CN116036129A

CN116036129A - Novel cGAS-STING agonist and application thereof

Info

Publication number: CN116036129A
Application number: CN202310015563.7A
Authority: CN
Inventors: 刘庄; 程亮; 雷华俐; 李衢广
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2023-01-04
Filing date: 2023-01-04
Publication date: 2023-05-02

Abstract

The invention discloses a novel cGAS-STING agonist and application thereof, and provides a series of metal cations and anions with the activation capacity of a cGAS-STING channel, wherein the cations and anions can be combined in pairs to construct a nano material with the activation potential of the cGAS-STING channel. The invention also provides a method for screening materials with the activation potential of the cGAS-STING pathway. The novel cGAS-STING pathway activator manganese molybdate nano-particles can effectively kill tumor cells, activate the cGAS-STING pathway, stimulate dendritic cells to mature, and enhance tumor metal immunotherapy. Therefore, the manganese molybdate nano-particles can simultaneously play the roles of killing tumor cells and activating immune response.

Description

Novel cGAS-STING agonist and application thereof

Technical Field

The invention relates to a novel cGAS-STING agonist and application thereof, belonging to the technical field of nano-drugs.

Background

Cancer immunotherapy is the inhibition of tumor growth and metastasis by activating the host's immune system, thereby achieving an anti-tumor immune response. Although cancer immunotherapy has recently achieved some clinical success, only a small fraction of patients with solid tumors respond to immunotherapy. One key cause of failure in cancer immunotherapy is the immunosuppressive tumor microenvironment, which is characterized by poor infiltration of proinflammatory immune cells, including tumor infiltrating cytotoxic T lymphocytes, proinflammatory M1 phenotype macrophages and dendritic cells. In order to enhance the anti-tumor immune response, several cancer treatments have been developed. Among them, the cGAS-STING pathway has been found and widely used for immune activation in cancer immunotherapy. When cyclic GMP-AMP synthetase (cGAS) detects double stranded DNA that should not be present in the cytoplasm, it catalyzes the production of cyclic small molecules of adenosine monophosphate (cGAMP). Meanwhile, dimeric interferon gene (STING) binds to cGAMP, changes STING conformation, recruits and phosphorylates TANK binding kinase 1 (TBK 1) protein, further activates interferon regulatory factor 3 (IRF 3), induces secretion of type I interferon, and activates innate immunity. Since malignant tumors are usually accompanied by cytoplasmic chromatin fragmentation and micronuclei formation, the DNA leakage of cancer cells is significantly higher than that of normal cells, and thus activation of cGAS-STING is of particular importance in cancer immunotherapy

In recent years, metal cations have been of interest for their particular biological effects in tumor therapy. More importantly, manganese ions (Mn ²⁺ ) And zinc ion (Zn) ²⁺ ) Has the ability to activate the cGAS-STING pathway, and is likely to act as an immune activator. Thus, designAnd construct various Mn-containing compositions ²⁺ Or Zn ²⁺ Further allowing these metal ions to be transported into the tumor along with other therapeutic molecules. However, although the role of metal cations in cGAS-STING pathway activation has been widely demonstrated, it is largely unknown whether metal anions have similar activity in immune activation. In recent years, unique biological processes and protein crown bridge type transport-conversion-bioavailability chains induced by molybdenum in molybdenum enzyme nano materials prove the unique fate and important roles of metal anions in organisms. However, none of the nano-drugs having an immune activating metal cation and an anion in the prior art has been designed, and it is significant to use the nano-drugs having an immune activating metal cation and an anion for anti-tumor metal immunotherapy.

Disclosure of Invention

Aiming at the defect that metal anions are used as the cGAS-STING pathway activator in the prior art, the invention provides a series of metal cations and anions with the activation potential of the cGAS-STING pathway, and synthesizes a novel cGAS-STING pathway activator which is used for synergistic tumor metal immunotherapy.

A first object of the present invention is to provide a cGAS-STING pathway activator comprising a metal cation selected from Mn and/or a metal anion ²⁺ 、Zn ²⁺ And Ga ³⁺ The metal anion is selected from MoO ₄ ^2- 、WO ₄ ^2- And VO (Voice over Internet protocol) ₄ ^3- 。

Further, the cGAS-STING pathway activator is a manganese molybdate nanoparticle.

Further, the cationic metal source in the manganese molybdate nano-particles is an Mn source, and the anionic metal source is an Mo source.

Further, the molar ratio of the Mn source to the Mo source is 1: (0.5-1.5).

In the invention, the chemical formula of the manganese molybdate nano-particles is MnMoO ₄ Wherein the cation Mn ²⁺ With anions MoO ₄ ^2- Bonding is by covalent bonds.

