Polyethylene glycol modified manganese chloride nanosheet as well as preparation method and application thereof

Abstract

The invention discloses a polyethylene glycol modified manganese chloride nanosheet as well as a preparation method and application thereof, the manganese chloride nanosheet is prepared by taking oleylamine as a surfactant, n-hexane as a solvent and manganese chloride crystals as a raw material through a simple ultrasonic assistance-liquid phase stripping method, and the polyethylene glycol modified manganese chloride nanosheet is prepared by taking polyethylene glycol modified manganese chloride as the raw material. Then, the hydrophilicity of the manganese chloride nanosheet is improved through distearoyl phosphatidyl ethanolamine-polyethylene glycol-hydroxyl modification. The prepared manganese chloride nanoparticles can significantly reduce the dissolution and release rate, realize rapid adhesion on cell surfaces, efficiently deliver manganese ions into tumor cells, specifically activate a cGAS-STING pathway, cooperatively enhance anti-tumor immune response by inducing tumor cell pan-apoptosis, and significantly inhibit tumor growth. The nanosheet disclosed by the invention not only can remarkably inhibit tumor growth, but also can induce a long-acting immune memory effect, effectively prevents tumor recurrence, and shows a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antitumor drugs, and particularly relates to a polyethylene glycol modified manganese chloride nanosheet as well as a preparation method and application thereof. BACKGROUND

[0002] As a serious health challenge, the global cancer incidence rate has shown a rising trend in recent years. According to statistics, the global new cancer cases will rise to 35 million by 2050. The current clinical treatment strategy mainly relies on radiotherapy combined with chemotherapy, but there are problems such as poor treatment tolerance, significant systemic toxicity and inhibition of immune system function. Unlike traditional tumor treatment which directly kills tumor cells, tumor immunotherapy activates the body's specific immune response to achieve precise recognition and elimination of tumor cells, thus having better specificity and safety, and having more significant effect in preventing tumor cell metastasis and recurrence.

[0003] Metal ions, as key effector molecules of immune regulation, play an important role in innate immunity and adaptive immunity. Mn 2+ With unique immune regulation ability and redox properties, it becomes a promising candidate for the next generation of cancer treatment. Mn 2+ It can enhance the sensitivity of cGAS to cytoplasmic double-stranded DNA (dsDNA) and enhance the binding affinity of STING to its ligand cGAMP, thereby activating the cGAS-STING signaling pathway. Mn 2+ By coordinating the powerful immune cascade of dendritic cell maturation, cytotoxic T cell activation and interferon secretion, it effectively converts "cold" tumors into "hot" immune microenvironments, thereby further enhancing the anti-tumor immune response. Manganese chloride is an excellent cGAS-STING pathway agonist, and its nanization is beneficial to cell uptake by endocytosis to enhance its effect. Conventional manganese chloride nanoparticles will dissolve in an instant when they come into contact with water, resulting in excessive release before cell uptake, which seriously limits the development of efficient Mn 2+ delivery systems. Therefore, rational design of particle structure to achieve its slow release in water is the key to realize its antitumor function.

[0004] Based on this, the application innovatively provides a simple, efficient and safe new strategy for tumor immunotherapy. Manganese chloride nanoparticles are synthesized in n-hexane by an ultrasonic-assisted solvent stripping method, and the slow release of manganese chloride in water is achieved by means of a reasonable surface modification strategy. The prepared manganese chloride nanoparticles can effectively inhibit the growth of various tumor models through the synergistic mechanism of Mn 2+ mediated pan-apoptosis effect and immune regulation, not only for Mn 2+The application in tumor immune combination therapy provides a new theoretical basis, and more importantly, a technical scheme with significant clinical transformation potential is developed. SUMMARY

[0005] The technical problem solved by the present application is to provide a polyethylene glycol modified manganese chloride nanosheet and a preparation method and application thereof. 2+ Specifically activates the cGAS-STING pathway, synergistically enhances the anti-tumor immune response by inducing tumor cell pan-apoptosis, and significantly inhibits tumor growth.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: a polyethylene glycol modified manganese chloride nanosheet, characterized in that: soluble manganese chloride crystals are converted into manganese chloride nanosheets by an ultrasonic-assisted liquid phase exfoliation method in a mixed solution of solvent n-hexane and surfactant oleylamine, and then the hydrophilicity of the manganese chloride nanosheets is increased by surface modification with distearoylphosphatidyl ethanolamine-polyethylene glycol-hydroxyl to finally obtain the polyethylene glycol modified manganese chloride nanosheet.

