CN114949186A - POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, preparation method and application - Google Patents

POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, preparation method and application Download PDF

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CN114949186A
CN114949186A CN202210324561.1A CN202210324561A CN114949186A CN 114949186 A CN114949186 A CN 114949186A CN 202210324561 A CN202210324561 A CN 202210324561A CN 114949186 A CN114949186 A CN 114949186A
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mesoporous silicon
heme
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hemin
msn
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张俊
蒋大振
吕梦
曹振
谢丛华
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Zhongnan Hospital of Wuhan University
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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/02—Silicon
    • C01B33/021—Preparation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

Abstract

The invention discloses a POD-GOD synergistically modified mesoporous silicon nanomaterial, a preparation method and application thereof. The nano-particles improve the content of active oxide in tumor cells based on the tumor microenvironment, increase the Reactive Oxide (ROS) through the cascade of chemical reactions, overcome the hypoxic condition in the tumor cells, increase the sensitivity of radiotherapy, improve the killing rate of the tumor cells, and provide a new idea for the treatment of tumors.

Description

POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, preparation method and application
Technical Field
The invention belongs to the field of medical materials, and relates to a preparation method of a mesoporous silicon nano-carrier, in particular to a POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nano-material, a preparation method and application thereof.
Background
Compared with the traditional enzyme, the nano enzyme has the characteristics of mimic enzyme, and shows huge potential in clinical diagnosis, biological analysis, biosensors and disease treatment due to the easy synthesis, adjustable catalytic activity and high stability. Reactive Oxygen Species (ROS) including
Figure BDA0003571312760000011
OH, OOH, free radicals and the like are involved in the cellular metabolic process. The nanoenzyme can regulate the intracellular ROS level, and provides possibility for mediating the ROS to achieve the expected treatment effect. Tumor Microenvironment (TME) has mild acidity and hydrogen peroxide (H) 2 O 2 ) Overexpression and the like. Therefore, on the one hand, because TME is highly complex, catalytic efficiency of nanoenzymes is insufficient, and it is difficult to obtain satisfactory therapeutic effects. On the other hand, response H 2 O 2 The nano enzyme can well utilize the characteristics to realize specific tumor treatment. These nanoenzymes react H under acidic conditions by mimicking Peroxidase (POD) 2 O 2 Decomposed into ROS. However, H in tumor cells 2 O 2 The amount of ROS is still insufficient to produce the desired therapeutic effect. Therefore, both ROS production and H production are considered in tumor therapy 2 O 2 Is generated.
Radiotherapy, one of the most widespread and effective methods for the clinical treatment of malignant tumors, is still facingSome problems. During radiotherapy, tumor cells undergo apoptosis by inducing both direct and indirect damage to DNA, where the indirect damage is the generation of ROS by radiation during radiotherapy. Due to the dose limitation of normal tissues, the radiation dose cannot be provided to the tumor region enough, and the effect of radiotherapy is seriously influenced. How to improve the sensitivity of the tumor to radiotherapy is always a prominent problem in clinic. Therefore, increasing the energy deposition of radiation ionization to tumor tissue is imminent. In recent years, the nano radiosensitizer with remarkable physicochemical properties plays an increasingly important role in tumor radiotherapy in enhancing the killing effect of radiation on tumor cells. Provides important opportunity for tumor radiotherapy sensitization. The radiosensitizer with specific targeting ability can not only accumulate in tumor cells, but also avoid side effects on normal tissue cells. Some nano radiation sensitizers use high atomic number (Z) elements to enhance the energy deposition of tumor tissue and increase the photoelectric effect of the radiation. Hypoxia is a distinct feature of TME that impedes the therapeutic effects of radiation therapy, thereby creating radiation resistance. Thus, another class of radiosensitizers focuses on the DNA free radical that modifies TME to stabilize it. For example, H overexpressed in the tumor region 2 O 2 Can be MnO of 2 The nano enzyme radiation sensitizer catalyzes to generate oxygen. In addition, radiation therapy is often combined with other therapies such as photothermal therapy, photodynamic therapy, sonodynamic therapy to achieve significant therapeutic effects. These radiosensitizers directly or indirectly promote the production of ROS and induce apoptosis of tumor cells, thereby improving the radiotherapy performance.
