A carbon monoxide generator with both stability and radiation responsiveness, and its preparation method and application

Abstract

The present invention provides a stable and radiation-responsive carbon monoxide generator, its preparation method, and application. The carbon monoxide generator comprises mesoporous silica nanoparticles and a tumor-targeting, cell-penetrating peptide modified on the surface of the mesoporous silica nanoparticles, wherein the silica in the mesoporous silica nanoparticles is bridged by carbonyl iron. This carbon monoxide generator degrades under radiation exposure and continuously and sequentially releases metal ions and carbon monoxide. This not only ensures spatial consistency between radiotherapy and gas therapy, but also prolongs the duration of carbon monoxide action and enhances the synergy between carbon monoxide and metal ions, enabling efficient and safe carbon monoxide-based combined radiotherapy for tumors. The preparation method of the carbon monoxide generator provided by the present invention is simple and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a carbon monoxide generator and a preparation method and application thereof, and in particular to the preparation and application of a carbon monoxide generator having both stability and radiation responsiveness. Background Art

[0002] Carbon monoxide gas can significantly increase the sensitivity of tumors to radiotherapy and reduce the DNA damage of normal tissue cells caused by radiation side effects, making it an ideal adjuvant drug for radiotherapy. Researchers have improved the targeting of gas delivery and the controllability of release by designing smart responsive nanocarriers and prodrug molecular structures. However, the stability of gas precursors is poor and they are prone to leaking before reaching the lesion, which reduces the effectiveness of gas therapy and poses safety risks. In addition, the duration of carbon monoxide gas release by current carbon monoxide carriers is short, and CO has low solubility and high diffusivity, so it cannot be retained in the lesion area for a long time and at high concentrations. Insufficient CO will even promote tumor growth. Therefore, how to develop CO nanocarriers with high stability and strong controllable release has become an important challenge in the field of gas therapy.

[0003] Silica is a non-toxic, non-irritating inorganic material widely used as a pharmaceutical excipient in oral and topical formulations, such as ibuprofen and simethicone tablets. Mesoporous silica nanoparticles, due to their unique advantages of superior morphology, stable structure, and excellent strength, play a significant role in various fields, including adsorption, catalysis, and biomedicine. However, mesoporous silica nanoparticles suffer from low drug loading rates and poor responsiveness, and also face compatibility and degradation issues in diagnostics, therapy, and tissue engineering. To address the shortcomings of traditional mesoporous silica nanoparticles, researchers have introduced active organic functional groups into the inorganic silicon-oxygen backbone of mesoporous silica nanoparticles to construct bridged organic-inorganic hybrid mesoporous silica nanoparticles. In these bridged organic-inorganic hybrid mesoporous silica nanoparticles, the organic groups are uniformly distributed throughout the mesoporous silica nanoparticle backbone. These organic groups not only avoid pore blockage and pore volume occupation but also imbue the inert silicon-based material with activity, thereby enabling responsive and controllable degradation of the mesoporous silica nanoparticle backbone.