[0007] The preparation method of the polyethylene glycol modified manganese chloride nanosheet comprises the following specific preparation steps: manganese chloride crystals are added to a mixed solution containing n-hexane and oleylamine, ultrasonic dispersion is performed for uniform mixing, the sample is then collected by centrifugation, the unreacted manganese chloride crystals are removed by washing with n-hexane for multiple times, the product is then dispersed in n-hexane and ultrasonically mixed with a distearoylphosphatidyl ethanolamine-polyethylene glycol-hydroxyl solution, and a rotary evaporator is used to remove the solvent at 30-60 DEG C under reduced pressure to obtain the polyethylene glycol modified manganese chloride nanosheet.

[0008] Further limited, the mass ratio of the manganese chloride crystals to n-hexane is 1:20-80, and the volume ratio of oleylamine to n-hexane is 1:8-32.

[0009] Further limited, the ultrasonic temperature is 20~40℃, the ultrasonic power is 200~400W, and the centrifugal condition is 10000~14000rpm centrifugation for 8~12min.

[0010] The application of the polyethylene glycol modified manganese chloride nanosheet as a gene or drug delivery carrier.

[0011] The application of the polyethylene glycol modified manganese chloride nanosheet in the preparation of a drug for treating or / and preventing malignant tumors.

[0012] Compared with the prior art, the present application has the following advantages and beneficial effects: The manganese chloride nanosheet is constructed by mainly double surface modification of oleylamine and DSPE-PEG-OH, which ensures the dispersibility in water and reduces the dissolution speed of the manganese chloride nanoparticles in water, so that the manganese chloride nanosheet can be phagocytosed by cells in a stable nano structure in water, and the cell uptake is increased.

[0013] The polyethylene glycol modified manganese chloride nanosheet constructed in the present application exhibits excellent environmental-independent Mn 2+ Release behavior: the release process is not affected by the concentration of H + or glutathione (GSH), and the sustained and controllable intracellular Mn 2+ release can be realized.

[0014] The polyethylene glycol modified manganese chloride nanosheet constructed in the present application activates the cGAS-STING pathway in tumor cells specifically, induces tumor cell pan-apoptosis, and synergistically enhances the anti-tumor immune response. In a CT26 colon cancer mouse model, the treatment strategy can achieve 57% complete tumor cure, induce long-term immune memory effect, and effectively prevent tumor recurrence. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a preparation process schematic diagram of the polyethylene glycol modified manganese chloride nanosheet.

[0016] Figure 2Transmission electron microscopy image of polyethylene glycol modified manganese chloride nanosheets.

[0017] Figure 3 Adhesion of polyethylene glycol modified manganese chloride nanosheets to CT26 cells and cell uptake.

[0018] Figure 4 Toxicity of polyethylene glycol modified manganese chloride nanosheets to different tumor cells.

[0019] Figure 5 Effect of polyethylene glycol modified manganese chloride nanosheets on promoting the maturation of bone marrow-derived dendritic cells (BMDCs) in vitro.

[0020] Figure 6 Effect of polyethylene glycol modified manganese chloride nanosheets on promoting cytokines in vitro.

[0021] Figure 7 Activation of polyethylene glycol modified manganese chloride nanosheets on the cGAS-STING pathway in bone marrow-derived dendritic cells (BMDCs).

[0022] Figure 8 Activation of polyethylene glycol modified manganese chloride nanosheets on the expression of pan-apoptosis related proteins in CT26 cells.

[0023] Figure 9 Inhibitory effect of polyethylene glycol modified manganese chloride nanosheets on CT26 model tumors.

[0024] Figure 10 Biological safety of polyethylene glycol modified manganese chloride nanosheets in vivo.

[0025] Figure 11 Activation of polyethylene glycol modified manganese chloride nanosheets on anti-tumor immune response. DETAILED DESCRIPTION

[0026] The above content of the present application is further described in detail through the following examples, but this should not be understood as limiting the scope of the above subject matter of the present application to only the following examples. Any technology realized based on the above content of the present application falls within the scope of the present application. Example 1

[0027] Preparation of polyethylene glycol modified manganese chloride nanosheets (MnCl2 NSs) (see FIG. 1) Figure 1 ) 0.1 g of MnCl2 .4 H2O was added to a mixed solution containing 4 mL of n-hexane and 160 μL of oleylamine, and dispersed uniformly at 30 °C by ultrasonic treatment (power 300 W). The product was centrifuged (12000 rpm for 10 min) and washed with n-hexane for 3 times to remove unreacted MnCl2 . 4 H2O, and the product was dispersed in 2 mL of n-hexane to obtain manganese chloride nanosheets, which were mixed with 10 μL of a 4 mg / mL DSPE-PEG-OH solution, uniformly mixed at 30 °C by ultrasonic treatment (power 300 W), and subjected to solvent removal at 40 °C by rotary evaporation under reduced pressure to obtain polyethylene glycol modified manganese chloride nanosheets (MnCl2 NSs). The polyethylene glycol modified manganese chloride nanosheets were ultrasonically dispersed in 1 mL of deionized water to obtain a mixed solution. The transmission electron micrograph of the obtained polyethylene glycol modified manganese chloride nanosheets is shown in FIG. 1. Figure 2 Example 2

