CN113975291B - Iron death inducer and preparation method and application thereof - Google Patents
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Abstract
The invention provides an iron death inducer and a preparation method and application thereof. The iron death inducer provided by the invention comprises vermiculite nano sheets, wherein the thickness of the vermiculite nano sheets is 1.0-1.3nm, the length of the vermiculite nano sheets is 305-335nm, and the width of the vermiculite nano sheets is 305-335nm. The iron death inducer provided by the invention has good biocompatibility and strong cancer cell killing effect, can be used for synthesizing tumor treatment medicines, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to an iron death inducer as well as a preparation method and application thereof.
Background
With increasing morbidity and mortality, cancer has become a major global public health problem. At present, the main treatment modes of cancers comprise operations, chemotherapy, radiotherapy, immunotherapy and the like, but the common treatment modes also have some defects, for example, the operations are easy to relapse, the radiotherapy effect is not ideal due to the problem of radiation resistance, and the chemotherapy brings serious side effects due to poor drug selectivity. Therefore, new cancer treatment modalities such as biotherapy are also emerging to solve the problems of the prior art.
Iron death is a novel programmed cell death mode that leads to cell death by the accumulation of iron-dependent lipid peroxides, first proposed in 2012 by the teaching of Brent r. Recent studies have shown that induction of iron death can be used in cancer therapy, particularly in eradication of aggressive malignancies resistant to traditional therapies. With the development of nano biotechnology, iron death-based anticancer nano-drugs have also made important progress, and are mainly divided into two major categories, namely iron-based nano-materials and non-iron-based nano-materials.
The iron-based nano material can enrich iron ions in cells and accelerate Fenton (Fenton) reaction, so that the level of Reactive Oxygen Species (ROS) in the cells is improved, and iron death is induced. Cisplatin-loaded iron oxide nano prodrug (FePt NP 2) constructed by Ma and the like can release cisplatin and Fe at specific tumor sites 2+ /Fe 3+ And Fenton reaction is generated in situ, so that the ROS level in the cells is obviously improved, the death of tumor cells by iron is induced, and the anticancer activity is enhanced. Pu et al reported iron-chelating semiconductor multi-composite nanoparticles (spfens) combined photothermal therapy with iron-death therapy to enhance cancer treatment. However, iron-based nanomaterials such as cisplatin-loaded iron oxide nanoprecursors and iron-chelating semiconductor multi-composite nanoparticles enhance the anti-tumor effect by inducing the iron death effect, but often need to use a higher iron dose or be used in combination with other treatment modes, so that the iron-based nanomaterials have complex nanostructures and multi-metal components, and are low in biological safety.
The non-iron-based nano material can inhibit glutathione peroxidase (GPX 4) or induce iron death by exogenously regulating and increasing the degree of lipid peroxidation of tumor cells. Gao et al use amphiphilic polymer micelles to deliver GPX4 inhibitor RSL3, and synergistically induce iron death to reverse multidrug resistance through GPX4 inhibition, glutathione (GSH) attenuation and lipid peroxidation. The arginine-rich manganese-silicon nanobubble prepared by Wang and the like can be used as an iron death inducer to realize tumor targeted diagnosis and treatment through intracellular GSH depletion. However, the polymer micelle encapsulating the small-molecule iron death inducer and the amorphous calcium carbonate composite nano-drug disclosed in the prior art can effectively reverse drug resistance by inducing iron death or further kill tumor cells by combined chemotherapy, but the risks of drug leakage and toxic and side effects exist. The manganese-silicon nanobubbles with GSH consumption capability can realize diagnosis and treatment of tumors at the same time, but only can be used for tumor cells with arginine succinate synthetase deficiency, and have no universality for the tumor cells. In addition, the residual nano-materials in the body may have a risk of long-term toxicity, etc.
