CN114904011A - Non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material and preparation method and application thereof - Google Patents
Non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material and a preparation method and application thereof. The non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material comprises cobalt molybdate-phosphomolybdic acid composite nanosheets and a biocompatible polymer with the surface of the composite nanosheets modified; the mass ratio of cobalt molybdate to phosphomolybdic acid is 1: 2-1: 4; the mass ratio of the cobalt molybdate-phosphomolybdic acid composite nanosheet to the biocompatible polymer is 1: 5-1: 15. the non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material can react with hydrogen peroxide to generate OH with the same activity, and can reduce GSH antioxidant in a tumor environment to achieve effective lipid peroxide accumulation.
Description
Technical Field
The invention belongs to the technical field of biological nano materials, and particularly relates to a non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material, and a preparation method and application thereof.
Background
The cell death modes mainly include apoptosis and non-apoptosis. Iron death, a non-apoptotic cell death mode, is of greater concern because of the increased iron accumulation and lipid metabolism required for tumor self anti-apoptotic capacity and tumor growth. Oncogenic signals stimulate oxidative activity-related enzymes to increase Reactive Oxygen Species (ROS), and are often also accompanied by the metabolism of antioxidant Glutathione (GSH) to eliminate highly expressed reactive oxygen species. Utilizing abundant hydrogen peroxide, Fe in the tumor microenvironment 2+ The guided Fenton (Fenton) reaction generates highly toxic hydroxyl radicals (· OH) that promote the production of lipid peroxides leading to iron death. However, the presence of GSH, which has reactive oxygen scavenging capacity, hinders the ROS-induced application of therapeutic approaches such as chemokinetics, sonokinetics, and photodynamic therapy, and iron-based materials are susceptible to normal tissue iron allergy. And the presence of GSH maintains the activity of glutathione peroxidase 4(GPX4) to reduce toxic lipid peroxides to less toxic lipools, resulting in diminished iron-death efficacy. Therefore, the development of alternatives to other metal compounds to enable their use in iron death therapy is a technical problem that is currently urgently needed to be solved.
Disclosure of Invention
In view of the above problems, the present invention provides a non-iron-based glutathione consumption and active oxygen species-synergistic composite material, which can react with hydrogen peroxide to generate OH with the same activity and simultaneously reduce GSH antioxidant in tumor environment to achieve effective lipid peroxide accumulation, and a preparation method and applications thereof.
In a first aspect, the present invention provides a non-iron based glutathione depletion synergistic active oxygen species enhancing composite. The non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material comprises cobalt molybdate-phosphomolybdic acid composite nanosheets and a biocompatible polymer with the surface of the composite nanosheets modified; the mass ratio of cobalt molybdate to phosphomolybdic acid is 1: 2-1: 4; the mass ratio of the cobalt molybdate-phosphomolybdic acid composite nanosheet to the biocompatible polymer is 1: 5-1: 15. by controlling the mass ratio within the range, the biocompatibility of the material and the glutathione consumption synergistic active oxygen species performance can be ensured.
Preferably, the biocompatible polymer comprises at least one of polyether F127, soya lecithin, distearoylphosphatidylethanolamine-polyethylene glycol, preferably polyether F127.
Preferably, the cobalt molybdate-phosphomolybdic acid composite nanosheets are composites that are uniformly dispersed and interwoven in the nanosheets.
Preferably, the non-iron-based glutathione consumption synergistic active oxygen species enhancement composite material has both glutathione consumption capacity and active oxygen generation capacity; preferably, the non-iron-based glutathione consumption is coordinated with the active oxygen species enhancement composite material to perform a redox reaction with glutathione overexpressed in the tumor to eliminate glutathione and increase the production of active oxygen during the redox reaction with glutathione.
Preferably, the non-iron-based glutathione consumption is cooperated with the product obtained after the redox reaction of the active oxygen species reinforced composite material and the glutathione, and the hydrogen peroxide are further reacted to generate singlet oxygen through a rosmarin mechanism 1 O 2 An active oxygen species.
