CN113975291B - Iron death inducer and preparation method and application thereof - Google Patents

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

A kind of ferroptosis inducer and its preparation method and application

technical field

The invention belongs to the technical field of preparation of antitumor drugs, and in particular relates to a ferroptosis inducer and its preparation method and application.

Background technique

With increasing morbidity and mortality, cancer has become a major global public health problem. At present, the main treatment methods for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, etc., but these commonly used treatment methods also have some disadvantages, such as surgery is prone to recurrence, radiotherapy is not ideal due to radiation resistance, and chemotherapy is due to drug selection. Poor sex can have serious side effects. Therefore, emerging cancer treatment methods such as biological therapy are also constantly emerging in order to solve the problems existing in the prior art.

Ferroptosis is a new type of programmed cell death caused by the accumulation of iron-dependent lipid peroxides, which was first proposed by Professor Brent R. Stockwell in 2012. Recent studies have shown that induction of ferroptosis can be used in cancer therapy, especially in the eradication of aggressive malignancies that are resistant to conventional therapies. With the development of nanobiotechnology, ferroptosis-based anticancer nanomedicine has also made important progress, which are mainly divided into two categories: iron-based nanomaterials and non-iron-based nanomaterials.

Iron-based nanomaterials can enrich iron ions in cells and accelerate the Fenton reaction, thereby increasing the level of reactive oxygen species (ROS) in cells and inducing ferroptosis. The cisplatin-loaded iron oxide nanoprodrug (FePt NP2) constructed by Ma et al. can release cisplatin and Fe ²⁺ /Fe ³⁺ at a specific tumor site, and the Fenton reaction occurs in situ, which significantly increases the intracellular ROS level, thereby Induces tumor cell ferroptosis and enhances anticancer activity. The iron-chelating semiconductor polycomposite nanoparticles (SPFeN) reported by Pu et al. combine photothermal therapy with ferroptosis therapy to enhance the therapeutic effect of cancer. However, although iron-based nanomaterials such as iron oxide nano-prodrugs loaded with cisplatin and iron-chelated semiconductor multi-composite nanoparticles enhance the anti-tumor effect by inducing ferroptosis, they often need to use higher iron doses or combine them with other treatments. It is used in combination with various methods, so it has a complex nanostructure and multi-metal components, and its biological safety is not high.

Non-iron-based nanomaterials can inhibit glutathione peroxidase (GPX4) or induce ferroptosis by increasing the degree of lipid peroxidation in tumor cells through exogenous regulation. Gao et al. used amphiphilic polymer micelles to deliver the GPX4 inhibitor RSL3, and reversed multidrug resistance by synergistically inducing ferroptosis through GPX4 inhibition, glutathione (GSH) attenuation, and lipid peroxidation. Arginine-rich manganese-silicon nanobubbles prepared by Wang et al. can be used as a ferroptosis inducer to achieve tumor-targeted diagnosis and treatment through intracellular GSH depletion. However, although polymer micelles loaded with small molecule ferroptosis inducers and amorphous calcium carbonate composite nano-drugs disclosed in the prior art can effectively reverse drug resistance or further kill tumor cells by inducing ferroptosis, there is drug leakage. with the risk of toxic side effects. Although manganese-silicon nanobubbles with GSH depletion ability can realize tumor diagnosis and treatment at the same time, they can only target arginine succinate synthase-deficient tumor cells, and are not universal to tumor cells. In addition, the residual nanomaterials in the body may have the risk of long-term toxicity and so on.

Therefore, research and development of an ultra-thin vermiculite nanosheet that is universal to tumor cells and has no risk of cytotoxicity to induce ferroptosis will have good application prospects and broad applicability.

Contents of the invention

In order to overcome the defects in the prior art, the present invention proposes a ferroptosis inducer and its preparation method and application. Once the ferroptosis inducer provided by the present invention is absorbed by hypoxic tumor cells, the redox couple (Fe ²⁺ /Fe ³⁺ ) can generate OH and O ₂ through the disproportionation reaction of hydrogen peroxide and the Fenton reaction, and can supply oxygen independently . In addition, the ferroptosis inducer of the present invention can regulate the tumor microenvironment (TME) by consuming glutathione, which can induce ferroptosis in tumor cells, and thus is suitable for preparing ferroptosis-based anticancer nano-medicines.

