (200-400):(50-150):(3-9), and the concentration of ammonia water is 28%; the mass volume ratio of CTAB to deionized water in the total mixed solution is (1.2-2.4):(220-440) g / mL; the mass volume ratio of CTAB to TESPTS is (1.2-2.4):(5-10) g / mL; the stirring reaction temperature is continued at 25-80°C and the time is 12-30 h.

[0018] Preferably, in step 2), solvent 3 is a mixed solvent consisting of ethanol and hydrochloric acid, wherein the volume ratio of ethanol to hydrochloric acid is (80-120):(10-15), and the concentration of hydrochloric acid is 37.5%; the mass volume ratio of tetrasulfide bond-doped silica material to solvent 3 is (300-500):(90-135) mg / mL; the stirring reaction temperature is 60-80°C, and the stirring reaction time is 12-36 h.

[0019] Preferably, in the step 3), the mass volume ratio of S4MSN to 3-aminopropyltriethoxysilane (APTES) is (200-300):(0.75-1.25) mg / mL; the mass volume ratio of S4MSN to toluene is (200-300):(40-60) mg / mL; the stirring reaction temperature is 90-110°C, and the stirring reaction time is 12-36h.

[0020] Preferably, in step 3), GOx, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are dissolved in deionized water and stirred to activate the carboxyl groups on GOx; during the activation of the carboxyl groups, the mass volume ratio of GOx to deionized water is (5-20):(5-10) mg / mL; the mass ratio of GOx, EDC and NHS is (5-20):(20-30):(10-20).

[0021] Preferably, in step 3), the mass volume ratio of NH2-S4MSN to deionized water is (20-40):(10-20) mg / mL; the mass ratio of NH2-S4MSN to GOx is (20-40):(5-20); the stirring reaction temperature is 20-30°C, and the stirring reaction time is 12-36h.

[0022] Preferably, in step 4), the mass ratio of GOx-S4MSN to L-Arg is (20-40):(20-40); the mass volume ratio of GOx-S4MSN to deionized water is (20-40):(5-20) mg / mL; the stirring reaction temperature is 20-30°C, and the stirring reaction time is 12-36 h.

[0023] The GOx-S4MSN@L-Arg nanomaterial is used as a nano drug carrier.

[0024] Principle of the Invention: The present invention provides a tetrasulfide-doped mesoporous silica nanomaterial. By adding a silicon source, TESPTS, to the self-assembly process of CTAB to form a micelle-like structure, the TESPTS undergoes hydrolysis and polycondensation under reaction conditions and deposits on the surface of the CTAB micelle-like structure, resulting in a CTAB micelle structure coated with a tetrasulfide-doped silica layer. The CTAB micelle structure with the tetrasulfide-doped silica layer is then dispersed in a mixed solvent of ethanol and hydrochloric acid, and the CTAB is removed by ion exchange extraction, thereby obtaining the S4MSN nanomaterial. Furthermore, amino groups are added to the surface of the S4MSN to achieve amination, and GOx is surface-modified via an amide reaction. Finally, L-Arg is loaded into the pores of the tetrasulfide-doped mesoporous silica by charge adsorption, resulting in the final product, GOx-S4MSN@L-Arg.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides an intelligent nanosystem GOx-S4MSN@L-Arg, which has the characteristics of GSH consumption, starvation therapy and NO generation for multimodal cancer treatment. In this cascade process, the tetrasulfide bonds in the nanosystem can preferentially consume GSH, thereby increasing the cellular ROS concentration, while triggering particle biodegradation and exposing L-Arg. In addition, GOx consumes glucose to initiate starvation therapy and produces a large amount of H2O2. Importantly, a large amount of H2O2 can not only stimulate L-Arg to release NO to achieve gas therapy, but also further increase the level of ROS in the cell and promote cell ferroptosis. In in vitro treatment, the huge synergistic effect of ferroptosis / starvation / gas therapy of this nanosystem can significantly kill cancer cells and significantly inhibit tumor growth without obvious side effects.

[0027] The present invention provides a preparation process of an intelligent nanosystem GOx-S4MSN@L-Arg nanomaterial, which has simple operation, good repeatability, short preparation cycle, low cost, and is easy to promote for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the TEM image of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1.

[0029] Figure 2 This is the nitrogen adsorption-desorption curve of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1.

[0030] Figure 3 This is the Raman spectrum of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1.