In a second aspect, the present invention provides a method for preparing the non-iron-based glutathione consumption and active oxygen species reinforced composite material as described in any one of the above. The preparation method comprises the following steps: mixing a molybdenum source, a cobalt source and an organic solvent to obtain a mixed solution; transferring the mixed solution into a high-pressure kettle for reaction, centrifugally washing after the reaction is finished, and collecting a reaction product; and dispersing and mixing the reaction product with a biocompatible polymer to obtain the non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material.
Preferably, the molybdenum source is polyoxometallate containing molybdenum, and the concentration of the molybdenum source is 0.01-0.02 mM; the cobalt source is cobalt salt, and the concentration of the cobalt source is 0.06-0.07 mM; the organic solvent is a mixture of acetone, ethanolamine and oleylamine, and preferably the volume ratio of the acetone to the ethanolamine to the oleylamine is (1-4): (1-4): (1-4).
Preferably, the reaction temperature is 160-200 ℃, and the reaction time is 3-12 h.
Preferably, the reaction product is mixed with the biocompatible polymer in an organic solvent in a dispersion and the organic solvent is subsequently removed; the organic solvent comprises at least one of dichloromethane, ethanol, trichloromethane and cyclohexane.
In a third aspect, the present invention provides the biological use of a non-iron-based glutathione depletion and reactive oxygen species enhanced composite as described in any one of the above as an iron death inducer.
Drawings
FIG. 1 is a transmission electron micrograph of cobalt molybdate-phosphomolybdic acid composite nanosheets (CPMNSs);
fig. 2 is a scanning electron micrograph of a cobalt molybdate-phosphomolybdic acid composite nanosheet;
FIG. 3 is a Mo, P, Co, O energy spectrum element analysis spectrogram of the cobalt molybdate-phosphomolybdic acid composite nanosheet; the length of the scale and the unit of the scale in each figure are kept consistent, and the unit of the scale is 2.5 mu m;
FIG. 4 is an X-ray diffraction (XRD) spectrum of a cobalt molybdate-phosphomolybdic acid composite nanosheet;
FIG. 5 (a) is a schematic diagram of hydroxyl radical detection by electron paramagnetic resonance spectroscopy of cobalt molybdate-phosphomolybdic acid composite nanosheets, (b) is a diagram of content of GSH in a reaction system of CPMNSs and GSH detected by a 5,5' -dithiobis (2-nitrobenzoic acid (DTNB) indicator, wherein the content is characterized by absorbance, (c) is an ultraviolet absorption spectrum of Methylene Blue (MB) degradation caused by active oxygen, and each curve sequentially represents MB, 2mM GSH, 0mM GSH, 0.5mM GSH and 1mM GSH from top to bottom;
FIG. 6 shows (a) X-ray photoelectron (XPS) spectra of Mo valence state change after GSH reduction, and (b) ESR spectra of the reduced CPMNSs (RecPMSs) and hydrogen peroxide reacted with singlet oxygen;
FIG. 7 shows (a) the effect of Ferrostatin-1(Fer-1) on cell survival of mouse breast cancer (4T1) induced by CPMNSs and (b) the effect of Liproxstatin-1(Lip-1) on cell survival of mouse breast cancer (4T1) induced by CPMNSs; (c) measuring the intracellular GSH content of the CPMNSs after the CPMNSs are cultured for different concentrations, (d) measuring the intracellular GSH content of the CPMNSs after the CPMNSs are cultured for different times;