During radiotherapy, ROS production indirectly induces apoptosis. However, ROS can be cleared by tumor cells, resulting in radiation tolerance and reduced effectiveness of radiation therapy. Therefore, due to the self-repair of tumor cells, the amount of ROS is insufficient, and more ROS are needed to enhance the effect of radiotherapy. Here, the present invention designs a biodegradable nano hybrid, which enhances radiosensitization by fixing Glucose Oxidase (GOD) and heme (Hemin) on the surface of Mesoporous Silicon Nanosphere (MSN), the principle is shown in (fig. 1). Initially, the nanohybrids were delivered to the tumor site by enhancing the permeability and retention effects. Then GOD fully catalyzes intracellular glucose on the nano hybrid and heme per se has POD enzyme activity, and a large amount of ROS is generated through decomposition, so that chemokinetic treatment is realized. The silicon dioxide nano microspheres are degradable and metabolizable in vivo, and show that the silicon dioxide nano microspheres have biocompatibility. Abundant ROS are generated in the process, and the shortage of ROS caused by radiotherapy is counteracted, so that the effect of ionizing radiation is improved, and the tumor is ablated without damaging adjacent normal tissues.
Disclosure of Invention
The invention aims to provide a POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial for improving the content of active oxides in tumor cells based on the microenvironment of the tumor and overcoming the defect of low lethality of the tumor cells under the condition of hypoxia in the existing radiotherapy process.
The second purpose of the invention is to provide a preparation method of POD-GOD (peroxidase-induced degradation) -synergistically modified mesoporous silicon nanomaterial, which is capable of reducing the preparation cost and improving the content of active oxides in tumor cells based on the microenvironment of the tumor.
The third purpose of the invention is to provide the use of the POD-GOD synergistically modified mesoporous silicon nanomaterial for improving the content of active oxides in tumor cells based on the microenvironment of the tumor itself.
The above object of the present invention is achieved by the following technical solutions:
a POD-GOD synergistically modified mesoporous silicon nanomaterial is characterized by comprising a mesoporous silicon nanomaterial simultaneously loaded with Glucose Oxidase (GOD) and heme (Hemin).
Preferably, the mesoporous silicon nano material is silicon dioxide nano particles, and the particle size of the mesoporous silicon nano material is 100-300 nm.
More preferably, the particle size of the mesoporous silicon nanomaterial is 160-250 nm.
The invention also provides a preparation method of the mesoporous silicon nanomaterial, which is characterized by comprising the following steps of:
step 1, slowly pouring heme (Hemin), a coupling agent of a hapten and N-hydroxysuccinimide (NHS) into a solvent, mixing and stirring until a mixture is dissolved, adding a first silane coupling agent, and continuously stirring at room temperature for a period of time to obtain a heme (Hemin) -silane precursor solution;
step 2, dissolving the ionic surfactant and the stabilizer in ultrapure water, stirring to fully mix the ionic surfactant and the stabilizer, then placing the mixture in an oil bath, and adding tetraethyl orthosilicate; after reacting for a period of time, adding a mixture of a second silane coupling agent and tetraethyl orthosilicate (TEOS) and the heme (Hemin) -silane precursor solution obtained in the step 1, and stirring to react under an inert gas atmosphere to obtain a heme (Hemin) -loaded mesoporous silicon nanoparticle material;
step 3, centrifugally collecting the mesoporous silicon nanoparticle material loaded with the heme (Hemin), washing the mesoporous silicon nanoparticle material with ethanol for several times, and then removing the ionic surfactant through extraction to obtain a pure mesoporous silicon nanoparticle material loaded with the heme (Hemin);
and 4, adding the heme (Hemin) -loaded mesoporous silicon nano-particle material obtained in the step 3 and Glucose Oxidase (GOD) into ultrapure water, stirring, centrifugally collecting, and cleaning with ultrapure water to obtain the heme (Hemin) -and Glucose Oxidase (GOD) -loaded mesoporous silicon nano-material.