[0004] Carbon monoxide gas prodrugs are mostly metal carbonyl complexes, which are a type of metal organic complex formed by transition metal elements (iron, manganese, ruthenium, etc.) with multiple carbon monoxide molecules and other functional ligands. They can release carbon monoxide gas through ligand exchange or under conditions such as X-rays, pH, enzymes, and oxidation. The toxicity of metal carbonyl complexes depends on the toxicity of carbon monoxide, the toxicity of the metal in the compound, and the instability of the carbonyl itself. Currently, research on the use of mesoporous silica nanoparticles to achieve carbon monoxide gas delivery is not uncommon, but most of them load metal carbonyl complexes into the pores of mesoporous silica nanoparticles through physical adsorption or chemical coupling. There are problems such as low drug loading, easy drug leakage, explosive release of carbon monoxide, and non-degradable mesoporous silica nanoparticle skeletons. Based on the advantages of high rigidity and stability of the solid skeleton of inorganic mesoporous silica nanoparticles. Inspired by the unique advantages of organic-inorganic hybrid materials, which combine stability and responsiveness, a metal carbonyl-coordinated organic precursor silicon source was introduced into the framework of mesoporous silica nanoparticles to construct metal carbonyl-bridged mesoporous silica nanoparticles that are both stable and responsive. This approach fully exploits the "cage effect" of the mesoporous silica nanoparticle framework, increasing drug loading while enhancing the stability of the metal carbonyl group, thereby preventing leakage of the carbon monoxide prodrug during storage, transport, and plasma. Furthermore, the metal carbonyl-bridged mesoporous silica nanoparticles can also degrade in response to endogenous and exogenous stimuli. By regulating the degradation rate of the mesoporous silica nanoparticle framework, a constant rate of carbon monoxide gas release can be achieved, thereby enhancing the durability of gas therapy. In summary, integrating active carbon monoxide precursors into the framework of inert mesoporous silica has the potential to create stable and responsive carbon monoxide generators, potentially addressing the current challenges facing nanogas generators in gas therapy and promoting the clinical application of carbon monoxide gas therapy. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention provides a carbon monoxide generator and a preparation method and application thereof, and in particular relates to the preparation and application of a carbon monoxide generator having both stability and radiation responsiveness.

[0006] In a first aspect, the present invention provides a carbon monoxide generator having both stability and radiation responsiveness, the carbon monoxide generator comprising: mesoporous silica nanoparticles and a tumor-targeting transmembrane peptide modified on the surface of the mesoporous silica nanoparticles.

[0007] The silicon dioxide in the mesoporous silicon dioxide nanoparticles is bridged by carbonyl iron.

[0008] Most studies on using mesoporous silica nanoparticles to achieve carbon monoxide gas delivery are to load metal carbonyl complexes into the pores of mesoporous silica nanoparticles through physical adsorption or chemical coupling. However, these studies have disadvantages such as low drug loading, easy drug leakage, explosive release of carbon monoxide, and non-degradable mesoporous silica nanoparticle skeleton.

[0009] This invention creatively introduces a metal carbonyl-coordinated organic precursor silicon source into the mesoporous silica nanoparticle framework, constructing metal carbonyl-bridged mesoporous silica nanoparticles that are both stable and responsive. This method fully utilizes the "cage effect" of the mesoporous silica nanoparticle framework, increasing drug loading while enhancing the stability of the metal carbonyl group, thereby preventing leakage of the carbon monoxide prodrug during storage, transportation, and plasma. Furthermore, the metal carbonyl-bridged mesoporous silica nanoparticles can also degrade in response to endogenous and exogenous stimuli. By regulating the degradation rate of the mesoporous silica nanoparticle framework, a constant rate of carbon monoxide gas release is achieved, thereby enhancing the durability of gas therapy.

[0010] Preferably, the tumor-targeting penetrating peptide is selected from iRGD, Arg9, Click EB1 or Pep-1, and more preferably iRGD.

[0011] iRGD is a targeting peptide for cancer imaging with the amino acid sequence: c(Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys / c(Cys-Arg-Gly-Asp-Arg-Gly-Pro-Asp-Cys); single-letter representation: c(CRGDKGPDC) or c(CRGDRGPDC). It can be effectively used to combine and deliver imaging agents or anticancer drugs into tumors. The iRGD peptide follows a multi-step tumor targeting process: first, it is proteolytically cleaved to generate CRGDK fragments (αvβ3 and αvβ5) by binding to the surface of protein-expressing cells. This fragment then binds to Neuropilin-1 and penetrates deeper into the tumor. . Compared with conventional RGD peptides, the affinity of iRGD for αv integrin is in the low to medium nanomolar range, and the affinity of the CRGDK fragment for Neuropilin-1 is stronger than that for α. Therefore, these advantages are conducive to the transfer of CRGDK fragments from integrins to Neuropilin-1, thereby penetrating deeper into tumors. Due to its specific binding and strong affinity, iRGD peptides can effectively and deeply deliver imaging agents and anticancer drugs into tumors, which has good effects on detecting tumors, blocking tumor growth and inhibiting tumor metastasis. The carbon monoxide generator in the present invention has a tumor-targeted transmembrane peptide iRGD modified on the surface of mesoporous silica nanoparticles, which can make the generator have excellent targeting effect on tumors.