FIG. 8 is a graph showing (a) an intracellular GPX4 activity assay after incubation of CPMNSs at different concentrations, (b) an intracellular GPX4 activity assay after incubation of CPMNSs for different periods, (c) an intracellular Malondialdehyde (MDA) content assay after incubation of CPMNSs at different concentrations, and (d) an intracellular Malondialdehyde (MDA) content assay after incubation of CPMNSs for different periods;
FIG. 9 is a plot of tumor volume versus time for the four treatment groups, Control, CPMNSs i.v., CPMNSs i.t., and CPMNSs i.t. + DFOM; the Control group does not perform any processing; CPMNSs i.v. group was injected with 10mg/kg CPMNSs in tail vein (i.v.) on days 0 and 7; cpmns i.t. group injected 10mg/kg CPMNSs intratumorally (i.t.) on days 0 and 7; CPMNSs i.t. + DFOM group on day 0, 4, 8, 12 on CPMNSs i.t. group 20mg/kg of iron death inhibitor deferoxamine mesylate (DFOM) was intraperitoneally injected;
FIG. 10 is the in-tumor Reactive Oxygen Species (ROS) and lipid peroxidation Level (LPO) measurements for Control, CPMNSs i.v., CPMNSs i.t., and CPMNSs i.t. + DFOM four treatment groups; the length of the scale and the scale unit in each figure are kept consistent, and the scale unit is 200 mu m;
FIG. 11 is an intratumoral GPX4 immunohistochemical section of the four treatment groups, Control, CPMNSs i.v., CPMNSs i.t., and CPMNSs i.t. + DFOM; the scale units of the figures are consistent and are all 100 micrometers;
FIG. 12 is a section of tumor hematoxylin-eosin staining (H & E), deoxyribonucleotide terminal transferase (TdT) -mediated nick end labeling (TUNEL) and Ki-67 after treatment of the Control, CPMNSs i.v., CPMNSs i.t., and CPMNSs i.t. + DFOM four treatment groups; the scale units of the figures are consistent and are all 100 micrometers;
fig. 13 is a record of the growth curves, i.e. the growth rate over time, of experimental mice from the four treatment groups Control, CPMNSs i.v., CPMNSs i.t., and CPMNSs i.t. + DFOM.
Detailed Description
The present invention is further illustrated by the following examples, which are to be understood as merely illustrative of, and not restrictive on, the present invention. Unless otherwise specified, each percentage means a mass percentage.
The present disclosure provides a non-iron based glutathione depletion synergistic active oxygen species enhanced composite. Glutathione depletion and reactive oxygen species enhanced cobalt molybdate (CoMoO), also known as tumor microenvironment response 4 ) Phosphomolybdic acid (H) 3 PMo 12 O 40 ) Composite materials, or composite materials reinforced by degradable glutathione consumption and active oxygen species. The non-iron based glutathione depletion synergistic active oxygen species enhancement composite includes cobalt molybdate (CoMoO) with glutathione depletion and active oxygen species enhancement 4 ) Phosphomolybdic acid (H) 3 PMo 12 O 40 ) Composite nanosheets and biocompatible polymers modifying the composite nanosheets. The surface-modified biocompatible polymer is critical to impart biocompatibility and colloidal stability to the composite nanoplatelets.
Cobalt molybdate (CoMoO) 4 ) Phosphomolybdic acid (H) 3 PMo 12 O 40 ) The composite nanosheet is structurally characterized by two-dimensional nanosheets which are uniformly dispersed and interwoven with one another. The cobalt provided by the composite nano-sheet can generate highly toxic hydroxyl free radicals (OH) with over-expressed hydrogen peroxide in a tumor microenvironment, and promote the generation of cell membrane lipid peroxides; the provided molybdenum and the GSH over-expressed in the tumor simultaneously carry out oxidation-reduction reaction to eliminate the GSH, inhibit the activity of GPX4 enzyme and prevent the elimination of lipid peroxide. The effective accumulation of lipid peroxides promotes the execution of iron death.