Further, the manganese molybdate nano-particles are prepared by the following method:

a) Mixing an anionic metal source Mo source, dodecyl glycol and dibenzyl ether, and heating to 120-160 ℃;

b) Maintaining the temperature at 120-160deg.C, adding oleylamine and oleic acid;

c) Heating to 240-280 ℃, adding a cationic metal source Mn source, and reacting to obtain the manganese molybdate nano-particles.

Further, steps A) to C) are all carried out under nitrogen protection.

Further, in step A), the anionic metal source is 0.5 to 1.5 mmol/l, the dodecyl glycol is 0.75 to 2.25 g/l, and the dibenzyl ether is 10 to 30 ml/l.

Further, in step B), the molar ratio of oleylamine to oleic acid is 1 (0.5-1.5).

Further, in step C), the cationic metal source is 0.5-1.5 mmoles/liter, the temperature of the reaction is greater than 260 ℃, and the time of the reaction is 15-45 minutes.

Further, in step C), the reaction further includes: ethanol was added to the obtained product, and the precipitate was collected by centrifugation and washed.

It is a second object of the present invention to provide a tumor metal immunotherapeutic agent comprising the cGAS-STING pathway activator.

The third object of the present invention is to provide a screening method of cGAS-STING pathway activator, comprising the steps of:

s1, 293-Dual ^TM mSTING cells were inoculated in well plates, cGAS plasmid was added and transfected;

after S2, incubation, cGAS transfected 293-Dual was obtained ^TM mSTING cells;

s3, adding ion solutions with different concentrations to incubate, wherein the ion solutions are selected from Mn ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Zn ²⁺ 、Na ⁺ 、Cu ²⁺ 、Ga ³⁺ 、MoO ₄ ^2- 、NO ₃ ^- 、WO ₄ ^2- 、SO ₄ ^2- 、Cl ^- 、VO ₄ ^3- 、VO ₃ ^- One or two ions;

s4, collecting samples after incubation, centrifuging to obtain supernatant, incubating the supernatant with a luciferase detection reagent, detecting the bioluminescence signal intensity of each group by using an enzyme-labeled instrument or a small animal optical imaging system after incubation, and judging the activation capacity of the cGAS-STING channel according to the bioluminescence signal intensity.

Further, in the S1 step, 293-Dual ^TM The density of mSTING cells is 15-25 ten thousand cells/well and the concentration of cGAS plasmid is 300-700 ng/well.

Further, in the step S2, the incubation time is 16-48 hours.

Further, in the step S3, the ion concentration is 0 to 500. Mu. Mol.

Further, in the step S4, the incubation time is 16-30 hours.

The beneficial effects of the invention are as follows:

(1) The invention provides a screening method of a novel cGAS-STING pathway activator, which can be used for screening materials with the activation potential of the cGAS-STING pathway, including but not limited to various metal anions and cations, can intuitively judge the activation capacity of the cGAS-STING pathway by only utilizing bioluminescence intensity, and provides convenience for exploring the novel cGAS-STING pathway activator.

(2) The present invention provides a series of metal cations and anions having the ability to activate the cGAS-STING pathway, including but not limited to the cation Mn ²⁺ 、Zn ²⁺ And Ga ³⁺ MoO in anions ₄ ^2- 、WO ₄ ^2- And VO (Voice over Internet protocol) ₄ ^3- . The cations and anions can be combined in pairs to construct nanomaterials with the activation potential of the cGAS-STING pathway.

(3) Currently, most of the reported nanoparticles have only a function of killing tumor cells, and cannot provide an immune agonist function, whereas commercial immune agonists such as Cyclic Dinucleotides (CDNs) have only a function of activating immune responses, and do not have the capability of killing tumor cells per se. The novel cGAS-STING pathway activator manganese molybdate nano-particles can effectively kill tumor cells, activate the cGAS-STING pathway, stimulate dendritic cells to mature, and enhance tumor metal immunotherapy. Therefore, the manganese molybdate nano-particles can simultaneously play the roles of killing tumor cells and activating immune response.

Description of the drawings:

FIG. 1 is a schematic diagram of a novel screening method for cGAS-STING pathway activator in example 1 of the present invention;

FIG. 2 is a schematic diagram of the activation principle of the cGAS-STING pathway in example 1 of the present invention;

FIG. 3 is a schematic representation of 293-Dual after incubation with various metal cations with novel activation potential for the cGAS-STING pathway in example 2 of the present invention ^TM Bioluminescence intensity of mSTING cells;

FIG. 4 is a schematic representation of 293-Dual after incubation with various anions with novel activation potential of the cGAS-STING pathway according to example 2 of the present invention ^TM Bioluminescence intensity of mSTING cells;