[0012] Preferably, the tumor-targeting cell-penetrating peptide is conjugated with polyethylene glycol and modified on the surface of mesoporous silica nanoparticles.

[0013] Preferably, the carbon monoxide generator having both stability and radiation responsiveness is a spherical structure with a particle size of 30-60 nm and a pore size of 2-5 nm.

[0014] The particle size of the carbon monoxide generator can be 30 nm, 35 nm, 37 nm, 38 nm, 39 nm, 45 nm, 50 nm, 55 nm, 60 nm, etc. Other specific values ​​within the above numerical range can be selected and will not be described in detail here.

[0015] The pore size of the carbon monoxide generator can be 2 nm, 2.4 nm, 2.5 nm, 2.7 nm, 3 nm, 4 nm, 5 nm, etc. Other specific values ​​within the above numerical range can be selected and will not be described in detail here.

[0016] Preferably, the carbon monoxide gas loading of the carbon monoxide generator is 0.2-2.6 μmol / mg.

[0017] The carbon monoxide gas loading of the carbon monoxide generator can be 0.2 μmol / mg, 0.3 μmol / mg, 0.4 μmol / mg, 0.5 μmol / mg, 1.3 μmol / mg, 2.0 μmol / mg, 2.6 μmol / mg, etc. Other specific values ​​within the above numerical range can be selected and will not be detailed here.

[0018] The carbonyl iron coordination bonds within the stable and radiation-responsive carbon monoxide generator described herein can break in response to radiotherapy radiation, causing the generator to degrade and release carbon monoxide gas. The sustained release of carbon monoxide gas lasts for over 24 hours. The generator can release iron ions in response to radiotherapy radiation. The release of carbon monoxide and iron ions occurs sequentially: CO gas is released first under the action of free radicals, followed by the release of iron ions under the action of free radicals and X-rays. The released iron ions can produce a chemokinetic effect in the tumor microenvironment.

[0019] In a second aspect, the present invention provides a method for preparing the carbon monoxide generator having both stability and radiation responsiveness according to the first aspect, the preparation method comprising the following steps:

[0020] (1) Synthesis of organic precursor silicon source coordinated with carbonyl iron

[0021] Under nitrogen protection, an iron carbonyl compound and a silane coupling agent are reacted in an organic solution to obtain an organic precursor silicon source coordinated with carbonyl iron, and the mixture is concentrated under reduced pressure and purified to an oily liquid;

[0022] (2) Synthesis of carbonyl iron-bridged mesoporous silica nanoparticles

[0023] A mixed solution of an organic precursor silicon source and an organosilicon alkoxide silicon source is added to an aqueous solution containing a surfactant, ammonia water is added and stirred, and ethanol is used to wash away the surfactant to obtain carbonyl iron-bridged mesoporous silica nanoparticles.

[0024] Preferably, the iron carbonyl compound is selected from any one of triiron dodecacarbonyl, diiron nonacarbonyl or pentacarbonyl iron.

[0025] Preferably, the silane coupling agent is selected from any one of vinyltrichlorosilane, vinyltriethoxysilane, γ-mercaptopropyltrimethoxysilane or γ-mercaptopropyltriethoxysilane.

[0026] Preferably, the organosilicon alkoxide silicon source is selected from any one of methyl orthosilicate, ethyl orthosilicate or propyl orthosilicate.

[0027] Preferably, the surfactant is selected from cetyltrimethylammonium bromide, cetylpyridinium chloride or 5-bromo-5-nitro-1,3-dioxane.