In the experimental process, the change of the valence state of the molybdenum element in the reduction process of molybdenum and glutathione leads the degradation of the nano-sheet caused by the change of the chemical structure of the material, promotes the release of the cobalt source and increases the generation of active oxygen. Specifically, hexavalent molybdenum (in the polyoxometallate structure of phosphomolybdic acid) is reduced to pentavalent molybdenum, thereby enabling further reaction with hydrogen peroxide through the rhodinMechanism generates another singlet oxygen: ( 1 O 2 ) Active oxygen species, increasing the source of active oxygen.
It is also demonstrated herein that the dual GSH consumption and active oxygen production of cobalt molybdate and phosphomolybdic acid exacerbates the results of lipid peroxidation, resulting in more efficient occurrence of iron death compared to the single components.
The following also exemplifies a method for preparing the non-iron-based glutathione-depleted and active oxygen species-enhanced composite material according to the present invention.
Mixing a molybdenum source, a cobalt source and an organic solvent. The molybdenum source is a polyoxometallate containing molybdenum, and phosphomolybdic acid with the concentration of 0.01-0.02mM, preferably 0.016 mM. The cobalt source is cobalt salt, and the concentration is 0.6-0.7mM, preferably 0.68mM cobalt acetate. The specific chemical composition of cobalt acetate and phosphomolybdic acid favours the formation of cobalt molybdate (CoMoO) 4 ) Phosphomolybdic acid (H) 3 PMo 12 O 40 ) A two-dimensional nanosheet structure of a composite nanosheet. Wherein, part of phosphomolybdic acid reacts with cobalt acetate to form cobalt molybdate, and other phosphomolybdic acid participates in the construction of the two-dimensional nanosheet structure. The organic solvent is a mixture of acetone, ethanolamine and oleylamine. The organic solvent of the composition has the function of maintaining the directional growth of the two-dimensional nanosheets. Preferably, the volume ratio of acetone, ethanolamine and oleylamine is 1: 1: 1.
transferring the mixed solution into a high-pressure reaction kettle, and sealing for reaction. The reaction temperature is 160-200 ℃; the reaction time is 3 to 12 hours, preferably 6 to 12 hours. After the reaction, the reaction mixture is centrifuged to wash the organic solvent remaining on the surface of the product. The product was washed with ethanol and cyclohexane, respectively. The number of washing times may be 3 to 5 times each.
The resulting product is mixed with a biocompatible polymer in an organic solvent. The effect is that the surface of the product is effectively wrapped by the biocompatible polymer, and the good biocompatibility and stability of the system are ensured. The organic solvent comprises at least one of dichloromethane, ethanol, trichloromethane and cyclohexane. For example, methylene chloride. The biocompatible polymer includes, but is not limited to, any one of polyether F127, soybean phospholipid, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), preferably polyether F127. The organic solvent was then removed by rotary evaporation. The rotary evaporation temperature can be 40-60 ℃, and the evaporation time can be 1-2 h.
In some embodiments, the rotary evaporated product is dispersed in a physiological buffer solution. For example, the product is dispersed in a physiological buffer solution of sodium dihydrogen phosphate/disodium hydrogen phosphate at a pH of 7.4.
The non-iron-based glutathione consumption and active oxygen species reinforced composite material solves the technical problem of insufficient accumulation of lipid peroxide in the treatment of tumor iron death, and provides the cobalt molybdate (CoMoO) with GSH consumption and active oxygen generation capacity 4 ) Phosphomolybdic acid (H) 3 PMo 12 O 40 ) The non-iron-based composite material of the composite nanosheet is used as an iron death inducer for relevant application in tumor treatment.
The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
CoMoO 4 -H 3 PMo 12 O 40 Preparing a composite nanosheet: 0.016mM phosphomolybdic acid, 0.68mM cobalt acetate, 3mL acetone, 3mL oleylamine, and 3mL ethanolamine were mixed and stirred for 10 minutes. The mixed solution was transferred to a 25mL stainless steel reaction vessel lined with p-phenylene and sealed at 180 ℃ for 6 h. After the reaction is finished, the reaction product is centrifugally washed for 3min by ethanol and cyclohexane at 10000 r/min. Collecting the product after centrifugal separation to obtain CoMoO 4 -H 3 PMo 12 O 40 Composite nanosheets (CPMNSs).