FIG. 5 is a schematic diagram of novel cGAS-STING pathway activator Mn in example 3 of the present invention ²⁺ And MoO ₄ ^2- 293-Dual after treatment ^TM Bioluminescence intensity of mSTING cells;

FIG. 6 is a schematic diagram of novel cGAS-STING pathway activator Mn in example 3 of the present invention ²⁺ And MoO ₄ ^2- Changes in key protein phosphorylation levels in the cGAS-STING pathway following treatment;

FIG. 7 is a schematic diagram of novel cGAS-STING pathway activator Mn in example 3 of the present invention ²⁺ And MoO ₄ ^2- Is effective in stimulating dendritic cell maturation;

FIG. 8 is a schematic diagram of novel cGAS-STING pathway activator Mn in example 3 of the present invention ²⁺ And MoO ₄ ^2- Content of ifnβ in the supernatant of dendritic cells after treatment;

FIG. 9 is a schematic diagram showing the synthesis and surface modification of novel cGAS-STING pathway activator manganese molybdate nanoparticles according to example 4 of the present invention;

FIG. 10 is a transmission electron microscope image of novel cGAS-STING pathway activator manganese molybdate nanoparticles synthesized in example 4 of the present invention;

FIG. 11 is an X-ray diffraction pattern of novel manganese molybdate nanoparticles for the cGAS-STING pathway activator synthesized in example 4 of the present invention;

FIG. 12 is a graph showing the ability of manganese molybdate nanoparticles, a novel cGAS-STING pathway activator synthesized in example 5 of the present invention, to stimulate dendritic cell maturation;

FIG. 13 is a graph showing the change in key protein phosphorylation levels in the cGAS-STING pathway after treatment with manganese molybdate nanoparticles, a novel cGAS-STING pathway activator synthesized in example 5 of the present invention;

FIG. 14 is a graph showing the ability of a DTNB probe to detect glutathione consumption of novel cGAS-STING pathway activator manganese molybdate nanoparticles in example 6 of this invention;

FIG. 15 shows the killing effect of the novel cGAS-STING pathway activator manganese molybdate nanoparticle of example 7 of the present invention on three mouse tumor cells;

FIG. 16 is a graph showing malondialdehyde content in colon cancer cells of mice treated with manganese molybdate nanoparticle as a novel cGAS-STING pathway activator in example 8 of the present invention;

FIG. 17 is a graph showing the amount of glutathione peroxidase 4 expressed in colon cancer cells of mice after treatment with the novel cGAS-STING pathway activator manganese molybdate nanoparticle of example 8 of the present invention;

FIG. 18 is a graph showing tumor growth in mice after intratumoral injection of novel cGAS-STING pathway activator manganese molybdate nanoparticles in example 9 of the present invention;

FIG. 19 is a graph showing tumor growth in mice after tail vein injection of novel cGAS-STING pathway activator manganese molybdate nanoparticles in example 9 of the present invention;

FIG. 20 is the content of mature dendritic cells in the draining lymph nodes of mice tumor after intratumoral injection of novel cGAS-STING pathway activator manganese molybdate nanoparticle in example 10 of the present invention;

FIG. 21 is a graph showing the content of mature dendritic cells in tumor draining lymph nodes of mice after tail vein injection of novel cGAS-STING pathway activator manganese molybdate nanoparticle in example 10 of the present invention.

Detailed Description

The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.

Materials:

six carbon molybdenum (Mo (CO) ₆ ) Purchased from Sigma-Aldrich, CAS number 13939-06-5.

Manganese acetylacetonate (Mn (acac) ₃ ) Purchased from Sigma-Aldrich, CAS number 14284-89-0.

Benzyl ether (99%), purchased from Sigma-Aldrich, CAS number 103-50-4.

Oleic acid (90%), purchased from Sigma-Aldrich, CAS number 112-80-1.

Oleylamine (> 70%) from Sigma-Aldrich, CAS number 112-90-3.

Sodium molybdate (Na) ₂ MoO ₄ ) Purchased from Sigma-Aldrich, CAS number 7631-95-0.

Manganese (II) chloride (MnCl) ₂ ) Purchased from Sigma-Aldrich, CAS number 7773-01-5.

Anhydrous calcium chloride (CaCl) ₂ ) Purchased from Sigma-Aldrich, CAS number 10043-52-4.

Zinc chloride (ZnCl) ₂ ) Purchased from Sigma-Aldrich, CAS number 7646-85-7.

Sodium tungstate dihydrate (Na) ₂ WO ₄ ·2H ₂ O) from Sigma-Aldrich, CAS number 10213-10-2.

Copper sulfate (CuSO) ₄ ) Purchased from Alatine, CAS number 7758-98-7.

Magnesium chloride (MgCl) ₂ ) Purchased from Alatine, CAS number 7786-30-3.