[0028] Preferably, the molar ratio of the organic precursor silicon source to the organosilicon alkoxide silicon source is (1-4):1.

[0029] The molar ratio of the organic precursor silicon source to methyl orthosilicate can be 1:1, 2:1, 3:1, 7:2, 4:1, etc. Other specific values ​​within the above numerical range can be selected and will not be detailed here.

[0030] In a third aspect, the present invention provides a use of a carbon monoxide generator having both stability and radiation responsiveness as described in the first aspect in the preparation of a drug for combined tumor radiotherapy and gas therapy.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a stable and radiation-responsive carbon monoxide generator, its preparation method, and its application. The carbon monoxide generator comprises mesoporous silica nanoparticles and a tumor-targeting, cell-penetrating peptide modified on the surface of the mesoporous silica nanoparticles. This carbon monoxide generator degrades under radiation exposure, continuously and sequentially releasing metal ions and carbon monoxide. This not only ensures spatial consistency between radiotherapy and gas therapy, but also prolongs the duration of carbon monoxide action and enhances the synergy between carbon monoxide and metal ions, enabling efficient and safe carbon monoxide-based combined radiotherapy for tumors. The preparation method of the carbon monoxide generator provided by the present invention is simple and suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the synthesis route of the organic precursor silicon source coordinated with carbonyl iron;

[0034] Figure 2 This is a diagram of the appearance of an organic precursor silicon source solution;

[0035] Figure 3 This is a mass spectrometry detection result diagram of the organic precursor silicon source;

[0036] Figure 4 This is a transmission electron microscope image of a carbon monoxide nanogenerator;

[0037] Figure 5 It is the nitrogen adsorption-desorption curve of carbon monoxide nanogenerator;

[0038] Figure 6 is a transmission electron microscopy image of a carbon monoxide nanogenerator under X-ray irradiation and / or co-incubation with H2O2;

[0039] Figure 7 It is a graph of carbon monoxide gas released by a carbon monoxide nanogenerator;

[0040] Figure 8 Figure 2 is the cytotoxicity of carbon monoxide nanogenerators with / without X-ray irradiation and / or with / without H2O2 addition on (a) HIEC-6 and HUVECs cells and (b) MC38 cells;

[0041] Figure 9 This is the result of the co-incubation experiment of carbon monoxide nanogenerator (FeCO-NRs) and MC38 cells;

[0042] Figure 10 This is a graph showing the experimental results of carbon monoxide nanogenerators (FeCO-NRs) delivering CO in MC38 cells;

[0043] Figure 11 This is the experimental result of carbon monoxide nanogenerator (FeCO-NRs) producing ROS in MC38;

[0044] Figure 12 This is the result of a depolarization test using cyanine dye JC-1 to detect mitochondrial transmembrane potential;

[0045] Figure 13 This is a comparison of the orthotopic tumor sizes of mice in each group. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] The following method for preparing inorganic mesoporous silica nanocarriers loaded with carbonyl iron is as follows: 0.03 ml of methyl orthosilicate is slowly added to an aqueous solution containing the surfactant cetyltrimethylammonium bromide (5 mg / mL, 10 mL), followed by the addition of 500 μL of 25% aqueous ammonia. The mixture is stirred at 80°C for 30 minutes, and the surfactant is washed away with ethanol to obtain inorganic mesoporous silica nanocarriers. 1 mg of carbonyl iron is added to 10 mL of a 1 mg / mL aqueous solution of inorganic mesoporous silica nanoparticles, stirred at 25°C for 24 hours, to obtain inorganic mesoporous silica nanocarriers loaded with carbonyl iron, hereinafter referred to as FeCO-NPs.