Preferably, in step 1, the coupling agent of the hapten is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), the first silane coupling agent is (3-aminopropyl) triethoxysilane (APTES), and the solvent is N, N-Dimethylformamide (DMF).
Preferably, in the step 1, the amount of the heme (Hemin) is 25-75 mg, the amount of the EDC is 20-45 mg, the amount of the NHS is 15-25 mg, the solvent DMF (5-10 mL), and the first silane coupling agent APTES solution (15-30 μ L).
Preferably, in the step 1, the chemical reaction temperature of the mixed solution is (20-24 ℃), the stirring time is (6-8 h), and the volume of the obtained heme-silane precursor solution is (5-10 mL), and the solution concentration is (5-15 mg/mL).
Preferably, in step 2, the ionic surfactant is Cetyl Trimethyl Ammonium Chloride (CTAC), and the stabilizer is Triethanolamine (TEA).
Preferably, in the step 2, the oil bath temperature is 60-70 ℃, and the reaction time is 1-2 h.
Preferably, in the step 2, the second silane coupling agent is bis [3- (triethoxysilyl) propyl ] tetrasulfide (BTES), the inert gas atmosphere is nitrogen atmosphere, and the reaction time is 3-6 h.
Preferably, in the step 2, the dosage of the ionic surfactant CTAC is (1-3 g), the dosage of the stabilizer TEA is (0.1-0.2 g), and the dosage of the ultrapure water is (10-30 mL); the dosage of TEOS is 1-2 mL, and the dosage of BETS is 0.5-1.5 mL.
Preferably, in the step 3, the extraction process is to dissolve the heme (Hemin) -loaded mesoporous silicon nanoparticle material obtained in the step 2 in methanol, extract with NaCl, and finally obtain a pure heme (Hemin) -loaded mesoporous silicon nanoparticle material.
Preferably, in the step 4, the amount of the mesoporous silicon nanoparticle material carrying the heme is (50-70 mg), the amount of the glucose oxidase GOD is (70-100 mg), the amount of the used ultrapure water is (30-50 ml), and the stirring time is (18-30 h).
Preferably, the Zeta diameter mean values of the Mesoporous Silicon Nanoparticles (MSN), the mesoporous silicon nanoparticles (Hemin-MSN) with the surfaces modified with heme (Hemin), the mesoporous silicon nanoparticles (GOD @ Hemin-MSN) with the surfaces modified with heme (Hemin) and Glucose Oxidase (GOD) are respectively 180nm \200nm \190 nm.
Preferably, the Zeta potential mean values of the Mesoporous Silicon Nanoparticles (MSN), the mesoporous silicon nanoparticles (Hemin-MSN) with the surface modified with the heme (Hemin), the mesoporous silicon nanoparticles (GOD @ Hemin-MSN) with the surface modified with the heme (Hemin) and the Glucose Oxidase (GOD) are respectively-22 mV \ 25mV \ 24 mV.