Specifically, it is realized through the following technical solutions:

A ferroptosis inducer comprising vermiculite nanoplatelets.

Further, the thickness of the vermiculite nanosheets is 1.0-1.3 nm.

Further, the length of the vermiculite nanosheets is 305-335nm.

Further, the width of the vermiculite nanosheets is 305-335nm.

The present invention also provides a preparation method for the above-mentioned ferroptosis inducer, which includes obtaining vermiculite nanosheets; the vermiculite nanosheets are obtained by adding vermiculite to an alkali metal ion salt solution for intercalation and exfoliation; wherein, each mg of Vermiculite is added with 0.1-10 moles per liter of alkali metal salt modifier. In every milligram of vermiculite, if the concentration of the added alkali metal salt modifier is lower than 0.1 mole per liter, the thickness of the prepared vermiculite sheet will be thickened; if the concentration of the alkali metal modifier is higher than 10 moles per liter, longer wash times are required to remove the alkali metal modifier.

The concrete preparation method of described vermiculite nanosheet comprises the following steps:

S1: adding vermiculite to the alkali metal salt solution modifier to expand the vermiculite;

S2: separate after expansion, and collect the upper layer of colloidal slurry;

S3: Ultrasonic treatment is performed on the colloidal slurry in the upper layer, and the vermiculite nanosheets are obtained by peeling off.

Preferably, the alkali metal salt solution modifier is one or more of lithium salt solution, potassium salt solution or sodium salt solution.

More preferably, the alkali metal salt solution modifier is one or more of lithium chloride solution, lithium ethylenediaminetetraacetate solution or lithium citrate solution. The chemically expanded vermiculite treated with the lithium salt modifier, compared with the thermally expanded or hydrogen peroxide-expanded physically expanded vermiculite, the vermiculite flakes can be exfoliated more completely and thinner. Lithium ethylenediamine tetraacetate and lithium citrate modifiers have the best expansion effect on vermiculite, which can reduce the order degree of vermiculite-phlogopite mixed layer minerals and phlogopite crystals in vermiculite, and vermiculite can be stripped more completely.

Further, the condition of step S1 is heating under reflux at a temperature of 80-90° C. for 24-36 hours.

Further, in step S3, the duration of ultrasonic treatment is 0.3-0.6h.

The present invention also provides the application of the above-mentioned ferroptosis inducer as an antitumor drug in regulating tumor microenvironment.

The beneficial effects of the present invention include:

The ferroptosis inducer provided by the invention has good biocompatibility and strong cancer cell killing effect, can be used in the synthesis of tumor treatment drugs, and has good application prospects.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

Figure 1 is _the absorption spectrum of TMB under different _H2O2 concentrations;

Figure 2 shows the change of absorbance at _650nm under different _H2O2 concentrations;

Figure 3 is the absorption spectrum of DTNB under different GSH concentrations;

Fig. 4 is the change of absorbance at 412nm under different GSH concentrations;

Fig. 5 is the iron content measurement result in the MC38 cell of embodiment 4;

Fig. 6 is the detection result of GPX4 activity in the MC38 cell of embodiment 5;

FIG. 7 is the result of tumor tissue analysis in Example 6. FIG.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

A ferroptosis inducer comprising vermiculite nanoplatelets. The vermiculite nano sheet is obtained by adding vermiculite into alkali metal ion salt solution for intercalation treatment and stripping, wherein 0.1-10 moles per liter of alkali metal salt modifier is added to every milligram of vermiculite. The vermiculite nanosheets have a thickness of 1.0 nm, a length of 310 nm, and a width of 315 nm.