[0048] Example

[0049] Preparation of carbon monoxide nanogenerator (hereinafter referred to as FeCO-NRs):

[0050] Preparation process as Figure 1 As shown, the following steps are included:

[0051] (1) Under nitrogen protection, 10 mg of triiron dodecacarbonyl and 40 mg of γ-mercaptopropyltriethoxysilane were reacted in tetrahydrofuran at 80°C for 30 min. Under reduced pressure, the mixture was concentrated by rotary evaporation at 200°C to purify it into an oily liquid, thereby obtaining an organic precursor silicon source coordinated with carbonyl iron, such as Figure 2 The prepared carbonyl iron coordinated organic precursor silicon source was characterized by mass spectrometry, and the results were as shown in Figure 3 shown.

[0052] (2) A mixture of an organic precursor silicon source and methyl orthosilicate with a total volume of 0.03 ml (wherein the volume of the organic precursor silicon source is 0.01 ml and the volume of methyl orthosilicate is 0.02 ml) was slowly added to an aqueous solution containing a surfactant, hexadecyltrimethylammonium bromide (5 mg / mL, 10 mL), and then 500 μL of 25% ammonia water was added. The mixture was stirred at 80°C for 30 min, and the surfactant was washed away with ethanol to obtain carbonyl iron-bridged mesoporous silica nanoparticles.

[0053] (3) Maleimide-polyethylene glycol-carboxylic acid was modified on the surface of amino-carbonyl iron-bridged mesoporous silica nanoparticles, and then iRGD was coupled on the surface. The specific operation was as follows: 10 mg of carbonyl iron-bridged mesoporous silica nanoparticles were dispersed in 10 ml of deionized water, and 20 mg of EDC and 40 mg of NHS were added. After stirring for 30 minutes, 1 mg of maleimide-polyethylene glycol-carboxylic acid was added and the reaction was carried out at room temperature for 6 hours. 0.05 mg of iRGD was added and the reaction was carried out at 25°C for 24 hours to finally obtain a carbon monoxide nanogenerator.

[0054] The obtained carbon monoxide nanogenerator was observed by electron microscopy, and the characterization results were as follows: Figure 4 As shown, Figure 4 The results show that the synthesized carbon monoxide nanogenerator has a uniform morphology and spherical structure with a diameter of about 40nm. The obtained carbon monoxide nanogenerator was subjected to nitrogen adsorption-desorption test, and the characterization results are as follows Figure 5 As shown, Figure 5 This shows that the carbon monoxide nanogenerator has a mesoporous structure.

[0055] Experimental Example 1

[0056] Degradation and drug release analysis of carbon monoxide nanogenerator:

[0057] Ten milligrams of the prepared carbon monoxide nanogenerators were dispersed in 10 mL of release medium (aqueous solution containing 100 μM or 0 μM H₂O₂) and irradiated with different doses of X-rays (0.5, 1, 2, 4, or 8 Gy). A non-irradiated control group was also established. CO, iron, and silicon release experiments were conducted at 37°C. 0.2 mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours, and an equal amount of blank release medium was added. The cumulative CO release in the various simulated media and conditions was measured using the myoglobin (Mb) assay, and the release of metals and silicon was determined using inductively coupled plasma mass spectrometry (ICP-MS). Samples were taken at different time points and observed for changes in nanoparticle morphology and degradation using transmission electron microscopy to determine the correlation between the degradation of the carbon monoxide nanogenerators and their drug release behavior.

[0058] The results are as follows Figure 6 and Figure 7 As shown, Figure 6 It is a transmission electron microscopy image of the carbon monoxide nanogenerator under X-ray irradiation for 5 minutes and / or co-incubation with H2O2, indicating that the carbon monoxide nanogenerator can respond to X-rays and H2O2 degradation. Figure 7This is a graph of carbon monoxide gas released by a carbon monoxide nanogenerator. Compared with the prior art FeCO-NPs, this carbon monoxide nanogenerator can respond to X-rays and H2O2 to continuously release sustainable carbon monoxide, while FeCO-NPs will quickly release CO. Compared with the carbon monoxide generator provided by the present invention, the action time is significantly shorter.