The principle of the invention is as follows:
on the basis of the traditional sol-gel method, the invention loads the hemoglobin and modifies the surface of the silicon nanoparticles of the mesoporous silicon while generating the mesoporous silicon by an in-situ synthesis mode, thereby providing a process for synthesizing the heme-mesoporous silicon nanomaterial (Hemin-Silane precursor) with heme (Hemin) participating in the field. The specific principle of preparing the mesoporous silicon by the sol-gel method is that a first silane coupling agent, an ionic surfactant and a stabilizer are dissolved in ultrapure water and stirred to be fully mixed, then the mixture is placed in an oil bath, and tetraethyl orthosilicate is added; after reacting for a period of time, adding a mixture of a second silane coupling agent and tetraethyl orthosilicate (TEOS) to synthesize the mesoporous silicon nanoparticles. While the mesoporous silicon material is prepared in the manner, heme (Hermin), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N-Dimethylformamide (DMF) and (3-aminopropyl) triethoxysilane (APTES) are doped in the mixing step of the first silane coupling agent for stirring, so that the mesoporous silicon nanoparticles are subjected to surface modification while carrying heme, and the in-situ synthesized heme-carrying mesoporous silicon nanoparticle material (Hemin-MSN) is obtained. After the mesoporous silicon nanoparticle material loaded with heme is purified, Glucose Oxidase (GOD) is mixed with the mesoporous silicon nanoparticle material to obtain the mesoporous silicon nanoparticle material (GOD @ Hemin-MSN) loaded with heme and glucose oxidase, namely the biodegradable mesoporous silicon nanoparticle modified with heme and glucose oxidase, so that the aim of simultaneously loading heme and glucose oxidase is fulfilled, and the loaded heme and glucose oxidase have a good slow-release effect.
The invention provides a mesoporous silicon nano material for improving the content of active oxides in tumor cells based on the microenvironment of the tumor, which is composed of mesoporous silicon nanoparticles (Hemin-MSN) with heme modified on the surface and Glucose Oxidase (GOD) encapsulated in the mesoporous silicon nanoparticles. In the nanoparticle for improving the content of active oxides in tumor cells based on the tumor microenvironment, the mesoporous silicon nanoparticle with the surface modified with heme is used as a carrier of Glucose Oxidase (GOD), and the H is converted into H by the simulated Peroxidase (POD) under the acidic condition of the hemoglobin in the tumor cells 2 O 2 Decomposing into ROS. The Glucose Oxidase (GOD) reacts with glucose in tumor cells to promote H in tumor 2 O 2 And (4) concentration. The inventionThe nano-particles can effectively improve ROS in a tumor microenvironment, thereby improving the killing efficiency of X-rays on tumor cells. Specifically, the nanoparticles are delivered to the tumor site by enhancing the permeability and retention effects by intravenous injection into the body. Then GOD fully catalyzes intracellular glucose on the nano hybrid, and heme per se has POD enzyme activity, so that a large amount of ROS is generated through decomposition, and chemokinetic treatment is realized. The silicon dioxide nano microspheres are degradable and metabolizable in vivo, and show that the silicon dioxide nano microspheres have biocompatibility. Abundant ROS are generated in the process, and the shortage of ROS caused by radiotherapy is counteracted, so that the ionizing radiation effect is improved, and the tumor is ablated without damaging adjacent normal tissues.
The invention has the following beneficial effects:
the invention provides a POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, which is a nanoparticle for improving the content of active oxides in tumor cells based on the tumor microenvironment, has controllable particle diameter, good biocompatibility and biodegradability, can be used for carrying out in-situ catalysis by utilizing the tumor microenvironment, consuming glucose in the tumor cells and improving H in the tumor cells 2 O 2 The concentration, the effect of heme POD enzyme is utilized to generate ROS, the radiosensitization is promoted, the lethality rate of tumor cells is improved, and therefore the tumor control rate is improved.
Drawings
FIG. 1 is a schematic diagram of a biodegradable peroxidase mimicking GOD @ Hemin-MSN for use in chemokinetic therapy to achieve radiosensitization.
FIG. 2 shows the physical properties of the mesoporous silicon nanoparticles obtained in examples 1, 2 and 3 of the present invention;
wherein FIG. 2(A) is a TEM image (from left to right) of MSN, Hemin-MSN and GOD @ Hemin-MSN;
FIG. 2(B) is a Zeta potential diagram of MSN, Hemin-MSN and GOD @ Hemin-MSN;
FIG. 2(C) is a plot of the z-average diameter measurements of MSN, Hemin-MSN and GOD @ Hemin-MSN;
FIG. 2(D) is a graph of the UV-Vis spectra of TMB solution reacted with GOD @ Hemin-MSN + glucose as a function of time;
FIG. 2(E) is the absorbance of TMB solution at 652nm as a function of time for different solutions.