Gallium (III) nitrate nonahydrate (Ga (NO) ₃ ) ₃ ·9H ₂ O) was purchased from Allatin, CAS number 7789-02-8.

Potassium nitrate (KNO) ₃ ) The CAS number is 7757-79-1, national pharmaceutical Congress chemical Co., ltd.

Sodium vanadate (v) (NaVO ₄ ) Purchased from Sigma-Aldrich, CAS number 13718-26-8.

1, 2-dodecanediol (> 93%) was purchased from Techniaria (Shanghai) chemical industry development Co., ltd., CAS number 1119-87-5 (TCI). All chemicals were analytical grade and were used without further purification.

Luciferase assay reagent (QUANTI-Luc) ^TM ) Purchased from InvivoGen, cat. Code: rep-qlc1.

TBK1/NAK (E8I 3G) Rabbit mAb was purchased from Cell Signaling Technology (CST), material code 38066S.

STING (D2P 2F) Rabbit mAb was purchased from Cell Signaling Technology (CST), material code 13647S.

The Phospho-STING (Ser 366) (E9 A9K) rabit mAb was purchased from Cell Signaling Technology (CST), material code 50907S.

Phospho-TBK1/NAK(Ser172)(D52C2)XP ^R The rabit mAb was purchased from Cell Signaling Technology (CST), material code 5483S.

And (3) cells:

4T1 mouse breast cancer cells (SCSP-5056), CT26 colon cancer cells (TCM 37) and B16F10 mouse melanoma cells were from Shanghai institute of bioscience cell bank at the national academy of sciences. 293-Dual ^TM mSTING cells were from InvivoGen, USA.

Mice:

Balb/C mice and C57 female mice were purchased from Kwangsi laboratory animals Inc. All animal experiments were performed according to the experimental protocol approved by the university of su laboratory animal center. Mice were housed in a single ventilated cage with 5 mice per group, and the room temperature was constant (21.+ -. 1 ℃) and the relative humidity was constant (40-70%) in the 12 hours light-dark cycle (8:00-20:00 light; 20:00-8:00 days black). All mice were free to gain access to food and water.

Definition of terms

And (3) calculating statistics: calculating P values shows component statistical differences, where "×" in the figures is a sign of significant differences, indicating P values <0.05, × indicating P values <0.01, × indicating P values <0.001, × indicating P values <0.0001.

Example 1: screening method of novel cGAS-STING pathway activator

As shown in FIG. 1, first, 293-Dual is applied ^TM mSTING cells at 2 x 10 ⁵ Cells/ml were seeded in 24-well plates, incubated with cGAS plasmid for 24 hours, and transfected. The second step involves combining a substance having the potential to activate the cGAS-STING pathway with 293-Dual which was successfully transferred into the cGAS plasmid ^TM The mSTING cells were incubated for an additional 24 hours. In the third step, the above cells were centrifuged at 1800 rpm for 5 minutes with the medium, 100. Mu.l of the centrifuged cell supernatant was taken and 25. Mu.l of luciferase assay reagent (QUANTI-Luc) was added ^TM ) Co-incubation was performed in 96-well plates. And finally, detecting the intensity of the bioluminescence signal by using an enzyme-labeled instrument.

The principle is as shown in fig. 2:

1. when cyclic GMP-AMP synthetase (cGAS) detects double stranded DNA or certain metal ions that should not be present in the cytoplasm, it catalyzes the production of small cyclic adenosine monophosphate molecules (cGAMP). Meanwhile, dimeric interferon gene (STING) binds to cGAMP, changes STING conformation, recruits and phosphorylates TANK binding kinase 1 (TBK 1) protein, further activates interferon regulatory factor 3 (IRF 3), induces secretion of type I interferon, and activates innate immunity.

2、293-Dual ^TM mSTING cells are unable to stably express the cGAS gene. And 293-Dual ^TM Incubation of mSTING cells with the cGAS plasmid and transfection with lipo3000 transfection kit allowed 293-Dual ^TM The mSTING cell can stably express the cGAS gene, and can activate 293-Dual after being incubated with metal ion with the activation capability of the cGAS-STING channel ^TM The cGAS-STING pathway in mssting cells, secretion of luciferase into cell supernatants, and QUANTI-Luc ^TM The contained coelenterazine substrates react to form bioluminescence resembling firefly light and can therefore be used to detect metal ions with cGAS-STING pathway activation capability.

Example 2: novel cGAS-STING pathway activators

The novel screening method for cGAS-STING pathway activators in example 1 was used to explore the activation of cGAS-STING pathway for a variety of anions and cations.