[0031] Figure 4 This is the infrared-visible spectrum of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1.

[0032] Figure 5 This is the thermogravimetric analysis diagram of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1.

[0033] Figure 6 This is the UV-visible spectrum of GSH after the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 consumes GSH under a pH 4.5 environment.

[0034] Figure 7 This is a curve of the H2O2 release ability of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 in a glucose solution with a pH of 4.5.

[0035] Figure 8 This is a graph showing the NO release capacity of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 in a glucose solution at pH 4.5.

[0036] Figure 9 This is a diagram of the therapeutic evaluation of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 on 4T1 tumor cells.

[0037] Figure 10 This is a diagram of the tumor tissue accumulation of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 after intravenous injection into tumor-bearing mice.

[0038] Figure 11 This is the excretion amount of the smart nanosystem GOx-S4MSN@L-Arg nanomaterial prepared in Example 1 after intravenous injection into rats through urine and feces. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0040] In the following examples, the concentration of the aqueous ammonia used was 28%; the concentration of the hydrochloric acid was 37.5%.

[0041] Example 1

[0042] In this embodiment, a preparation process of a smart nanosystem GOx-S4MSN@L-Arg nanomaterial includes:

[0043] 1.2 g of CTAB and 5 mL of TESPTS were added to a mixed solvent consisting of 20 mL of deionized water and 10 mL of ethanol, stirred for 30 min until the CTAB was completely dissolved and the TESPTS was evenly dispersed. The mixture was then added to a mixed solution consisting of 300 mL of deionized water, 100 mL of ethanol, and 6 mL of ammonia water, and stirred in a 30°C water bath for 24 h. After the reaction was completed, the product was filtered and washed three times with deionized water and ethanol, then dried in a 60°C oven. Finally, fine and uniform particles were obtained by grinding, namely tetrasulfide-doped silica nanoparticles. 500 mg of tetrasulfide-doped silica was placed in 135 mL of a mixed solvent consisting of 90% ethanol and 10% hydrochloric acid, refluxed at 70°C for 24 h, and the supernatant was removed by centrifugation. The solution was washed several times with ethanol and deionized water, and vacuum-dried to obtain tetrasulfide-doped mesoporous silica (S4MSN) nanomaterials. 250 mg of S4MSN was dispersed in 40 mL of toluene, and 1 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was stirred in an oil bath at 110°C for 24 h, the supernatant was removed by centrifugation, and the mixture was washed three times with ethanol and dried in vacuum to obtain amino-modified S4MSN (NH2-S4MSN). 10 mg of GOx, 24 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 18 mg of N-hydroxysuccinimide (NHS) were added to 5 mL of deionized water and stirred at room temperature for 30 min to fully activate the carboxyl groups on GOx. 10 mL of deionized water and 20 mg of NH2-S4MSN were then added. The mixture was stirred in a water bath at 30°C for 24 h. After the reaction was completed, the mixture was washed with water and ethanol several times and dried in vacuum to obtain pure GOx-modified S4MSN (GOx-S4MSN). 20 mg of GOx-S4MSN was dispersed in 10 mL of deionized water containing 20 mg of L-Arg, and then stirred in a 30°C water bath for 24 h. The mixture was then washed with water and alcohol several times and dried in vacuum to obtain GOx-S4MSN loaded with L-Arg (GOx-S4MSN@L-Arg).

[0044] In the smart nanosystem GOx-S4MSN@L-Arg nanomaterial in this embodiment, the mass ratio of added S4MSN, GOx and L-Arg is 20:10:20.

[0045] The volume ratio of deionized water, ethanol, and ammonia water in this embodiment is 320:110:6.

[0046] The mass-to-volume ratio of CTAB to TESPTS in this example is 1.2:5 g / mL.

[0047] The mass volume ratio of S4MSN to 3-aminopropyltriethoxysilane in this embodiment is 250:1 mg / mL.

[0048] The stirring reaction temperature of the tetrasulfide bond-doped silica in this embodiment is 30° C., and the reaction time is 24 h.