[0059] Experimental Example 2

[0060] Cytotoxicity evaluation of carbon monoxide nanogenerators:

[0061] Normal human intestinal epithelial cells HIEC-6, human umbilical vein endothelial cells HUVECs and mouse colon cancer cells MC38 were used as research objects. The cytotoxicity of bridged mesoporous silica nanoparticles with different metal carbonyl complex doping contents and types was studied by SRB method at 24, 48 and 72 hours. The effects of the doping types and doping contents of a series of carbon monoxide nanogenerators on cell survival were explored, and safe doses were screened for subsequent experiments. The cytotoxicity evaluation results are shown in Figure 2. Figure 8 As shown, the cytotoxicity of the carbon monoxide nanogenerator was low, while its cytotoxicity was significantly increased under X-ray irradiation or with or without H2O2 incubation.

[0062] Experimental Example 3

[0063] Cellular internalization of carbon monoxide nanogenerators:

[0064] The experiment was terminated after the carbon monoxide nanogenerator was co-incubated with MC38 cells for 6 hours. The nanoparticles, lysosomes, mitochondria and cell nuclei were labeled respectively. The efficiency of mesoporous silica nanoparticles in cell endocytosis was observed using laser confocal fluorescence microscopy. The fluorescence co-localization of mesoporous silica nanoparticles and organelles was used to explore the nanoparticle endocytosis process and intracellular distribution. Subsequently, the average fluorescence intensity of the carbon monoxide nanogenerator in tumor, normal and immune cells was detected by flow cytometry to verify the tumor cell targeting of the nanoparticles. The results are shown in Figure 2. Figure 9 As shown, the carbon monoxide nanogenerator can be effectively taken up by MC38 cells through the lysosomal pathway.

[0065] Experimental Example 4

[0066] Intracellular delivery of CO gas:

[0067] MC38 cells were plated at 10 per well 5The cells were seeded at a density of 100 μM H2O2 in a 12-well plate, and 0 or 100 μM H2O2 was added to the cell culture medium and incubated for 24 hours. The carbon monoxide nanogenerator (FeCO-NRs) prepared in the above example and the inorganic mesoporous silica nanocarrier loaded with carbonyl iron (FeCO-NPs) were then added to each well, and the cells were co-incubated for 2, 6, 24, and 48 hours, respectively. The X-ray irradiation group was given different doses (0.5, 1, 2, 4, or 8 Gy) of X-ray irradiation, and a non-irradiated control group was set up. The cells were incubated with the CO fluorescent probe FL-CO-1 (1 μM) at 37°C for 30 minutes. Finally, the cells were washed three times with PBS, and the production of CO was observed on a laser confocal microscope. At the same time, the same method was used to detect the intracellular delivery of CO gas by the inorganic mesoporous silica nanoparticle nanodrug delivery system loaded with metal carbonyl complexes as a control. The results of intracellular delivery are shown in Figure 2. Figure 10 As shown, compared with the currently reported inorganic mesoporous silica nanocarriers loaded with carbonyl iron (FeCO-NPs), FeCO-NRs have less CO leakage in the absence of X-ray irradiation and H2O2 addition, indicating better stability; while in the presence of X-ray irradiation and H2O2 addition, the CO release efficiency is higher, proving its strong controllability.

[0068] Experimental Example 5

[0069] Generation of reactive oxygen species (ROS):

[0070] MC38 cells were plated at 10 per well 5 The cells were seeded at a density of 100 μM in laser confocal microplate culture dishes, and 0 or 100 μM H2O2 was added to the cell culture medium and cultured overnight. FeCO-NRs (50 μg / mL) and FeCO-NPs (50 μg / mL) were added to the above cell culture dishes and continued to be co-incubated for 2, 6, 24, and 48 hours. The X-ray irradiation group was given different doses of X-ray irradiation (0.5, 1, 2, 4, or 8 Gy), and a non-irradiated control group was set up. Fresh culture medium was replaced in the cell culture dishes of each group, and 2',7'-dichlorofluorescein (DCF), SOSG singlet oxygen fluorescent probe, 3,3',5,5'-tetramethylbenzidine (TMB) and other reagents were used to detect the total ROS and singlet oxygen ( 1 In addition, the same method was used to detect the ROS generation of FeCO-NPs and FeCO-NRs. The results of ROS generation are shown in Figure 2. Figure 11As shown in the results, compared with FeCO-NPs, FeCO-NRs produced less ROS in the absence of X-ray irradiation and H2O2 addition, indicating that FeCO-NRs were safer, while in the presence of X-ray irradiation and H2O2 addition, the ROS production efficiency was higher, proving that FeCO-NRs had stronger X-ray / H2O2 responsiveness.