The first step: 293-Dual which successfully transferred the cGAS plasmid ^TM mSTING cells were treated with various concentrations of ionic solutions (Mn ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Zn ²⁺ 、Na ⁺ 、Cu ²⁺ 、Ga ³⁺ 、MoO ₄ ^2- 、NO ₃ ^- 、WO ₄ ^2- 、SO ₄ ^2- 、Cl ^- 、VO ₄ ^3- And VO (Voice over Internet protocol) ₃ ^- ) Incubation was performed.

Second, after 24 hours, 100. Mu.l of the cell supernatant and 25. Mu.l of luciferase assay reagent (QUANTI-Luc) were taken ^TM ) And (3) co-incubating, and detecting the intensity of each group of bioluminescence signals by using an enzyme-labeled instrument, wherein the stronger the bioluminescence signals are, the stronger the activation capability of the cGAS-STING channel of the ion is.

As shown in FIG. 3 and Table 1, the cations are mixed with Mn ²⁺ 、Zn ²⁺ And Ga ³⁺ 293-Dual after incubation ^TM mSTING cells showed strong bioluminescence signals, indicating that all three had the ability to activate the cGAS-STING pathway, wherein Mn ²⁺ The highest bioluminescence signal of (C) indicates that the cGAS-STING pathway is most active. And in the anions shown in FIG. 4 and Table 2, with MoO ₄ ^2- 、WO ₄ ^2- And VO (Voice over Internet protocol) ₄ ^3- 293-Dual after incubation ^TM mSTING cells showed strong bioluminescence signals, indicating that all three had the ability to activate the cGAS-STING pathway, moO ₄ ^2- Is the most powerful.

Table 1: 293-Dual after incubation with Metal cations ^TM Relative bioluminescence intensity of mSTING cells

Table 2: 293-Dual after incubation with anions ^TM Relative bioluminescence intensity of mSTING cells

Example 3: novel activation mechanism of cGAS-STING pathway activator and ability to stimulate dendritic cell maturation

(1) Novel mechanism of activation of cGAS-STING pathway activators

The cation Mn with the strongest cGAS-STING pathway activation ability selected in example 2 was selected ²⁺ And an anionic MoO ₄ ^2- The activation mechanism of cGAS-STING pathway and the ability to stimulate dendritic cell maturation after the combination of both were studied separately.

Experimental grouping:

example 3.1: blank control group;

example 3.2: moO (MoO) ₄ ^2- Ionic solution (200 μm concentration);

example 3.3: mn (Mn) ²⁺ Ionic solution (200 μm concentration);

example 3.4: moO (MoO) ₄ ^2- Ion and Mn ²⁺ Ion mixed solution (both concentrations are 200 μm);

first, the cation Mn was screened using the novel cGAS-STING pathway activator of example 1 ²⁺ And an anionic MoO ₄ ^2- The activation ability of the cGAS-STING pathway was validated after each and the combination of the two studies, respectively. As shown in FIG. 5, the fluorescence signal intensities of examples 3.2 and 3.3 are much greater than those of example 3.1, illustrating Mn in examples 3.2 and 3.3 ²⁺ And MoO ₄ ^2- The cGAS-STING pathway is well activated. Further, the fluorescence signal intensity of example 3.4 was higher than that of examples 3.2 and 3.3, indicating Mn ²⁺ And MoO ₄ ^2- After the two are combined, the activation capacity of the cGAS-STING channel is improved.

Next, mn was detected by immunoblotting ²⁺ 、MoO ₄ ^2- And the phosphorylation levels of the key proteins TANK-binding kinase 1 (TBK 1), interferon regulatory factor 3 (IRF 3) and interferon gene stimulating factor (STING) proteins in the cGAS-STING pathway after the combination of the two to characterize the activation capacity of the cGAS-STING pathway. From FIG. 6As can be seen, the protein content of p-TBK1, p-IRF3 and p-STING in examples 3.2 and 3.3 are significantly higher than in example 3.1, illustrating Mn in examples 3.2 and 3.3 ²⁺ And MoO ₄ ^2- Can effectively up-regulate the phosphorylation levels of TBK1, IRF3 and STING proteins. Furthermore, the protein expression levels of p-TBK1, p-IRF3 and p-STING in example 3.4 were significantly higher than those in examples 3.2 and 3.3, indicating Mn ²⁺ And MoO ₄ ^2- After the two are combined, the phosphorylation content of the three proteins can be further improved.