The CPMNSs are dispersed in ethanol, dripped on a copper mesh and observed in morphology by using a TEM, and the CPMNSs are in irregular nanosheet morphologies as can be seen from figures 1 and 2. The thickness of the composite nano-sheet is 3-4 nm.
CPMNSs are dispersed in ethanol and dripped on a silicon wafer for element energy spectrum analysis. From fig. 3, it can be seen that Mo, P, Co and O are uniformly distributed in the CPMNSs nanosheets.
CPMNSs were freeze dried for XRD measurements. XRD analysis of FIG. 4 illustrates CoMoO 4 -H 3 PMo 12 O 40 Composite nanosheet with CoMoO 4 And H 3 PMo 12 O 40 The complex exists in a form.
Modified CoMoO 4 -H 3 PMo 12 O 40 Composite nanosheet: 5mg of CoMoO 4 -H 3 PMo 12 O 40 Composite nanosheets) were mixed uniformly with 50mg of polyether F127 and added to 10mL of dichloromethane and rotary evaporated under vacuum at 45 ℃ for 2 h. After the rotary evaporation was completed, the resulting material was dispersed in a phosphate buffer solution (pH 7.4).
And (5) performance characterization of the CPMNSs. Modification of biocompatible polymers (e.g., polyether F127) did not affect the performance evaluation of cpmns. Performance measurements were performed in phosphate buffered solutions at pH 6.5 (mimicking the tumor microenvironment), not specifically described.
CPMNSs (100. mu.g/mL) were reacted with 4mM hydrogen peroxide and 10mM sodium bicarbonate for 10 minutes, and the generated OH was captured using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a capture reagent and detected by ESR spectroscopy. In fig. 5 (a), a typical 1: 2: 2: 1, indicating the generation of. OH. And adding HCO rich in physiological conditions 3 - After that, the radical signal is enhanced.
After 1mM GSH and CPMNSs (50. mu.g/mL) were reacted for 3h, the GSH content was measured using DTNB (5,5' -dithiobis (2-nitrobenzoic acid) (0.5 mM). As can be seen in FIG. 5 (b), the UV characteristic absorbance at 412nm of DTNB decreased after reaction with CPMNSs, indicating that GSH was consumed.
Reacting with GSH with different concentrations for 30min, and reducing RecPMSSs and H 2 O 2 (10mM) and HCO 3 - Reaction (25mM) for 10min, and Activity measurement with Methylene Blue (MB) degradation (10. mu.g/mL)The presence of oxygen. As can be seen from (c) in FIG. 5, when the GSH concentration was 0.5mM or 1mM, the methylene blue degradation ability of the RecPMNS was stronger than that of the CPMNSs, indicating that the addition of GSH accelerates the release of cobalt ions, which are then mixed with H 2 O 2 The fenton-like reaction of (1) is enhanced. When the GSH concentration was 2mM, the methylene blue-degrading ability was weakened because the excess unreacted GSH having the antioxidant ability eliminated the generated active oxygen.
After the reaction with 5mg of CPMNSs, 15mL of PBS (pH 6.5, 10mM) and GSH (10mM) and the set time point reached (10min, 0.5h, 1h, 2h, 4h, 6h, 12h, 24h, 36h, 48h, 72h), 1mL of the reaction solution was taken. The obtained solution was digested with aqua regia and the content of Co ions released was detected by Inductively Coupled Plasma (ICP). It can be seen from (d) in FIG. 5 that the addition of GSH accelerates the release of Co ions, making it more effective with H 2 O 2 The reaction produces active oxygen.