[0071] Experimental Example 6

[0072] Radiotherapy sensitization effect:

[0073] MC38 cells were used as the research object. FeCO-NPs and FeCO-NRs were treated with X-rays at doses of 0.5, 1, 2, 4, or 8 Gy, and their radiosensitization function was evaluated by colony formation assay. DNA fragmentation was evaluated by γ-H2AX staining. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of apoptosis-related genes such as JNK, ERK, p38, Bax, Bcl-2, CyclinD1, Caspase-9, and Caspase-3 was detected by Western Blot. The depolarization of mitochondrial transmembrane potential was detected using cyanine dye JC-1 to explore the mechanism of radiosensitization. Figure 12 As shown, Figure 12 This is a diagram of the radiosensitization effect of carbon monoxide nanogenerators (FeCO-NRs), indicating that FeCO-NRs can enhance the DNA breakage ability of MC38 cells induced by X-rays.

[0074] Experimental Example 7

[0075] Evaluation of the anti-tumor effectiveness of carbon monoxide nanogenerator in vivo:

[0076] (1) Constructing MC38 subcutaneous tumor model mice, the steps are as follows:

[0077] MC38 cells (1×10 7 The subcutaneous colon cancer model mice were established by injecting 6-week-old male C57BL / 6 mice (100 μL PBS / site, logarithmic growth phase) into the right hind leg.

[0078] (2) Construct a mouse model of peritoneal metastasis using the following steps:

[0079] CT26 cells in the logarithmic growth phase were taken and plated at 2×10 5 The cells were suspended at a density of 100 μg / mL in 0.1 mL of culture medium without fetal bovine serum and penicillin / streptomycin liquid, and the cell suspension was injected into the abdominal cavity of 6-week-old BALB / c mice to allow the tumor to grow for 7 days.

[0080] (3) MC38 subcutaneous tumor model mice and peritoneal metastasis model mice were treated in the following modes: a. Normal saline control group (PBS group); b. Radiotherapy control group (PBS+X-ray group); c. Carbon monoxide nanogenerator control group (FeCO-NRs group); d. Carbon monoxide nanogenerator combined with radiotherapy group (FeCO-NRs+X-ray group); In group c, only carbon monoxide nanogenerator was given without radiotherapy; in group d, carbon monoxide nanogenerator and radiotherapy were given. Group b was given a conventional radiotherapy regimen, with a total dose of 4Gy of X-rays irradiating the tumor site of the mouse for 4 minutes. The radiotherapy regimen in group d was the same as that in group b, and group c was given only carbon monoxide nanogenerator without radiotherapy. The size of the in situ tumor in each group of mice was examined. The results are shown in the figure below. Figure 13 As shown, Figure 13 This is a diagram showing the anti-tumor effect of carbon monoxide nanogenerator combined with radiotherapy, which shows that carbon monoxide nanogenerator combined with radiotherapy can effectively inhibit tumors and prove its radiotherapy sensitization effect.