Concrete preparation method comprises the following steps:

S1: Add 50 mg of commercial vermiculite powder to 5 moles per liter of lithium chloride solution, heat at 80°C for 24 hours under reflux, and intercalate the vermiculite powder to make it expand;

S2: After the expansion is completed, centrifuge at a speed of 3000rpm for 20 minutes to collect the upper colloidal slurry;

S3: Ultrasonic treatment is performed on the colloidal slurry in the upper layer for 0.5 hours several times, and the vermiculite nanosheets are peeled off.

The ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effect.

Example 2

A ferroptosis inducer comprising vermiculite nanoplatelets. The vermiculite nano sheet is obtained by adding vermiculite to alkali metal ion salt solution for intercalation treatment, wherein 0.1-10 moles per liter of alkali metal salt modifier is added to every milligram of vermiculite. The vermiculite nanosheets have a thickness of 1.2 nm, a length of 305 nm, and a width of 335 nm.

Concrete preparation method comprises the following steps:

S1: Add 50 mg of commercial vermiculite powder to 100 moles per liter of lithium ethylenediamine tetraacetate solution, heat at 85°C for 24 hours under reflux, and intercalate the vermiculite powder to make it expand;

S2: After the expansion is completed, centrifuge at a speed of 3500rpm for 30 minutes to collect the upper colloidal slurry;

S3: Ultrasonic treatment is performed on the upper colloidal slurry for 0.3 hours for several times, and the vermiculite nanosheets are obtained by exfoliation.

The ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effects.

Example 3

A ferroptosis inducer, including vermiculite nanosheets, vermiculite nanosheets are obtained by adding vermiculite to alkali metal ion salt solution for intercalation treatment, and exfoliating, wherein XX moles of alkali metal salt are added to every mg of vermiculite Modifier. The vermiculite nanosheets have a thickness of 1.3nm, a length of 335nm, and a width of 305nm.

Concrete preparation method comprises the following steps:

S1: Add 50 mg of commercial vermiculite powder to 500 moles per liter of lithium citrate solution, heat at 90°C for 32 hours under reflux, and intercalate the vermiculite powder to make it expand;

S2: After the expansion is completed, centrifuge at a speed of 3000rpm for 25 minutes to collect the upper colloidal slurry;

S3: Ultrasonic treatment is performed on the upper colloidal slurry for 0.6 hours for several times, and the vermiculite nanosheets are obtained by peeling off.

The ferroptosis inducer provided in this example can be used as an antitumor drug to regulate the tumor microenvironment to achieve antitumor effects.

Experimental Example 1 Evaluation of the Peroxidase Mimic Activity of Vermiculite Nanosheets

Degradation of H ₂ O ₂ by vermiculite nanosheets (NSs) in a PBS solution at a temperature of 37°C and a pH of 6.5, using 3,3',5,5'-tetramethylbenzidine (TMB) as a staining indicator Evaluation of peroxidase mimetic activity.

The detection principle is: vermiculite nanosheets act as a peroxidase, catalyzing TMB to produce a soluble blue product, and at the same time catalyzing H ₂ O ₂ into H ₂ O, the blue product of TMB can usually measure the absorbance at 620-650nm.

TMB solution (100 μg/mL) was mixed with _H2O2 (0, 1, 2.5, 5 _, 10 and 30 mM). Add NSs (200 μg/mL), react at 37°C for 15 minutes, and perform steady-state kinetic analysis. To do this, absorbance peaks are collected and _{the H2O2} concentration is plotted. In addition, linear Lineweaver-Burk plots were performed on oxTMB using ε = 39000 M ⁻¹ cm ⁻¹ to determine Km and Vmax.

See Figure 1 and Figure 2 for the results. In Figure 1, the numbers 1-6 represent the absorbance results of the test groups with H ₂ O ₂ concentrations of 30mM, 10mM, 5mM, 2.5mM, 1mM, and 0mM, respectively. The OH produced by the degradation of hydrogen peroxide catalyzed by vermiculite nanosheets can oxidize TMB, showing blue oxTMB, calculated by plotting the substrate concentration and reaction rate of absorbance at 650nm and fitting to Michaelis-Menton kinetic analysis, Km and max The reaction velocity (Vmax) is 3.4mM and 7.97×10 ^-8 Ms ^-1 , which proves that the enzyme activity of the vermiculite nanosheets is comparable to that of peroxidase mimetic enzymes in the prior art.