Therefore, the research and development of the ultrathin vermiculite nanosheet which is universal for tumor cells and has no cytotoxicity risk for inducing iron death has a good application prospect and wide applicability.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an iron death inducer as well as a preparation method and application thereof. The iron death inducer provided by the invention is oxidized and reduced to (Fe) once being absorbed by hypoxic tumor cells 2+ /Fe 3+ ) Formation of OH and O by disproportionation of hydrogen peroxide and Fenton reaction 2 And oxygen can be supplemented automatically. In addition, the iron death inducer of the present invention can regulate Tumor Microenvironment (TME) by consuming glutathione, which can induce iron droop of tumor cells, thus being suitable for preparing iron death-based anticancer nano-drugs.
The method is realized by the following technical scheme:
an iron death inducing agent comprising vermiculite nanoplates.
Further, the thickness of the vermiculite nanosheet is 1.0-1.3nm.
Further, the length of the vermiculite nanosheet is 305-335nm.
Further, the width of the vermiculite nano sheet is 305-335nm.
The invention also provides a preparation method of the iron death inducer, which comprises the steps of obtaining vermiculite nanosheets; the vermiculite nanosheets are obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment and stripping; wherein 0.1-10 mol/L alkali metal salt modifier is added per mg vermiculite. The concentration of the alkali metal salt modifier added per milligram of vermiculite, below 0.1 mole per liter, results in a thickening of the vermiculite flakes produced; if the concentration of alkali metal modifier is higher than 10 moles per liter, a longer washing time is required to wash the alkali metal modifier away.
The specific preparation method of the vermiculite nanosheet comprises the following steps:
s1: adding vermiculite into an alkali metal salt solution modifier to expand the vermiculite;
s2: separating after expansion, and collecting upper layer colloidal slurry;
s3: and carrying out ultrasonic treatment on the upper layer colloidal slurry, and stripping to obtain the vermiculite nanosheet.
Preferably, the alkali metal salt solution modifier is one or more of a lithium salt solution, a potassium salt solution or a sodium salt solution.
More preferably, the alkali metal salt solution modifier is one or more of a lithium chloride solution, an ethylenediaminetetraacetic acid lithium solution or a lithium citrate solution. Compared with the vermiculite obtained by a thermal expansion or hydrogen peroxide expansion physical method, the chemically-expanded vermiculite obtained by treatment with the lithium salt modifier can be completely peeled off and has a thinner thickness. The modifier of lithium ethylene diamine tetraacetate and lithium citrate has the best effect on the expansion of vermiculite, so that the order degree of crystals of vermiculite-phlogopite mixed layer minerals, phlogopite and the like in the vermiculite can be reduced, and the vermiculite can be more completely peeled.
Further, the condition of the step S1 is that the reflux heating is carried out for 24-36h at the temperature of 80-90 ℃.
Further, in step S3, the duration of ultrasonic treatment is 0.3-0.6h.
The invention also provides application of the iron death inducer as an anti-tumor medicament in regulating tumor microenvironment.
The beneficial effects of the invention include:
the iron death inducer provided by the invention has good biocompatibility and strong cancer cell killing effect, can be used for synthesizing tumor treatment medicines, and has good application prospect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows a case of a difference H 2 O 2 Absorption spectrum of TMB at concentration;
FIG. 2 shows a case of a difference H 2 O 2 Absorbance change at 650nm at concentration;
FIG. 3 is a graph of the absorption spectrum of DTNB at different GSH concentrations;
FIG. 4 is a graph of the change in absorbance at 412nm for different GSH concentrations;
FIG. 5 shows the results of the measurement of the intracellular iron content of MC38 in example 4;
FIG. 6 shows the results of the assay for GPX4 activity in MC38 cells of example 5;
FIG. 7 shows the results of tumor tissue analysis in example 6.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
An iron death inducer comprising vermiculite nanoplates. The vermiculite nanosheet is obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment and stripping, wherein 0.1-10 mol/L of alkali metal salt modifier is added into each milligram of vermiculite. The thickness of the vermiculite nano sheet is 1.0nm, the length is 310nm, and the width is 315nm.