[0049] Example 2

[0050] In this embodiment, a preparation process of a smart nanosystem GOx-S4MSN@L-Arg nanomaterial includes:

[0051] 1.6 g of CTAB and 5 mL of TESPTS were added to a mixed solvent consisting of 20 mL of deionized water and 10 mL of ethanol, stirred for 30 minutes until the CTAB was completely dissolved and the TESPTS was evenly dispersed. The above solution was then added to a mixed solution consisting of 300 mL of deionized water, 100 mL of ethanol, and 9 mL of ammonia water, and stirred in a 30°C water bath for 20 hours. After the reaction was completed, the product was filtered and washed three times with deionized water and ethanol, then dried in a 60°C oven. Finally, fine and uniform particles were obtained by grinding, namely tetrasulfide-doped silica nanoparticles. 500 mg of tetrasulfide-doped silica was placed in 135 mL of a mixed solvent consisting of 90% ethanol and 10% hydrochloric acid, refluxed at 70°C for 24 hours, and the supernatant was removed by centrifugation. The solution was washed several times with ethanol and deionized water, and vacuum-dried to obtain tetrasulfide-doped mesoporous silica (S4MSN) nanomaterials. 250 mg of S4MSN was dispersed in 40 mL of toluene, and 1.2 mL of APTES was added. The mixture was stirred in an oil bath at 110 °C for 24 h, the supernatant was removed by centrifugation, and the mixture was washed three times with ethanol and dried in vacuum to obtain amino-modified S4MSN (NH2-S4MSN). 15 mg of GOx, 24 mg of EDC, and 18 mg of NHS were added to 5 mL of deionized water and stirred at room temperature for 30 min to fully activate the carboxyl groups. Then 10 mL of deionized water was added, and 20 mg of NH2-S4MSN was added. The mixture was stirred in a water bath at 30 °C for 24 h. After the reaction was completed, the mixture was washed with water and ethanol several times and dried in vacuum to obtain pure GOx-modified S4MSN (GOx-S4MSN). 20 mg of GOx-S4MSN was dispersed in 10 mL of deionized water containing 30 mg of L-Arg, and then stirred in a 30°C water bath for 24 h. The mixture was then washed with water and alcohol several times and dried in vacuum to obtain GOx-S4MSN loaded with L-Arg (GOx-S4MSN@L-Arg).

[0052] In the smart nanosystem GOx-S4MSN@L-Arg nanomaterial in this embodiment, the mass ratio of added S4MSN, GOx and L-Arg is 20:15:30.

[0053] The volume ratio of deionized water, ethanol, and ammonia water in this embodiment is 320:110:9.

[0054] The mass volume ratio of CTAB to TESPTS in this example is 1.6:5 g / mL.

[0055] The mass volume ratio of S4MSN to APTES in this example is 250:1.2 mg / mL.

[0056] The stirring reaction temperature of the tetrasulfide bond-doped silica in this embodiment is 30° C., and the reaction time is 20 h.

[0057] Example 3

[0058] In this embodiment, a preparation process of a smart nanosystem GOx-S4MSN@L-Arg nanomaterial includes:

[0059] 1.6 g of CTAB and 8 mL of TESPTS were added to a mixed solvent consisting of 20 mL of deionized water and 10 mL of ethanol and stirred for 30 minutes until the CTAB was completely dissolved and the TESPTS was evenly dispersed. The above solution was then added to a mixed solution consisting of 350 mL of deionized water, 100 mL of ethanol, and 9 mL of ammonia water and stirred in a 30°C water bath for 20 hours. After the reaction was completed, the product was filtered and washed three times with deionized water and ethanol, then dried in a 60°C oven. Finally, fine and uniform particles were obtained by grinding, namely tetrasulfide-doped silica nanoparticles. 500 mg of tetrasulfide-doped silica was placed in 135 mL of a mixed solvent consisting of 90% ethanol and 10% hydrochloric acid, refluxed at 70°C for 24 hours, and the supernatant was removed by centrifugation. The solution was washed several times with ethanol and deionized water, and vacuum-dried to obtain tetrasulfide-doped mesoporous silica (S4MSN) nanomaterials. 300 mg of S4MSN was dispersed in 40 mL of toluene, and 1.2 mL of APTES was added. The mixture was stirred in an oil bath at 110 °C for 24 h, the supernatant was removed by centrifugation, and the mixture was washed three times with ethanol and dried in vacuum to obtain amino-modified S4MSN (NH2-S4MSN). 15 mg of GOx, 24 mg of EDC, and 18 mg of NHS were added to 5 mL of deionized water and stirred at room temperature for 30 min to fully activate the carboxyl groups. Then, 10 mL of deionized water was added, and 30 mg of NH2-S4MSN was added. The mixture was stirred in a water bath at 30 °C for 24 h. After the reaction was completed, the mixture was washed with water and ethanol several times and dried in vacuum to obtain pure GOx-modified S4MSN (GOx-S4MSN). 30 mg of GOx-S4MSN was dispersed in 10 mL of deionized water containing 30 mg of L-Arg, and then stirred in a 30°C water bath for 24 h. The mixture was then washed with water and alcohol several times and dried in vacuum to obtain GOx-S4MSN loaded with L-Arg (GOx-S4MSN@L-Arg).