(2) Novel cGAS-STING pathway activators' ability to stimulate dendritic cell maturation

Experimental grouping:

example 3.5: blank control group;

example 3.6: moO (MoO) ₄ ^2- An ionic solution;

example 3.7: mn (Mn) ²⁺ An ionic solution;

example 3.8: moO (MoO) ₄ ^2- Ion and Mn ²⁺ Mixing the ions;

next, novel cGAS-STING pathway activators were tested for their ability to stimulate dendritic cell maturation. Mn at different concentrations ²⁺ And MoO ₄ ^2- And detecting the proportion of mature dendritic cells 16 hours after incubation of the mixture of the two with dendritic cells. As shown in FIG. 7, the proportion of mature dendritic cells in examples 3.6 and 3.7 was significantly higher than in example 3.5, indicating Mn ²⁺ And MoO ₄ ^2- Can effectively increase the proportion of mature dendritic cells. Further examples 3.8, in which the proportion of mature dendritic cells was higher than examples 3.6 and 3.7, demonstrate Mn ²⁺ And MoO ₄ ^2- After combination, dendritic cells can be further stimulated to mature.

Finally, mn is detected ²⁺ And MoO ₄ ^2- And the content of interferon beta (IFNbeta) in the cell supernatant after 16 hours after incubation of the dendritic cells with the mixture of the two. As can be seen from FIG. 8, the content of interferon beta in the supernatant of dendritic cells in examples 3.2 and 3.3 was significantly higher than that in example 3.1, indicating Mn ²⁺ And MoO ₄ ^2- Can effectively activate the cGAS-STING pathway and promote the secretion of IFN beta by dendritic cells. Further, the highest content of interferon beta in the dendritic cell supernatant in example 3.4 indicates Mn ²⁺ And MoO ₄ ^2- The two can better stimulate dendritic cells to mature after being combined, and induce secretion of interferon beta.

Example 4: synthesis and characterization of novel cGAS-STING pathway activator manganese molybdate nanoparticles

The manganese molybdate nano-particles are synthesized by adopting a high-temperature pyrolysis method. As shown in FIG. 9, first, 1.5 g of dodecyl alcohol, 20 ml of dibenzyl ether, and 1 mmol of molybdenum hexacarbonyl were charged into a three-necked flask, and heated to 120 to 160℃under the protection of nitrogen gas, and kept for 20 minutes. In the second step, 1 ml of oleylamine and 1 ml of oleic acid are added, and the mixture is kept at 120-160 ℃ for 5 minutes. Third, heating to 260 ℃ and adding manganese acetylacetonate, and reacting for 30 minutes. And fourthly, adding absolute ethyl alcohol into the reaction product, centrifuging to obtain precipitate, and repeatedly washing with cyclohexane for multiple times to obtain manganese molybdate nano particles with the particle size of 5 nanometers.

The manganese molybdate nanoparticle was then characterized, and a transmission electron microscope image as shown in fig. 10, the manganese molybdate nanoparticle being a nanodot structure. The X-ray diffraction pattern is shown in FIG. 11 to show that manganese molybdate nano-particles exist obviously MnMo ₄ Characteristic peaks, which indicate that the novel cGAS-STING pathway activator manganese molybdate nanoparticle was successfully synthesized.

Example 5: novel cGAS-STING pathway activator manganese molybdate nanoparticle cGAS-STING pathway activation capability and mechanism

Experimental grouping:

example 5.1: blank control group, negative control;

example 5.2: manganese molybdate nano-particles with the concentration of 12.5 micromoles;

example 5.3: manganese molybdate nano particles with the concentration of 25 micromoles;

example 5.4: manganese molybdate nano-particles with the concentration of 50 micromoles;

example 5.5: manganese molybdate nano-particles with the concentration of 100 micromoles;

example 5.6: LPS, positive control;

the novel cGAS-STING pathway activator manganese molybdate prepared in example 4 was ultrasonically dispersed in methylene chloride, and DSPE-PEG was added _5k The reaction was stirred at room temperature for 30 minutes, and after removing methylene chloride, the mixture was ultrasonically dispersed in water to obtain an aqueous manganese molybdate solution. The proportion of mature dendritic cells was measured after incubating manganese molybdate nanoparticles with different concentrations with the dendritic cells for 16 hours. As shown in FIG. 12, the proportion of mature dendritic cells in examples 5.2, 5.3, 5.4 and 5.5 was significantly higher than that in example 5.1, which is comparable to that in example 5.6, indicating that manganese molybdate, a cGAS-STING pathway activator, was effective in stimulating dendritic cell maturation.

Further immunoblotting was used to detect the phosphorylation levels of manganese molybdate nanoparticles on the key proteins TBK1, IRF3 and STING in the cGAS-STING pathway, to explore the activation mechanism of the cGAS-STING pathway. As can be seen from fig. 13, the protein expression amounts of p-TBK1, p-IRF3 and p-STING were significantly increased after 16 hours of manganese molybdate nanoparticle treatment, and increased with increasing concentration of manganese molybdate nanoparticle, indicating that manganese molybdate nanoparticle was able to stimulate dendritic cell maturation by effectively up-regulating phosphorylation levels of TBK1, IRF3 and STING proteins.