The preparation method comprises the following steps:
s1: adding 50mg of commercial vermiculite powder into 5 mol/L lithium chloride solution, heating under reflux at 80 ℃ for 24h, and intercalating the vermiculite powder to expand the vermiculite powder;
s2: after the swelling is finished, centrifuging at the rotating speed of 3000rpm for 20 minutes, and collecting the upper layer colloidal slurry;
s3: and (4) carrying out ultrasonic treatment on the upper layer colloidal slurry for multiple times, wherein the ultrasonic treatment time is 0.5h, and stripping to obtain the vermiculite nanosheet.
The iron death inducer provided by the embodiment can be used as an anti-tumor drug for regulating the tumor microenvironment and realizing the anti-tumor effect.
Example 2
An iron death inducing agent comprising vermiculite nanoplates. The vermiculite nanosheet is obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment, wherein 0.1-10 mol/L of alkali metal salt modifier is added into each mg of vermiculite. The thickness of the vermiculite nano-sheet is 1.2nm, the length is 305nm, and the width is 335nm.
The preparation method comprises the following steps:
s1: adding 50mg of commercial vermiculite powder into 100 mol/L of lithium ethylenediamine tetraacetate solution, refluxing and heating at 85 ℃ for 24h, and performing intercalation treatment on the vermiculite powder to expand the vermiculite powder;
s2: after the expansion is finished, centrifuging for 30 minutes at the rotating speed of 3500rpm, and collecting the upper layer colloidal slurry;
s3: and (4) carrying out ultrasonic treatment on the upper layer colloidal slurry for multiple times, wherein the ultrasonic treatment time is 0.3h, and stripping to obtain the vermiculite nanosheet.
The iron death inducer provided by the embodiment can be used as an anti-tumor drug for regulating the tumor microenvironment and realizing the anti-tumor effect.
Example 3
The iron death inducer comprises vermiculite nanosheets, wherein the vermiculite nanosheets are obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment and stripping, wherein XX mol of alkali metal salt modifier is added into each mg of vermiculite. The thickness of the vermiculite nano-sheet is 1.3nm, the length is 335nm, and the width is 305nm.
The preparation method comprises the following steps:
s1: adding 50mg of commercial vermiculite powder into 500 mol/L lithium citrate solution, heating under reflux at 90 ℃ for 32h, and intercalating the vermiculite powder to expand the vermiculite powder;
s2: after the swelling is finished, centrifuging for 25 minutes at the rotating speed of 3000rpm, and collecting the upper layer colloidal slurry;
s3: and (4) carrying out ultrasonic treatment on the upper layer colloidal slurry for multiple times, wherein the ultrasonic treatment time is 0.6h, and stripping to obtain the vermiculite nanosheet.
The iron death inducer provided by the embodiment can be used as an antitumor drug for regulating the tumor microenvironment and realizing the antitumor effect.
Experimental example 1 evaluation of peroxidase simulation Activity of vermiculite nanosheets
Degrading H vermiculite nano-sheets (NSs) in PBS solution with the temperature of 37 ℃ and the pH of 6.5 by using 3,3', 5' -Tetramethylbenzidine (TMB) as a dyeing indicator 2 O 2 The peroxidase mimic activity evaluation of (4).
The detection principle is as follows: the vermiculite nanosheet acts as peroxidase, catalyzes TMB to generate a soluble blue product, and simultaneously reacts H 2 O 2 Catalytic formation of H 2 The blue product of O, TMB can usually be measured for absorbance at 620-650 nm.