[0060] In the smart nanosystem GOx-S4MSN@L-Arg nanomaterial in this embodiment, the mass ratio of added S4MSN, GOx and L-Arg is 30:15:30.

[0061] The volume ratio of deionized water, ethanol, and ammonia water in this embodiment is 370:110:9.

[0062] The mass volume ratio of CTAB to TESPTS in this embodiment is 1.6:8 g / mL.

[0063] The mass volume ratio of S4MSN to APTES in this example is 300:1.2 mg / mL.

[0064] The stirring reaction temperature of the tetrasulfide bond-doped silica in this embodiment is 30° C., and the reaction time is 20 h.

[0065] Example 4

[0066] In this embodiment, a preparation process of a smart nanosystem GOx-S4MSN@L-Arg nanomaterial includes:

[0067] 2.4 g of CTAB and 10 mL of TESPTS were added to a mixed solvent consisting of 20 mL of deionized water and 10 mL of ethanol, stirred for 30 minutes until the CTAB was completely dissolved and the TESPTS was evenly dispersed. The above solution was then added to a mixed solution consisting of 350 mL of deionized water, 150 mL of ethanol, and 9 mL of ammonia water, and stirred in a 30°C water bath for 30 hours. After the reaction was completed, the product was filtered and washed three times with deionized water and ethanol, then dried in a 60°C oven. Finally, fine and uniform particles were obtained by grinding, namely tetrasulfide-doped silica nanoparticles. 500 mg of tetrasulfide-doped silica was placed in 135 mL of a mixed solvent consisting of 90% ethanol and 10% hydrochloric acid, refluxed at 70°C for 24 hours, and the supernatant was removed by centrifugation. The solution was washed several times with ethanol and deionized water, and vacuum-dried to obtain tetrasulfide-doped mesoporous silica (S4MSN) nanomaterials. 400 mg of S4MSN was dispersed in 40 mL of toluene, and 1.2 mL of APTES was added. The mixture was stirred in an oil bath at 110 °C for 24 h, the supernatant was removed by centrifugation, and the mixture was washed three times with ethanol and dried in vacuum to obtain amino-modified S4MSN (NH2-S4MSN). 20 mg of GOx, 24 mg of EDC, and 18 mg of NHS were added to 5 mL of deionized water and stirred at room temperature for 30 min to fully activate the carboxyl groups. Then, 10 mL of deionized water was added, and 40 mg of NH2-S4MSN was added. The mixture was stirred in a water bath at 30 °C for 24 h. After the reaction was completed, the mixture was washed with water and ethanol several times and dried in vacuum to obtain pure GOx-modified S4MSN (GOx-S4MSN). 40 mg of GOx-S4MSN was dispersed in 10 mL of deionized water containing 40 mg of L-Arg, and then stirred in a 30 °C water bath for 24 h. The mixture was then washed with water and alcohol several times and dried in vacuum to obtain GOx-S4MSN loaded with L-Arg (GOx-S4MSN@L-Arg).

[0068] In the smart nanosystem GOx-S4MSN@L-Arg nanomaterial in this embodiment, the mass ratio of added S4MSN, GOx and L-Arg is 40:20:40.

[0069] The volume ratio of deionized water, ethanol, and ammonia water in this embodiment is 370:160:9.

[0070] The mass volume ratio of CTAB to TESPTS in this example is 2.4:10 g / mL.

[0071] The mass volume ratio of S4MSN to APTES in this example is 400:1.2 mg / mL.

[0072] The stirring reaction temperature of the tetrasulfide bond-doped silica in this embodiment is 30° C., and the reaction time is 30 h.

[0073] Example 5 Characterization and Analysis

[0074] Morphology, structural characterization and performance analysis of GOx-S4MSN@L-Arg nanomaterials of intelligent nanosystem:

[0075] Figure 1 This is the TEM image of the GOx-S4MSN@L-Arg nanomaterial in Example 1. It can be seen from the TEM image that the morphology of the nanomaterial is spherical, the particles are uniform, and the particle size is about 30 nm.