Example 6: glutathione consumption capability of novel cGAS-STING pathway activator manganese molybdate nanoparticle

The novel cGAS-STING pathway activator manganese molybdate nanoparticle prepared in example 4 (concentration 100 micromolar) was incubated with glutathione (concentration 1 millimolar) and detected with a 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) probe. As can be seen from fig. 14, the characteristic absorption peak of 5,5' -dithiobis (2-nitrobenzoic acid) at 412 nm is significantly reduced with the prolonged incubation time, which indicates that the manganese molybdate nanoparticle has a good glutathione consumption function.

Example 7: tumor cell killing ability of novel cGAS-STING pathway activator manganese molybdate nanoparticle

The novel cGAS-STING pathway activator manganese molybdate nanoparticle prepared in example 4 (concentrations of 0, 5, 10, 25, 50, 75 and 100 μg/ml, respectively) was incubated with three tumor cells, mouse colon cancer cell (CT 26), mouse breast cancer cell (4T 1) and mouse melanoma cell (B16F 10), respectively, for 12 hours, and cell viability was examined. As can be seen from fig. 15, the survival rates of CT26, 4T1 and B16F10 cells were all significantly reduced with increasing concentration of manganese molybdate nanoparticles, indicating that the manganese molybdate nanoparticles had a good killing effect on mouse tumor cells.

Example 8: novel ability of cGAS-STING pathway activator manganese molybdate nanoparticles to induce iron death of tumor cells

The novel cGAS-STING pathway activator prepared in example 4 was first incubated for 12 hours at concentrations of 0, 25, 50, 100 and 200 μg/ml respectively for manganese molybdate nanoparticles to show the level of lipid peroxidation in CT26 mouse colon cancer cells. As can be seen from fig. 16, after the manganese molybdate nanoparticle treatment, the expression level of malondialdehyde is significantly reduced, and the expression level of malondialdehyde is increased along with the increase of the concentration of the manganese molybdate nanoparticle, which indicates that the manganese molybdate nanoparticle can effectively increase the expression level of malondialdehyde in colon cancer cells of mice, and increase the lipid peroxidation level in the cells.

And then detecting the content of glutathione peroxidase 4 in the colon cancer cells of the mice after the manganese molybdate nano-particles are incubated for 12 hours by using immunoblotting. As can be seen from fig. 17, after the manganese molybdate nanoparticle (concentration of 100 μg/ml) was treated for 12 hours, the expression level of glutathione peroxidase 4 in the colon cancer cells of the mice was significantly reduced, which suggests that the manganese molybdate nanoparticle can effectively induce iron death of tumor cells of the mice by inhibiting the expression of glutathione peroxidase 4 in the cells and increasing the lipid peroxidation level in the cells.

Example 9: novel in-vivo tumor immunotherapy effect of cGAS-STING pathway activator manganese molybdate nano-particles

Experimental grouping:

example 9.1: blank control group, intratumoral injection of PBS, injection time of day 0, day 2 and day 4, volume 50 microliters;

example 9.2: intratumoral injection of cGAS-STING pathway activator manganese molybdate nanoparticles once, with injection time of day 0 and dosage of 5 mg/kg;

example 9.3: intratumoral injection of cGAS-STING pathway activator manganese molybdate nanoparticles twice at doses of 5 mg/kg at day 0 and day 2;

example 9.4: intratumoral injection of cGAS-STING pathway activator manganese molybdate nanoparticles three times at doses of 5 mg/kg for

days

0, 2 and 4;

example 9.5: blank control group, tail vein injected with PBS, injection time of 0 day, 2 days and 4 days, volume of 200 microliters;

example 9.6: the manganese molybdate nano-particles of the cGAS-STING pathway activator are injected into the tail vein three times, the injection time is 0 day, 2 days and 4 days, and the dosage is 10 mg/kg;

example 9.7: the manganese molybdate nano-particles of the cGAS-STING pathway activator are injected into the tail vein three times, the injection time is 0 day, 2 days and 4 days, and the dosage is 15 mg/kg;

example 9.8: the manganese molybdate nano-particles of the cGAS-STING pathway activator are injected into the tail vein three times, the injection time is 0 day, 2 days and 4 days, and the dosage is 20 mg/kg;

first, a mouse subcutaneous colon cancer cell was injected into a balb/c mouse to construct a mouse subcutaneous colon cancer model. Four experimental groups were set up, one control group (example 9.1), one intratumoral injection (example 9.2), two intratumoral injections (example 9.3) and three intratumoral injections (example 9.4) of manganese molybdate nanoparticles, respectively. The control group was intratumorally injected with PBS and the remaining groups were intratumorally injected with the novel cGAS-STING pathway activator manganese molybdate nanoparticle prepared in example 4 at a concentration of 2 mg/ml at a dose of 50 μl at a frequency of once every two days. Tumor volumes were measured at different time points in different experimental groups of mice using vernier calipers. The growth curves of the mouse tumors are shown in fig. 18, the growth rate of the mouse tumors in examples 9.2, 9.3 and 9.4 is obviously slower than that in example 9.1, and the more obvious the inhibition effect on the mouse tumors with the increase of the injection times, the better the tumor immunotherapy effect is shown by the intratumoral injection of the manganese molybdate nano particles at the living level.