FIG. 3 shows the cell experiments of examples 4 and 5 of the present invention;
FIG. 3(A) cell viability of NHLF cells after incubation with different concentrations of GOD @ Hemin-MSN;
FIG. 3(B) is the cell viability of A549 cells after different experiments;
FIG. 3(C) is a γ -H2AX stained CLSM image;
fig. 3(D) is a ROS-stained CLSM image;
FIG. 3(E) is colony formation;
FIG. 3(F) is a Transwell experiment after different experiments;
FIG. 3(G) shows the determination of injury (1. control, 2. Radiation Therapy (RT),3.Hemin-MSN,4.GOD @ Hemin-MSN, 5.Hemin-MSN + RT,6.GOD @ Hemin-MSN + RT). Data were expressed as mean ± standard deviation using one-way square difference analysis of Tukey multiple comparison test. P < 0.001; p <0.01 or p < 0.05.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available, and for convenience of expression, the following examples replace the corresponding raw materials with the abbreviations defined above.
Example 1: preparation method of heme and glucose oxidase doped mesoporous silicon nanoparticles (GOD @ Hemin-MSN)
Slowly pouring Hemin (25mg), EDC (20mg) and NHS (25mg) into a solvent DMF (5ml), mixing and stirring until the mixture is dissolved, adding APTES (15 mu L), and continuously stirring at room temperature for a period of time to obtain a hemoglobin-silane precursor solution;
CTAC (2g) and TEA (0.1g) were dissolved in 20mL of ultrapure water and stirred for 0.5 h. The solution was placed in a 65 deg.C oil bath, then 1mL TEOS was added. After 1 hour of reaction, a mixture of 1mL BTES and 0.5mL TEOS with 1mL of the above heme-silane precursor solution was added to the above solution and reacted under a nitrogen atmosphere for another 4 hours. The resulting Hemin-MSN product was collected by centrifugation and washed several times with ethanol. After extraction of CTAC with NaCl in methanol, the CTAC-free Hemin-MSN nanoparticles were dissolved in 10mL of ultrapure water.
Hemin-MSN and GOD synthesized above were added to 30mL of ultrapure water and stirred for 24 h. The resulting GOD @ Hemin-MSN product was collected by centrifugation and then washed with water.
Example 2 physical characterization of GOD @ Hemin-MSN
The diameter of GOD @ Hemin-MSN was determined by transmission electron microscopy (TEM; Tecnai G2F 20S-Twin, FEI, USA) under 100kev accelerated voltage conditions (FIG. 2A). The zeta potential (FIG. 2B) and zeta diameter (FIG. 2C) of GOD @ Hemin-MSN in 1 XPBS solution were measured by dynamic light scattering (DLS, Nano-Zen 3600, Malvern Instruments, UK). Surface chemistry was analyzed by XPS (ESCA-Lab250XI, Thermo Fisher Ltd, USA). The light absorption of NPs in the wavelength range of 300-. X-ray diffraction (XRD; Bruker D8 Advance, Germany) was used with copper ka radiation (λ 0.15406 nm).
Example 3 measurement of peroxidase Activity
Catalytic and comparative experiments were performed with the addition of H2O2(13mmol/L), Hemin-MSN, GOD @ Hemin-MSN, Hemin-MSN + H2O2(13mM), GOD @ Hemin-MSN + glucose (100. mu.g/mL) in phosphate buffer (PBS, ph5,3mL) containing TMB (0.1mmol/L) and monitored by measuring the absorbance at 652 nm. Absorption spectra were obtained as a function of time in PBS containing TMB and GOD @ Hemin-MSN + glucose (100. mu.g/mL). Hemin-MSN and GOD @ Hemin-MSN had the same concentration at 50 μ g/mL (FIG. 2D, E).