Products obtained by redox reaction of CPMNSs (100. mu.g/mL) with GSH (1mM) were lyophilized and analyzed by XPS. As can be seen from (a) in fig. 6, after the GSH reduction, a peak of lower valence Mo, represented by lower binding energy, appears, indicating that hexavalent molybdenum is reduced to pentavalent molybdenum.
Reducing RecPMSSs (100. mu.g/mL) and H 2 O 2 (10mM) reaction for 10min, and ESR spectroscopy was used to detect singlet oxygen production using 2,2,6, 6-Tetramethylpiperidine (TEMP) as a trapping agent. It can be seen from (b) in fig. 6 that the reaction of the recmnss with hydrogen peroxide produces a clear 1: 1: 1, triplet. This is because phosphomolybdic acid forms a tetraoxide with hydrogen peroxide by the rhodinic mechanism and is further converted into singlet oxygen.
Intracellular iron death assessment
4T1 cells were seeded in 96-well plates at a volume fraction of 5% CO 2 Incubated at 37 ℃ for 24 h. Adherent cells were washed with PBS and then incubated with media containing CPMNSs (150. mu.g/mL) and iron death inhibitors for 24h, and cell viability was measured using the CCK-8 kit.
As can be seen from (a) and (b) in FIG. 7, cell death by CPMNSs was significantly inhibited after co-incubation with either Fer-1 or Lip-1 iron death inhibitors.
4T1 cells were plated in 6-well plates for 24 hours, and then incubated with different concentrations of CPMNSs (0, 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL) for 12 hours or with the same concentration of CPMNSs (150. mu.g/mL) for different times (0, 3 hours, 6 hours, 9 hours), 4T1 cells were lysed with a lysate, and then the GSH content and the GPX4 activity were measured with a total glutathione assay kit (Shanghai Bin Yuntan Biotechnology Co., Ltd.) using a GSH assay kit and a glutathione peroxidase assay kit (Shanghai Bin Yuntan Biotechnology Co., Ltd.).
From (c) and (d) in FIG. 7, the GSH content becomes smaller as the concentration increases or the culture time is prolonged. As can be seen from (a) to (b) in FIG. 8, the GPX4 activity decreased with increasing concentration or with increasing incubation time. The content of Malondialdehyde (MDA), which is a lipid peroxidation product, under the same cell treatment conditions was detected by a lipid oxidation (MDA) detection kit (shanghai bi yuntian biotechnology limited). As can be seen from (c) and (d) in fig. 8, cellular MDA levels increased with increasing concentrations of CPMNSs and with increasing incubation times, indicating an increase in lipid peroxidation.
Evaluation of iron death treatment effect of CPMNSs in vivo
4T1 cells were implanted subcutaneously in Balb/c nude mice when the tumor volume reached about 100mm 3 Thereafter, the mice were randomly divided into four groups. The first group does not perform any processing (Control); the second group was injected with 10mg/kg CPMNSs (CPMNSs i.v.) in tail vein (i.v.) on days 0 and 7; the third group was intratumoral (i.t.) with 10mg/kg CPMNSs (CPMNSs i.t.) on days 0 and 7; the fourth group was administered with 20mg/kg of desferoxamine mesylate (DFOM), an iron death inhibitor, on day 0, 4, 8, 12 on a third group basis (CPMNSs i.t. + DFOM). Mice were continuously fed and analyzed for 16 days.
As can be seen from fig. 9, the CPMNSs-treated mice inhibited the tumor to a different extent compared to the control group, and the effect of the CPMNSs on tumor growth was significantly reduced after the introduction of the iron death inhibitor DFOM.
To observe the active oxygen and lipid peroxidation of CPMNSs in vivo, mice were injected with CPMNSs intratumorally or in tail vein (10mg/kg) for 12h, and then injected with 100. mu.L of DCFH-DA dye (alive)Sex oxygen indicator, 10. mu.M) or 100. mu. L C11-BODIPY 581/591 Dye (lipid peroxidation indicator, 10 μ M) was incubated for 30min, then tumors were taken for sectioning and fluorescence observation with confocal microscope. FIG. 10 shows that the fluorescence of active oxygen and lipid peroxidation increases with the addition of CPMNSs, indicating that CPMNSs can cause the generation of active oxygen in tumors and produce lipid peroxidation.