And then subcutaneously injecting colon cancer cells of the mice into the balb/c mice to construct a subcutaneous colon cancer model of the mice. Four experimental groups were set up, each control group (example 9.5), and manganese molybdate nanoparticles were injected three times into the tail vein at doses of 10 mg/kg (example 9.6), 15 mg/kg (example 9.7) and 20 mg/kg (example 9.8), each at a frequency of two days. Tumor volumes were measured at different time points in different experimental groups of mice using vernier calipers. Growth curve graph 19 of mouse tumor growth rate of mouse tumors in examples 9.6, 9.7 and 9.8 is significantly slower than that of example 9.5, and the more obvious the inhibition effect on mouse tumor with increasing injection dose, the better tumor immunotherapy effect is shown by manganese molybdate nanoparticle tail vein at living level.

Example 10: novel cGAS-STING pathway activator manganese molybdate nanoparticle ability to stimulate dendritic cell maturation at in vivo level

On the seventh day after treatment, the proportion of mature dendritic cells in the tumor draining lymph nodes of each group of mice in example 9 was examined separately. Fig. 20 shows that the proportion of mature dendritic cells in the tumor draining lymph nodes of mice in examples 9.2, 9.3 and 9.4 is significantly higher than in example 9.1, and increases with increasing intratumoral injection times. Meanwhile, as shown in fig. 21, the proportion of mature dendritic cells in the tumor drainage lymph nodes of the mice in the examples 9.6, 9.7 and 9.8 is significantly higher than that in the example 9.5, and the proportion of mature dendritic cells increases along with the increase of the tail vein injection dose, which indicates that the manganese molybdate nano particles of the cGAS-STING pathway activator can effectively stimulate the maturation of the dendritic cells at the living level, activate the anti-tumor immune response and enhance the anti-tumor effect.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A cGAS-STING pathway activator, wherein said cGAS-STING pathway activator comprises a metal cation selected from the group consisting of Mn and/or a metal anion ²⁺ 、Zn ²⁺ And Ga ³⁺ The metal anion is selected from MoO ₄ ^2- 、WO ₄ ^2- And VO (Voice over Internet protocol) ₄ ^3- 。

2. The cGAS-STING pathway activator according to claim 1, wherein the cGAS-STING pathway activator is a manganese molybdate nanoparticle.

3. The cGAS-STING pathway activator of claim 2, wherein the cationic metal source in the manganese molybdate nanoparticle is a Mn source and the anionic metal source is a Mo source.

4. The cGAS claimed in claim 3, wherein the molar ratio of Mn source to Mo source is 1: (0.5-1.5).

5. The cGAS-STING pathway activator according to claim 2, wherein the manganese molybdate nanoparticle is prepared by:

6. The cGAS claimed in claim 5, wherein in step a), the anionic metal source is 0.5 to 1.5 mmol/l, the dodecyl glycol is 0.75 to 2.25 g/l, and the dibenzyl ether is 10 to 30 ml/l.

7. The cGAS claimed in claim 5, wherein in step B) the molar ratio of oleylamine to oleic acid is 1 (0.5-1.5).

8. The cGAS claimed in claim 5, wherein in step C) the cationic metal source is 0.5 to 1.5 mmol/l, the reaction temperature is greater than 260 ℃ and the reaction time is 15 to 45 minutes.

9. A tumor metal immunotherapeutic agent comprising the cGAS-STING pathway activator according to any one of claims 1 to 8.

10. A method of screening for a cGAS-STING pathway activator according to any one of claims 1 to 8, comprising the steps of:

after S2, incubation, cGAS transfected 293-Dual was obtained ^TM mSTING cells;

s3, adding ion solutions with different concentrations to incubate, wherein the ion solutions are selected from Mn ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Zn ²⁺ 、Na ⁺ 、Cu ² ⁺ 、Ga ³⁺ 、MoO ₄ ^2- 、NO ₃ ^- 、WO ₄ ^2- 、SO ₄ ^2- 、Cl ^- 、VO ₄ ^3- 、VO ₃ ^- One or two ions;