EXAMPLE 4 cytotoxicity assay (CCK-8)
CCK-8 was used to evaluate the toxicity of different GOD @ Hemin-MSN concentrations on cells and the effect of different treatment groups on cell viability. First, a549 cells were seeded at a density of 5 × 103 cells/well in 96-well plates and incubated for 24h in 7 groups (5 cells per group). The untreated group served as a control group, and the remaining 6 groups were incubated with GOD @ Hemin-MSN at different concentrations (12.5, 25, 50, 100, 200, 500. mu.g/mL) for 24 h. Add 10. mu.L of CCK-8 reagent to each well. After 2 hours of incubation, the absorbance of the characteristic peak at 450nm was measured using a microplate analyzer (Rayto-6000system, Rayto, China). A549 cells were seeded in a 96-well plate at a density of 5X 103 cells/well and divided into 6 groups (5 wells per group) (1) control group, (2) Radiation Therapy (RT), (3) Hemin-MSN, (4) GOD @ Hemin-MSN, (5) Hemin-MSN + RT, (6) GOD @ Hemin-MSN + RT. Wherein the radiotherapy dose is 6Gy, and the equivalent concentration of MSN is 100 mug/mL. From (FIG. 3A) it can be seen that the cell viability decreased slightly with increasing concentrations of GOD @ Hemin-MSN. It is noted that the cell survival rate still reaches more than 60% under the condition of high concentration (500 mug/mL), which indicates that GOD @ Hemin-MSN has no obvious cytotoxicity to cells and has good biocompatibility.
Example 5GOD @ Hemin-MSN combination radiotherapy sensitization experiment
(FIG. 3B) wherein the radiation therapy dose was 6Gy and the hemin-msn equivalent concentration was 50. mu.g/mL. No obvious cytotoxicity is observed in the control group, the RT group and the Hemin-MSN group. While the Hemin-MSN + RT and GOD @ Hemin-MSN + RT treated groups induced significant cell death, the GOD @ Hemin-MSN + RT treated group showed more significant cytotoxicity than the Hemin-MSN + RT treated group. This result indicates that GOD can oxidize glucose to generate a large amount of H2O2, which together with endogenous H2O2, generates ROS under hemin catalysis, and achieves chemo-kinetic treatment, thereby killing cells to a greater extent. Moreover, after 6Gy radiotherapy, the killing rate of GOD @ Hemin-MSN + RT group cells is obviously higher than that of single radiotherapy, which shows that the GOD @ Hemin-MSN can be used together with radiotherapy to improve the treatment effect.
Radiation therapy can also cause cell death by inducing DNA double strand breaks. Gamma-H2 AX is a sensitive fluorescent staining marker for DNA damage molecules. As shown (fig. 3C), foci fluorescence of γ -H2AX (red) was observed in the nuclei. The GOD @ Hemin-MSN + RT group has obvious DNA damage. It is worth mentioning that the damage rate of the Hemin-MSN mediated RT to DNA is 34.4%; GOD @ Hemin-MSN + RT, however, retained 78.2% of the γ -H2AX lesion fluorescence, showing impressive radiosensitizing ability. In addition, to further confirm and expand these results, cells were incubated with DCFH-DA and the production of different sets of ROS were examined (FIG. 3D). The GOD @ Hemin-MSN group showed green fluorescence, confirming that GOD @ Hemin-MSN can consume glucose to produce H2O2, and then produce ROS. The GOD @ Hemin-MSN + RT group has stronger fluorescence and generates a large amount of ROS. That is, GOD @ Hemin-MSN + RT can be used in combination with RT to improve therapeutic effect. The colony formation results are shown in FIG. 3E. The GOD @ Hemin-MSN + RT group has obvious inhibition effect on tumor cell proliferation. Meanwhile, the sensitization enhancement rate of GOD @ Hemin-MSN is 1.60, which is obviously higher than that of Hemin-MSN (1.32). The above results demonstrate that GOD @ Hemin-MSN mediated chemokinetic treatment can significantly enhance radiotherapy. Invasion and scratch experiments (FIGS. 3F and G-2) also show the same experimental results, that GOD @ Hemin-MSN + RT group has the strongest inhibition on A549 cell invasion and the lowest cell fusion rate. The data show that GOD @ Hemin-MSN inhibits metastasis of A549 cells by affecting their adhesion, invasion and migration.