[0028] Adhesion of MnCl2 NSs to CT26 cells and uptake of the cells CT26 cells were co-incubated with FITC-labeled MnCl2 NSs (500 μM), and the adhesion of the cells and the uptake of the nanomaterials were observed by confocal laser scanning microscopy (CLSM). The experimental results showed that the MnCl2 NSs could quickly adhere to the surface of tumor cells in a short time and enter the tumor cells by endocytosis, as shown in FIG. 2. Figure 3 . Example 3

[0029] Anti-tumor activity experiment The cytotoxicity of MnCl2 NSs on different cell lines was determined by MTT method. CT26 (mouse colon cancer cells), 4T1 (mouse breast cancer cells), A549 (human lung cancer cells), MDA-MB-231 (human breast cancer cells), HepG2 (human liver cancer cells), A375 (human melanoma cells), and B16-F10 (mouse melanoma cells) were used as test cell lines. Different cells were co-incubated with different concentrations of PBS, MnCl2 (aq), and MnCl2 NSs at 37 °C in a CO2 incubator for 24 h. Then 15 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) solution with a concentration of 5 mg / mL was added to each well, and the 96-well plate was placed in the incubator for another 4 h. The culture medium was aspirated, 100 μL of DMSO was added to each well, and the optical density of each well was determined at 570 nm by an enzyme marker.

[0030] The inhibition rate of the drug on tumor cell growth was calculated according to the following formula: Tumor cell growth inhibition rate % = (1-OD experiment / OD control) x 100% ​The experimental results show that compared with manganese chloride aqueous solution (MnCl2(aq)), MnCl2 NSs exhibit enhanced cytotoxicity in all tested cell lines, indicating that the nanofabrication strategy can effectively enhance the tumor cell killing effect of MnCl2, see Figure 2. Figure 4 . Example 4

[0031] Activation of BMDCs in vitro BMDCs were incubated with PBS, MnCl2(aq) and MnCl2 NSs, respectively, for 12 h, and the sample concentration was 6.25 μg / mL. After incubation, the surface of BMDCs was stained with anti-CD11c-APC, anti-CD80-FITC and anti-CD86-PE / Cyanine7 antibodies, and the expression of surface markers of BMDCs in each group was detected by flow cytometry. The expression levels of CD80 and CD86 were analyzed to evaluate the maturation of BMDCs. The experimental results show that MnCl2 NSs can well promote the maturation of BMDCs, and the effect is better than that of MnCl2(aq), see Figure 3. Figure 5 . Example 5

[0032] Cytokine secretion in vitro CT26 cells were incubated with PBS, MnCl2(aq) and MnCl2 NSs, respectively, for 24 h, 5 x 10 5 cells per well, and the sample concentration was 6.25 μg / mL. After incubation, the supernatant was collected, and the concentrations of cytokines IFN-α, IL-6 and IL-1β were detected by enzyme-linked immunosorbent (ELISA) kit. The experimental results show that MnCl2 NSs can well promote the production of type I interferon (IFN-α) and proinflammatory cytokines (IL-6, IL-1β), see Figure 4. Figure 6 . Example 6

[0033] Activation of cGAS-STING pathway MnCl2 NSs were co-incubated with bone marrow-derived dendritic cells (BMDCs), and the expression of cGAS-STING pathway-related proteins in BMDCs after MnCl2 NSs treatment was evaluated from the cell level by Western blot (WB) method. The experimental results show that the efficient accumulation of Mn 2+ in the MnCl2 NSs treatment group activates the cGAS-STING pathway by up-regulating cGAS, p-IRF3 and p-STING, see Figure 5. Figure 7 . Example 7

[0034] Expression of pan-apoptosis related proteins MnCl2 NSs were co-incubated with CT26 cells, and the expression of apoptosis executioner Cleaved caspase-3, necroptosis marker phosphorylated MLKL (p-MLKL), and pyroptosis effector Gasdermin E-N terminal fragment (GSDME-N) proteins in CT26 cells after MnCl2 NSs treatment were evaluated from cell layer by WB method. The experimental results showed that Cleaved caspase-3, p-MLKL and GSDME-N were synchronously up-regulated in the MnCl2 NSs treatment group, which confirmed the occurrence of panoptosis through the synergistic cell death program integrating the apoptosis, necroptosis and pyroptosis pathways. Example 8