[0076] Figure 2 The nitrogen adsorption-desorption curve of the S4MSN nanomaterial in Example 1 has a specific surface area of ​​211 m 2 / g, pore volume is 0.77cm 3 / g, and the pore size is 2.98nm.

[0077] Figure 3 The Raman spectrum of the S4MSN nanomaterial in Example 1 shows that compared with MSN, the SS bonds in S4MSN are at 438 cm -1 and 488cm -1 , CS key at 638cm -1 The strong stretching vibration of can clearly indicate the successful doping of tetrasulfide bonds.

[0078] Figure 4 This is the infrared visible spectrum of the GOx-S4MSN@L-Arg nanomaterial in Example 1. The figure shows the infrared visible spectrum at 1654 cm -1 The strong stretching vibration at 1420 cm indicates the successful conjugation of GOx; and -1 At , the characteristic peak of L-Arg also has obvious stretching vibration, indicating that L-Arg is also successfully loaded.

[0079] Figure 5 This is the thermogravimetric analysis diagram of the GOx-S4MSN@L-Arg nanomaterial in Example 1. It can be seen from the figure that the loading rates of GOx and L-Arg are 10.48% and 10.21%, respectively.

[0080] Figure 6 This is the UV-visible spectrum of GSH after the GOx-S4MSN@L-Arg nanomaterial consumes GSH at pH 4.5 in Example 1. The decrease in the absorption peak in the figure indicates a decrease in GSH concentration and demonstrates the GSH consumption ability of the GOx-S4MSN@L-Arg nanomaterial.

[0081] Figure 7 This graph shows the H₂O₂ release capacity of the GOx-S₄MSN@L-Arg nanomaterial from Example 1 in a pH 4.5 glucose solution. The graph demonstrates that GOx-S₄MSN@L-Arg rapidly degrades glucose and releases a significant amount of H₂O₂. Notably, the glucose decomposition activity of GOx-S₄MSN@L-Arg did not decrease significantly.

[0082] Figure 8 Figure 1 shows the NO release capacity of the GOx-S4MSN@L-Arg nanomaterial in a pH 4.5 glucose solution. The figure shows that GOx-S4MSN@L-Arg can utilize H2O2 released by glucose decomposition to catalyze the release of NO from L-Arg, achieving a cascade effect.

[0083] Figure 9 This is a diagram showing the therapeutic evaluation of GOx-S4MSN@L-Arg nanomaterials on 4T1 tumor cells in Example 1. The figure shows that GOx-S4MSN@L-Arg nanomaterials can deliver drugs into tumor cells and exert a killing effect.

[0084] Figure 10 This is a diagram showing the accumulation of GOx-S4MSN@L-Arg nanomaterials prepared in Example 1 in tumor tissue after intravenous injection into tumor-bearing mice. Figure 10 As shown, GOx-S4MSN@L-Arg nanomaterials injected into tumor-bearing mice via the tail vein quickly accumulated in tumor tissue, reaching a maximum accumulation value within 24 hours. After 48 hours, the accumulation value decreased significantly, and the accumulation value in organs also decreased significantly, indicating that GOx-S4MSN@L-Arg has excellent biodegradability in mice.

[0085] Figure 11The amount of GOx-S4MSN@L-Arg nanomaterials prepared in Example 1 excreted through urine and feces after intravenous injection into rats. Figure 11 It can be seen that 72 hours after intravenous injection, about 80% of the particles can be excreted from the body through feces and urine. This shows that the mesoporous silica-lipid composite nanomaterial has a good in vivo clearance rate, which can effectively reduce the biological toxicity caused by particle retention.

[0086] The above-described embodiments merely represent four implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An enzyme-modified tetrasulfide bond-doped mesoporous silica nanoparticle GOx-S4MSN@L-Arg, characterized in that: The GOx-S4MSN@L-Arg is composed of tetrasulfide bond-doped mesoporous silica S4MSN nanomaterial, GOx and L-Arg. The GOx is loaded on the surface of the S4MSN nanomaterial, and the L-Arg is loaded in the pores of the S4MSN nanomaterial.

2. The GOx-S4MSN@L-Arg according to claim 1, characterized in that The specific surface area of ​​the S4MSN nanomaterial is 200~500 m 2 / g, pore volume of 0.5~0.8 cm 3 / g, with a pore size of 2~5 nm; the average particle size of the GOx-S4MSN@L-Arg is 20~50 nm.