TMB solution (100. Mu.g/mL) with H 2 O 2 (0, 1, 2.5, 5, 10 and 30 mM) were mixed. NSs (200. Mu.g/mL) was added and reacted at 37 ℃ for 15 minutes for steady state kinetic analysis. For this, the absorbance peak was collected and plotted as H 2 O 2 And (4) concentration graph. Also, with ε =39000M -1 cm -1 Linear Lineweaver-Burk mapping was performed on oxTMB to determine Km and Vmax.
Results referring to FIGS. 1 and 2, in FIG. 1, reference numerals 1 to 6 denote H, respectively 2 O 2 Absorbance results for test groups at concentrations of 30mM, 10mM, 5mM, 2.5mM, 1mM, 0 mM. TMB can be oxidized by OH generated by degrading hydrogen peroxide under the catalysis of vermiculite nanosheets, blue oxTMB is presented, the substrate concentration and reaction rate calculation value of absorbance are drawn to be 650nm, and the kinetic analysis is fitted to Michaelis-Menton,km and the maximum reaction velocity (Vmax) were 3.4mM and 7.97X 10 -8 Ms -1 The enzyme activity of the vermiculite nanosheet is proved to be comparable to peroxidase mimic enzyme in the prior art.
Experimental example 2 evaluation of glutathione oxidase mimic Activity of vermiculite nanosheets
Evaluation of glutathione oxidase mimic activity of vermiculite nanosheets the evaluation of glutathione oxidase mimic activity of vermiculite nanosheets was carried out with 5' -dithiobis (2-nitrobenzoic acid) (DTNB) as an indicator and Glutathione (GSH) as a substrate.
The detection mechanism is as follows: the vermiculite nano-sheets play a role of glutathione oxidase, and DTNB will appear yellow when glutathione GSH is oxidized.
DTNB was mixed with GSH (0, 0.0625,0.125,0.25,0.5 and 1 mM) in DMSO (300. Mu.g/mL). NSs (200. Mu.g/mL) was added and reacted at 37 ℃ for 5 minutes for steady state kinetic analysis. Absorbance was collected for 0-15 minutes and a glutathione concentration chart was drawn. Also, epsilon =13600M using TNB -1 cm -1 Linear Lineweaver-Burk plots were performed to determine Km and Vmax.
The results are shown in FIGS. 3 and 4, and in FIG. 3, reference numerals 1 to 6 represent the results of absorbance in the test groups having GSH concentrations of 0mM, 0.0625mM, 0.125mM, 0.25mM, 0.5mM, and 1mM, respectively. Km and maximum reaction velocity (Vmax) were 1.34mM and 1.35X 10 by plotting substrate concentration versus reaction rate calculations of absorbance at 412nm and fitting to Michaelis-Menton kinetic analysis -4 Ms -1 Proves that the enzyme activity of the vermiculite nanosheet is comparable to that of glutathione oxidation mimic enzyme in the prior art
Experimental example 3 measurement of intracellular iron content
The iron content in the MC38 cells incubated with the different treatments was determined using an iron colorimetric kit (Applygen, E1042). NSs medium (100. Mu.g/mL) was added to the 6-well plates and the cells were incubated for 12h. And finally, detecting the iron content according to a kit using method.
The results are shown in fig. 5, and the NSs-related group detected more iron ions than either PBS (control group) or DCPy group, indicating that NSs induced intracellular iron overload, inducing iron death.
EXAMPLE 4 intracellular GPX4 Activity assay
GPX4 activity in MC38 cells incubated with different treatments was detected using a cell peroxidase detection kit (Beyotime, S0056). Vermiculite nanoplatelet media (100 μ g/mL) was added to 6-well plates and incubated for 0-12h. Finally, the GPX4 activity was detected according to the kit method.
As a result, as shown in fig. 6, glutathione peroxidase 4 (GPX 4), which is a central regulatory factor of iron droop, has an important role in a lipid repair system, and glutathione depletion inactivates GPX4, thereby inducing iron death. NSs are a glutathione oxidase mimic enzyme, leading to glutathione depletion. In addition, NSs can also cause intracellular iron overload, inducing iron death. Cellular GPX4 activity was detected using a cellular glutathione peroxidase assay kit. The activity of GPX4 in the NSs-related group was significantly reduced compared to the control group (control group) and DCPy group. These results indicate that NSs significantly induced iron death by inhibiting GPX4 activity.