After treatment, tumors were harvested for GPX4 immunohistochemistry, hematoxylin-eosin staining (H & E), transferase-mediated deoxyguanosine triphosphate-biotin nicked end labeling (TUNEL), and Ki-67 section analysis. GPX4 expression was significantly reduced in CPMNSs-treated group compared to control group (fig. 11) and tumor tissues were damaged, apoptosis appeared, and cell proliferation ability was decreased (fig. 12).
To further evaluate the efficacy of the CPMNSs tumor treatments, the survival curves of the mice were recorded, and the tumor volumes of the mice were recorded every two days when the tumor volumes reached 1500mm 3 When it is, it is considered to be dead. As can be seen in fig. 13, the survival of mice treated with CPMNSs was greater than that of untreated controls, and decreased after inhibition of iron death with DFOM, suggesting that CPMNSs can act as potent iron death inducers.
Claims (10)
1. A non-iron-based glutathione consumption synergistic active oxygen species enhanced composite material, which is characterized by comprising cobalt molybdate-phosphomolybdic acid composite nanosheets and a biocompatible polymer with the surface of the composite nanosheets modified; the mass ratio of cobalt molybdate to phosphomolybdic acid is 1: 2-1: 4; the mass ratio of the cobalt molybdate-phosphomolybdic acid composite nanosheet to the biocompatible polymer is 1: 5-1: 15.
2. the non-iron-based glutathione depletion synergistic active oxygen species-enhanced composite material according to claim 1, wherein said biocompatible polymer comprises at least one of polyether F127, soy phospholipid, distearoylphosphatidylethanolamine-polyethylene glycol, preferably polyether F127.
3. The non-iron based glutathione depleting synergistic active oxygen species reinforced composite according to claim 1 or 2, wherein the cobalt molybdate-phosphomolybdic acid composite nanosheets are composites uniformly dispersed and interwoven among the nanosheets.
4. The non-iron based glutathione-consuming co-reactive oxygen species-enhancing composite material according to any one of claims 1 to 3, wherein the non-iron based glutathione-consuming co-reactive oxygen species-enhancing composite material has both glutathione-consuming capability and reactive oxygen species-producing capability; preferably, the non-iron-based glutathione consumption is coordinated with the active oxygen species enhancement composite material to perform a redox reaction with glutathione overexpressed in the tumor to eliminate glutathione and increase the production of active oxygen during the redox reaction with glutathione.
5. The non-iron-based glutathione consumption and active oxygen species enhancement composite material as claimed in claim 4, wherein the product of the non-iron-based glutathione consumption and active oxygen species enhancement composite material after the oxidation-reduction reaction with glutathione is further reacted with hydrogen peroxide to generate singlet oxygen by the rosmarin mechanism 1 O 2 An active oxygen species.
6. The method for preparing a non-iron-based glutathione consumption and active oxygen species enhancement composite according to any one of claims 1 to 5, wherein the method for preparing comprises: mixing a molybdenum source, a cobalt source and an organic solvent to obtain a mixed solution; transferring the mixed solution into a high-pressure kettle for reaction, centrifugally washing after the reaction is finished, and collecting a reaction product; and dispersing and mixing the reaction product with a biocompatible polymer to obtain the non-iron-based glutathione consumption synergistic active oxygen species reinforced composite material.
7. The method according to claim 6, wherein the molybdenum source is a polyoxometallate containing molybdenum, and the concentration of the molybdenum source is 0.01 to 0.02 mM; the cobalt source is cobalt salt, and the concentration of the cobalt source is 0.06-0.07 mM; the organic solvent is a mixture of acetone, ethanolamine and oleylamine, and preferably the volume ratio of the acetone to the ethanolamine to the oleylamine is (1-4): (1-4): (1-4).