3. A method for preparing GOx-S4MSN@L-Arg as claimed in claim 1, comprising the following steps: 1) Preparation of tetrasulfide-doped silica: CTAB and bis-[γ-(triethoxysilyl)propyl]-tetrasulfide (TESPTS) are added to solvent 1 and stirred until the CTAB is dissolved and the TESPTS is evenly dispersed. The dissolved CTAB solution and the evenly dispersed TESPTS are then added to solvent 2 and the reaction is continued with stirring. After the reaction is completed, tetrasulfide-doped silica is obtained. 2) Preparation of tetrasulfide bond-doped mesoporous silica S4MSN: Tetrasulfide bond-doped silica material was dispersed in solvent 3 and stirred to remove CTAB to obtain S4MSN nanomaterials; 3) Preparation of GOx-surface-modified S4MSN: S4MSN nanomaterials were dispersed in a toluene solution, and 3-aminopropyltriethoxysilane (APTES) was added. After heating and stirring, the reaction was completed to obtain amino-modified S4MSN, namely NH2-S4MSN. The carboxyl groups on GOx were activated and added to deionized water containing NH2-S4MSN. The reaction was stirred and completed to obtain GOx-surface-modified S4MSN (GOx-S4MSN). 4) Preparation of L-Arg-loaded GOx-S4MSN: GOx-S4MSN was dispersed in deionized water containing L-Arg and stirred overnight at room temperature. After stirring, the mixture was washed and vacuum-dried to obtain L-Arg-loaded GOx-S4MSN (GOx-S4MSN@L-Arg).

4. The preparation method according to claim 3, characterized in that In the step 1), solvent 1 is a mixed solvent consisting of deionized water and ethanol, wherein the volume ratio of deionized water to ethanol is (20-40):(10-20); the stirring temperature is 25-80°C, and the stirring time is 20-50 min; solvent 2 is a mixed solvent consisting of deionized water, ethanol, and ammonia water, wherein the volume ratio of deionized water, ethanol, and ammonia water is (200-400):(50-150):(3-9), and the concentration of ammonia water is 28%; the mass volume ratio of CTAB to deionized water in the total mixed solution is (1.2-2.4):(220-440) g / mL; the mass volume ratio of CTAB to TESPTS is (1.2-2.4):(5-10) g / mL; the stirring reaction temperature is continued at 25-80°C, and the time is 12-30 h.

5. The preparation method according to claim 3, characterized in that In step 2), solvent 3 is a mixed solvent consisting of ethanol and hydrochloric acid, wherein the volume ratio of ethanol to hydrochloric acid is (80-120):(10-15), and the concentration of hydrochloric acid is 37.5%; the mass volume ratio of tetrasulfide bond-doped silica material to solvent 3 is (300-500):(90-135) mg / mL; the stirring reaction temperature is 60-80°C, and the stirring reaction time is 12-36 h.

6. The preparation method according to claim 3, characterized in that In step 3), the mass volume ratio of S4MSN to 3-aminopropyltriethoxysilane (APTES) is (200-300): (0.75-1.25) mg / mL; the mass volume ratio of S4MSN to toluene is (200-300): (40-60) mg / mL; the stirring reaction temperature is 90-110°C, and the stirring reaction time is 12-36 h.

7. The preparation method according to claim 3, characterized in that In step 3), GOx, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) are dissolved in deionized water and stirred to activate the carboxyl groups on GOx. During the activation of the carboxyl groups, the mass-to-volume ratio of GOx to deionized water is (5-20):(5-10) mg / mL; and the mass ratio of GOx, EDC, and NHS is (5-20):(20-30):(10-20).

8. The preparation method according to claim 3, characterized in that In step 3), the mass volume ratio of NH2-S4MSN to deionized water is (20-40): (10-20) mg / mL; the mass ratio of NH2-S4MSN to GOx is (20-40): (5-20); the stirring reaction temperature is 20-30°C, and the stirring reaction time is 12-36 h.

9. The preparation method according to claim 3, characterized in that In step 4), the mass ratio of GOx-S4MSN to L-Arg is (20-40):(20-40); the mass volume ratio of GOx-S4MSN to deionized water is (20-40):(5-20) mg / mL; the stirring reaction temperature is 20-30°C, and the stirring reaction time is 12-36 h.

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