[0081] The applicant declares that the present invention uses the above-described embodiments to illustrate the detailed process flow of the present invention, but the present invention is not limited to the above-described detailed process flow, that is, it does not mean that the present invention must rely on the above-described detailed process flow to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A carbon monoxide generator having both stability and radiation responsiveness, characterized in that: The carbon monoxide generator comprises: mesoporous silica nanoparticles and tumor-targeting transmembrane peptides modified on the surface of the mesoporous silica nanoparticles; The silica in the mesoporous silica nanoparticles is bridged by carbonyl iron; The carbon monoxide generator having both stability and radiation responsiveness is prepared by the following method, which comprises the following steps: (1) Synthesis of organic precursor silicon source coordinated with carbonyl iron Under nitrogen protection, an iron carbonyl compound and a silane coupling agent are reacted in an organic solution to obtain an organic precursor silicon source coordinated with carbonyl iron, and the mixture is concentrated under reduced pressure and purified to an oily liquid; (2) Synthesis of carbonyl iron-bridged mesoporous silica nanoparticles A mixed solution of an organic precursor silicon source and an organosilicon alkoxide silicon source is added to an aqueous solution containing a surfactant, ammonia water is added and stirred, and the surfactant is washed away with ethanol to obtain carbonyl iron-bridged mesoporous silica nanoparticles; The iron carbonyl compound is selected from any one of triiron dodecacarbonyl, diiron nonacarbonyl or pentacarbonyl iron; the silane coupling agent is selected from any one of vinyltrichlorosilane, vinyltriethoxysilane, γ-mercaptopropyltrimethoxysilane or γ-mercaptopropyltriethoxysilane; and the organosilicon alkoxide silicon source is selected from any one of methyl orthosilicate, ethyl orthosilicate or propyl orthosilicate.

2. The carbon monoxide generator having both stability and radiation responsiveness according to claim 1, characterized in that: The tumor-targeting cell-penetrating peptide is selected from iRGD, Arg9, Click EB1 or Pep-1.

3. The carbon monoxide generator having both stability and radiation responsiveness according to claim 1, characterized in that: The tumor-targeting cell-penetrating peptide is conjugated and modified on the surface of mesoporous silica nanoparticles through polyethylene glycol.

4. The carbon monoxide generator having both stability and radiation responsiveness according to claim 1, characterized in that: The carbon monoxide generator is a spherical structure with a particle size of 30-60 nm and a pore size of 2-5 nm.

5. The carbon monoxide generator having both stability and radiation responsiveness according to claim 1, characterized in that: The carbon monoxide gas loading capacity of the carbon monoxide generator is 0.2-2.6 μmol / mg.

6. The method for preparing a carbon monoxide generator having both stability and radiation responsiveness according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) Synthesis of organic precursor silicon source coordinated with carbonyl iron Under nitrogen protection, an iron carbonyl compound and a silane coupling agent are reacted in an organic solution to obtain an organic precursor silicon source coordinated with carbonyl iron, and the mixture is concentrated under reduced pressure and purified to an oily liquid; (2) Synthesis of carbonyl iron-bridged mesoporous silica nanoparticles A mixed solution of an organic precursor silicon source and an organosilicon alkoxide silicon source is added to an aqueous solution containing a surfactant, ammonia water is added and stirred, and the surfactant is washed away with ethanol to obtain carbonyl iron-bridged mesoporous silica nanoparticles; The iron carbonyl compound is selected from any one of triiron dodecacarbonyl, diiron nonacarbonyl or pentacarbonyl iron; the silane coupling agent is selected from any one of vinyltrichlorosilane, vinyltriethoxysilane, γ-mercaptopropyltrimethoxysilane or γ-mercaptopropyltriethoxysilane; and the organosilicon alkoxide silicon source is selected from any one of methyl orthosilicate, ethyl orthosilicate or propyl orthosilicate.

7. The preparation method according to claim 6, characterized in that The surfactant is selected from any one of cetyltrimethylammonium bromide, cetylpyridinium chloride or 5-bromo-5-nitro-1,3-dioxane.

8. The preparation method according to claim 6, characterized in that The molar ratio of the organic precursor silicon source to the organosilicon alkoxide silicon source is (1-4):

1.

9. Use of the stable and radiation-responsive carbon monoxide generator according to any one of claims 1 to 5 in the preparation of a drug for combined radiotherapy and gas therapy of colon cancer.

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