8. The method as set forth in claim 6 or 7, wherein the reaction temperature is 160-200- o And C, the reaction time is 3-12 h.
9. The method of any one of claims 6 to 8, wherein the reaction product is dispersedly mixed with the biocompatible polymer in an organic solvent and the organic solvent is subsequently removed; the organic solvent comprises at least one of dichloromethane, ethanol, trichloromethane and cyclohexane.
10. The biological use of a non-iron based glutathione depleting synergistic reactive oxygen species enhancing composite according to any one of claims 1 to 5 as iron death inducer.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104511290A (en) * | 2014-12-31 | 2015-04-15 | 中国地质大学（武汉） | Preparation method of visible-light-driven photocatalyst nano spherical MoSe2 material |
| CN108133829A (en) * | 2017-12-21 | 2018-06-08 | 哈尔滨师范大学 | Co(OH)2@CoMoO4The preparation method of composite nano plate |
| US20180338935A1 (en) * | 2015-12-07 | 2018-11-29 | General Oncology, Inc. | Combination For The Effective Treatment Of Metastatic Cancer In Patients |
| WO2019079841A1 (en) * | 2017-10-26 | 2019-05-02 | University Of South Australia | Nanoparticle cancer therapy |
| CN110585238A (en) * | 2018-06-11 | 2019-12-20 | 沈阳药科大学 | Antitumor drug composition with synergistic effect and application thereof |
| CN111017996A (en) * | 2019-09-25 | 2020-04-17 | 青岛大学 | Synthesis of MoO with double simulated enzyme activity3-XMethod for producing antimicrobial material |
-
2021
- 2021-07-06 CN CN202110763296.2A patent/CN114904011B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104511290A (en) * | 2014-12-31 | 2015-04-15 | 中国地质大学（武汉） | Preparation method of visible-light-driven photocatalyst nano spherical MoSe2 material |
| US20180338935A1 (en) * | 2015-12-07 | 2018-11-29 | General Oncology, Inc. | Combination For The Effective Treatment Of Metastatic Cancer In Patients |
| WO2019079841A1 (en) * | 2017-10-26 | 2019-05-02 | University Of South Australia | Nanoparticle cancer therapy |
| CN108133829A (en) * | 2017-12-21 | 2018-06-08 | 哈尔滨师范大学 | Co(OH)2@CoMoO4The preparation method of composite nano plate |
| CN110585238A (en) * | 2018-06-11 | 2019-12-20 | 沈阳药科大学 | Antitumor drug composition with synergistic effect and application thereof |
| CN111017996A (en) * | 2019-09-25 | 2020-04-17 | 青岛大学 | Synthesis of MoO with double simulated enzyme activity3-XMethod for producing antimicrobial material |
Non-Patent Citations (3)
| Title |
|---|
| JINPING SHI ET AL: "Recent advances in MoS 2 -based photothermal therapy for cancer and infectious disease treatment", 《JOURNAL OF MATERIALS CHEMISTRY B》, vol. 8, pages 5793 * |
| MAQSOOD A. SIDDIQUI ET AL: "Molybdenum nanoparticles-induced cytotoxicity, oxidative stress, G2/Marrest, and DNA damage in mouse skin fibroblast cells (L929)", 《COLLOIDS AND SURFACES B: BIOINTERFACES》, vol. 125, no. 2015, pages 73 - 81, XP029125405, DOI: 10.1016/j.colsurfb.2014.11.014 * |
| 邹卫国等: "氯化钴通过活性氧诱导PC12细胞凋亡并伴有AP-1的活化", 《中国生物化学与分子生物学会第八届会员代表大会暨全国学术会议论文摘要集》, pages 421 - 422 * |
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