The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art will appreciate that various combinations, modifications and equivalents can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is encompassed by the claims of the present invention.

Claims (10)

1. A POD-GOD synergistically modified mesoporous silicon nanomaterial is characterized by comprising a mesoporous silicon nanomaterial loaded with glucose oxidase and heme simultaneously.
2. The mesoporous silicon nanomaterial according to claim 1, characterized in that: the mesoporous silicon nano material is silicon dioxide nano particles, and the particle size of the mesoporous silicon nano material is 100-300 nm.
3. A method for preparing the mesoporous silicon nanomaterial of claim 1 or 2, comprising the following steps:
step 1, slowly pouring heme, a coupling agent of hapten and N-hydroxysuccinimide into a solvent, mixing and stirring until a mixture is dissolved, then adding a first silane coupling agent, and continuously stirring at room temperature for a period of time to obtain a heme-silane precursor solution;
step 2, dissolving the ionic surfactant and the stabilizer in ultrapure water, stirring to fully mix the ionic surfactant and the stabilizer, then placing the mixture in an oil bath, and adding tetraethyl orthosilicate; after reacting for a period of time, adding a second silane coupling agent, a mixture of tetraethyl orthosilicate and the heme-silane precursor solution obtained in the step 1, and stirring to react in an inert gas atmosphere to obtain a heme-loaded mesoporous silicon nanoparticle material;
step 3, centrifugally collecting the heme-loaded mesoporous silicon nanoparticle material, washing the heme-loaded mesoporous silicon nanoparticle material with ethanol for several times, and then removing the ionic surfactant through extraction to obtain a pure heme-loaded mesoporous silicon nanoparticle material;
and 4, adding the heme-loaded mesoporous silicon nanoparticle material obtained in the step 3 and glucose oxidase into ultrapure water, stirring, centrifugally collecting, and cleaning with ultrapure water to obtain the heme-and glucose oxidase-loaded mesoporous silicon nanoparticle material.
4. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 1, the coupling agent of the hapten is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the first silane coupling agent is (3-aminopropyl) triethoxysilane, and the solvent is N, N-dimethylformamide.
5. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 1, the chemical reaction temperature of the mixed solution is 20-24 ℃, and the stirring time is 6-8 h.
6. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 2, the ionic surfactant is cetyl trimethyl ammonium chloride, and the stabilizer is triethanolamine.
7. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 2, the oil bath temperature is 60-70 ℃, and the reaction time is 1-2 h.
8. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 2, the second silane coupling agent is bis [3- (triethoxysilyl) propyl ] tetrasulfide, the inert gas atmosphere is nitrogen atmosphere, and the reaction time is 3-6 h.
9. The method for preparing the mesoporous silicon nanomaterial according to claim 3, wherein the method comprises the following steps: in the step 3, the extraction process is to dissolve the heme-loaded mesoporous silicon nanoparticle material obtained in the step 2 in methanol, and extract the material with NaCl to obtain a pure heme-loaded mesoporous silicon nanoparticle material.
10. Use of the mesoporous silicon nanomaterial of claim 1 or 2, wherein: used in tumor radiotherapy to raise the content of active oxide in tumor cell based on the tumor microenvironment.
CN202210324561.1A 2022-03-29 2022-03-29 POD-GOD (peroxidase-GOD) synergistically modified mesoporous silicon nanomaterial, preparation method and application Pending CN114949186A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116327979A (en) * 2023-05-25 2023-06-27 西南石油大学 Transition metal-based mesoporous nano catalytic medicine, preparation method and application

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116327979A (en) * 2023-05-25 2023-06-27 西南石油大学 Transition metal-based mesoporous nano catalytic medicine, preparation method and application

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