Experimental example 2 Evaluation of glutathione oxidase mimetic activity of vermiculite nanosheets

Evaluation of glutathione oxidase mimetic activity of vermiculite nanosheets with 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as indicator and glutathione (GSH) as substrate for vermiculite Evaluation of glutathione oxidase-mimicking activity of nanosheets.

The detection mechanism is: vermiculite nanosheets act as glutathione oxidase, and when glutathione GSH is oxidized, DTNB will appear yellow.

DTNB was mixed with GSH (0, 0.0625, 0.125, 0.25, 0.5 and 1 mM) in DMSO (300 μg/mL). Add NSs (200 μg/mL), react at 37°C for 5 minutes, and perform steady-state kinetic analysis. Absorbance was collected from 0-15 min and plotted for glutathione concentration. In addition, linear Lineweaver-Burk plots were performed using ε=13600M ⁻¹ cm ⁻¹ of TNB to determine Km and Vmax.

The results are shown in Figures 3 and 4. In Figure 3, the numbers 1-6 represent the absorbance results of the test groups with GSH concentrations of 0mM, 0.0625mM, 0.125mM, 0.25mM, 0.5mM, and 1mM, respectively. By plotting the absorbance of the substrate concentration and the calculated value of the reaction rate at 412nm and fitting to the Michaelis-Menton kinetic analysis, the Km and the maximum reaction velocity (Vmax) are 1.34mM and 1.35×10 ^-4 Ms ^-1 , demonstrating that vermiculite nano The enzymatic activity of the tablet is comparable to that of glutathione oxidation mimetic enzymes in the prior art

Experimental Example 3 Determination of Intracellular Iron Content

Iron content in MC38 cells incubated with different treatments was detected by iron colorimetric kit (Applygen, E1042). NSs medium (100 μg/mL) was added to the 6-well plate, and the cells were incubated for 12 h. Finally, detect the iron content according to the method used in the kit.

The results are shown in Figure 5. Compared with the PBS (control group) or DCPy group, more iron ions were detected in the NSs-related group, indicating that NSs induced intracellular iron overload and induced ferroptosis.

Experimental Example 4 Intracellular GPX4 Activity Determination

Cell peroxidase detection kit (Beyotime, S0056) was used to detect GPX4 activity in MC38 cells incubated with different treatments. Vermiculite nanosheet medium (100 μg/mL) was added to the 6-well plate, and then incubated for 0-12h. Finally, detect GPX4 activity according to the method used in the kit.

The results are shown in Figure 6. As a central regulator of ferroptosis, glutathione peroxidase 4 (GPX4) plays an important role in the lipid repair system. Glutathione consumption can inactivate GPX4, thereby inducing ferroptosis . NSs is a glutathione oxidase-mimicking enzyme that leads to glutathione depletion. In addition, NSs can also cause intracellular iron overload and induce ferroptosis. Cellular GPX4 activity was detected using a cellular glutathione peroxidase assay kit. Compared with the control group (control group) and the DCPy group, GPX4 activity was significantly reduced in the NSs-related group. These results indicated that NSs significantly induced ferroptosis by inhibiting GPX4 activity.

Experimental example 5 Tumor tissue analysis

Four groups of experiments were set up, which were PBS group, PBS+light (light irradiation) group, NSs group and NSs+light (light irradiation) group, and GPX4 antibody stained tumor sections with different treatments. Nuclei were then stained with DAPI. These tumor sections were taken by Olympus microscope (SLIDEVIEW VS200).

The results are shown in Figure 7. Immunohistochemical staining of tumor tissue sections showed that GPX4 was significantly down-regulated in the NSs group (the brown part of the NSs group was significantly less than that in the PBS group), indicating that the therapeutic effect was significantly attributed to ferroptosis.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

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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