[0035] Evaluation of therapeutic effect of MnCl2 NSs in CT26 tumor-bearing mouse model Subcutaneous tumor model construction and treatment BALB / c female mice at the age of 6-8 weeks were subcutaneously injected with 2x10 6 CT26 cells on the right flank to construct the transplantation model (day -7). When the tumor volume grew to about 100 mm 3 , the mice were randomly divided into three groups: (i) control group, (ii) MnCl2 (aq) group, (iii) MnCl2 NSs group. The mice were intratumorally injected on day 0, day 2 and day 4, respectively. The tumor volume and the body weight of the mice were measured every 2 days. The tumor volume of the mice was calculated according to the following formula: Tumor volume = (tumor length) x (tumor width) / 2 2 H&E staining analysis After the end of the experiment, the main organs of the mice were subjected to histopathological examination. Hematoxylin and eosin (H&E) staining was used to observe the morphological changes of the liver, spleen, lung, kidney organs of the mice, and to judge the necrosis of the cells and the safety.

[0036] The experimental results showed that MnCl2 NSs could significantly inhibit the growth of tumors, and this multi-effect immunomodulatory effect could achieve complete local tumor eradication in 57% of the treated mice (see Figure 8), and had a certain safety (see Figure 9). Figure 9 Figure 10 Example 9

[0037] In vivo immune activation BALB / c female mice at the age of 6-8 weeks were subcutaneously injected with 2x10 6 CT26 cells on the right flank to construct the transplantation model (day -7). When the tumor volume grew to about 100 mm 3 ​​​At the end of the treatment, mice were randomly divided into three groups: (i) control group, (ii) MnCl2 (aq) group, (iii) MnCl2 NSs group. After 1 treatment, tumor-draining lymph nodes (TDLNs) and tumor tissues of mice in each group were collected, digested and homogenized into single-cell suspensions in PBS. To detect mature DCs, the collected cells were stained with anti-CDllc-APC, anti-CD80-FITC and anti-CD86-PE / Cyanine7 antibodies. To analyze helper T cells and cytotoxic T cells, the collected cells were stained with anti-CD3-PE, anti-CD4-FITC and anti-CD8a-APC antibodies. The analysis was performed by flow cytometry.

[0038] The experimental results show that MnCl2 NSs can well promote the maturation of DCs in the lymph nodes of mice, significantly increase the proportion of tumor-infiltrating CD8 + T cells, and the effect is better than that of MnCl2 (aq), see the attached Figure 11 .

[0039] The above examples describe the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples. The above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the scope of the principles of the present application, various changes and improvements can be made to the present application. These changes and improvements all fall within the scope of protection of the present application.

Claims

1. A polyethylene glycol-modified manganese chloride nanoplatelet, characterized by: The soluble manganese chloride crystals are converted into manganese chloride nanosheets by an ultrasonic-assisted liquid phase exfoliation method in a mixed solution of solvent n-hexane and surfactant oleylamine, and then the hydrophilicity of the manganese chloride nanosheets is increased by surface modification with distearoylphosphatidyl ethanolamine-polyethylene glycol-hydroxyl to finally obtain polyethylene glycol modified manganese chloride nanosheets, which can slowly dissolve in water and release manganese ions in a non-environment-dependent manner; by virtue of the rapid cell adhesion and non-environment-dependent release characteristics, the manganese ions are efficiently delivered into tumors, and the cGAS-STING pathway is specifically activated, while the tumor cells are induced to undergo pan-apoptosis, thereby synergistically enhancing the anti-tumor immune response.

2. A method of preparing the polyethylene glycol-modified manganese chloride nanosheet of claim 1, characterized by The specific preparation steps are as follows: manganese chloride crystals are added to a mixed solution containing n-hexane and oleylamine, ultrasonically dispersed and mixed uniformly, then the sample is collected by centrifugation, and the unreacted manganese chloride crystals are removed by washing with n-hexane for multiple times, then the product is dispersed in n-hexane and ultrasonically mixed with a distearoylphosphatidyl ethanolamine-polyethylene glycol-hydroxyl solution, and a rotary evaporator is used to remove the solvent at 30-60 DEG C under reduced pressure to obtain polyethylene glycol modified manganese chloride nanosheets.

3. The method for preparing polyethylene glycol-modified manganese chloride nanosheets according to claim 2, characterized in that: The mass ratio of the manganese chloride crystals to n-hexane is 1:20-80, and the volume ratio of oleylamine to n-hexane is 1:8-32.

4. The method for preparing polyethylene glycol-modified manganese chloride nanosheets according to claim 2, characterized in that: The ultrasonic temperature is 20-40 DEG C, the ultrasonic power is 200-400 W, and the centrifugation condition is 10000-14000 rpm of rotation speed for 8-12 min.

5. The polyethylene glycol modified manganese chloride nanosheets of claim 1 are used as gene or drug delivery carriers.

6. The polyethylene glycol modified manganese chloride nanosheets of claim 1 are used in the preparation of a drug for treating or / and preventing malignant tumors.

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