Experimental example 5 tumor tissue analysis
Set up 4 sets of experiments, PBS + light, NSs and NSs + light, respectively, and GPX4 antibody stained the differently treated tumor sections. The nuclei were subsequently stained with DAPI. These tumor sections were taken by Olympus microscope (SLIDEVIEW VS 200).
The results are shown in fig. 7, and immunohistochemical staining of tumor tissue sections showed significant down-regulation of GPX4 in the NSs group (significant reduction of brown fraction in the NSs group compared to the PBS group), indicating that the therapeutic effect was significantly attributed to iron death.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. An iron death inducer comprising vermiculite nanoplates;
the thickness of the vermiculite nanosheet is 1.0-1.3nm;
the length of the vermiculite nanosheet is 305-335nm;
the width of the vermiculite nanosheet is 305-335nm;
the preparation method of the iron death inducer comprises the steps of obtaining vermiculite nanosheets; the vermiculite nanosheets are obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment and stripping; wherein 0.1-10 mol/L alkali metal salt modifier is added into each milligram of vermiculite, and the alkali metal salt modifier is one or more of lithium chloride solution, ethylene diamine tetraacetic acid solution or lithium citrate solution.
2. A method of preparing an iron death inducer according to claim 1 comprising obtaining vermiculite nanoplates; the vermiculite nanosheets are obtained by adding vermiculite into an alkali metal ion salt solution for intercalation treatment and stripping; wherein, 0.1-10 mol of alkali metal salt modifier is added into each liter of vermiculite; the alkali metal salt solution modifier is one or more of a lithium chloride solution, an ethylene diamine tetraacetic acid lithium solution or a lithium citrate solution.
3. The method for producing an iron death inducer according to claim 2,
the specific preparation method of the vermiculite nanosheet comprises the following steps:
s1: adding vermiculite into an alkali metal salt solution modifier to expand the vermiculite;
s2: separating after expansion, and collecting upper layer colloidal slurry;
s3: and carrying out ultrasonic treatment on the upper layer colloidal slurry, and stripping to obtain the vermiculite nanosheet.
4. The method for preparing an iron death inducer according to claim 3, wherein the duration of the ultrasonic treatment in step S3 is 0.3 to 0.6h.
5. Use of an iron death inducer according to claim 1 or prepared by a method according to any one of claims 2 to 4 in the manufacture of a medicament for regulating the microenvironment of a tumor.
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| PCT/CN2021/138481 WO2023070885A1 (en) | 2021-10-27 | 2021-12-15 | Ferroptosis inducer, preparation method therefor and application thereof |
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| CN101658533A (en) * | 2008-08-29 | 2010-03-03 | 首都医科大学宣武医院 | Stem cell delivery of antitumor drugs |
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| CN113184866B (en) * | 2021-04-21 | 2022-07-15 | 北京科技大学 | Preparation method of high-temperature oxide nanoparticle spacing vermiculite micro nanosheet |
| CN113181211A (en) * | 2021-05-12 | 2021-07-30 | 南京大学深圳研究院 | Fe2O3@ TA-Pt nano composite material and preparation method and application thereof |
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| CN107815928A (en) * | 2017-10-24 | 2018-03-20 | 贵州大学 | A kind of preparation method of the fire retardant papers based on two-dimensional nano clay layer |
| CN110209013A (en) * | 2019-05-20 | 2019-09-06 | 李日生 | A kind of preparation method of light-sensitive material synergist |
| CN111233509A (en) * | 2020-01-17 | 2020-06-05 | 西南科技大学 | Vermiculite nanosheet and preparation method thereof |
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