CN111643454A

CN111643454A - Manganese-containing micro-precipitation liposome for immunotherapy and preparation method thereof

Info

Publication number: CN111643454A
Application number: CN202010573158.3A
Authority: CN
Inventors: 林志强; 游富平; 吕丹; 梁子超; 雒钰杰; 王晓
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2020-09-11
Anticipated expiration: 2040-06-22
Also published as: CN111643454B

Abstract

The invention discloses a manganese-containing micro-precipitation liposome for immunotherapy and a preparation method thereof. The liposome consists of a manganese ion-containing micro-precipitation core and a phospholipid bilayer shell, and can be prepared by combining an emulsion mixing method and a film dispersion method. The liposome has a spherical structure of 20-200nm, has a certain amount of positive charges on the surface, and can be degraded in a certain acidic environment. The liposome can stimulate innate immune system and/or adaptive immunity or be used as an immunologic adjuvant for treating related diseases such as antiviral, antibacterial, antitumor and autoimmune diseases.

Description

Manganese-containing micro-precipitation liposome for immunotherapy and preparation method thereof

Technical Field

The invention relates to a manganese-containing micro-precipitation liposome (NanoMn) for immunotherapy or cancer therapy and a preparation method thereof, belonging to the field of nano preparations.

Background

Manganese metal, one of trace elements essential to the human body, plays a variety of very important physiological functions in the body. In recent years, it has been reported in the literature that divalent manganese ions play an important immune function in vivo and are natural immune activators and alarms in cells. Natural immunity is the first line of defense against the invasion of pathogenic microorganisms by the body's host cells. A plurality of studies show that the cGAS-STING signal pathway is an important pathway for immune activation of organisms, can sense the invasion of viruses in antiviral immunity, activates transcription factors IRF3/7 and NF-kB, and generates various antiviral cytokines such as I-type Interferon (IFN); it also plays an important role in anti-tumor immunity and autoimmune diseases. The existing research shows that the divalent manganese ions can be used as a novel cGAS-STING activator to improve innate immunity and/or adaptive immunity.

However, the use of divalent manganese ions for immunotherapy still has the following problems: it can not realize specific distribution after in vivo administration, and is easily distributed to nervous system, which damages central nerve, and also has certain toxic and side effects on immune system. In addition, the exogenous manganese ions entering the cells are mainly diffused by means of concentration gradient and cannot specifically enter target cells but not non-target cells.

Disclosure of Invention

Aiming at the problems of nonspecific distribution, neurotoxicity and the like of bivalent manganese ions in immunotherapy, the invention aims to construct a liposome containing bivalent manganese ion micro-precipitates, and the liposome can be directionally distributed to tumor tissues or other target sites after intravenous administration, slowly degraded in acidic environments in tissues or cells, and released with manganese ions, thereby playing a role in immune activation; and the liposomes do not cross the blood brain barrier, thereby reducing central neurotoxicity.

The manganese-containing micro-precipitation liposome provided by the invention is a biodegradable manganese ion-coated precipitation liposome, and consists of a manganese ion-containing micro-precipitation inner core and a phospholipid bilayer shell, wherein the phospholipid bilayer shell has positive charges, has good biocompatibility in vivo and can be more easily endocytosed by cells; in a slightly acidic environment, the liposomes slowly degrade and release manganese ions.

The manganese ions are divalent manganese ions, and the micro-precipitation core containing the manganese ions is selected from one or more of the following compounds: manganese phosphate, manganese hydrogen phosphate, manganese carbonate, manganese oxalate, and the like.

The phospholipid bilayer shell is composed of a phospholipid material and cholesterol, and the phospholipid material can be selected from one or more of the following materials: DOPA (dioleoylphosphatidic acid), DOTAP ((2, 3-dioleoxypropyl) trimethylammonium chloride), DSPE-PEG (distearoylphosphatidylethanolamine-polyethylene glycol), and the like. In some embodiments of the invention, the molar percentage of DOPA in the phospholipid bilayer ranges from 20% to 80%, the molar percentage of DOTAP ranges from 8% to 40%, the molar percentage of cholesterol ranges from 8% to 40%, and the molar percentage of DSPE-PEG ranges from 2% to 20%.

Preferably, the manganese-containing micro-precipitation liposome is a nanoparticle with the particle size of 20-200nm, and the size of the nanoparticle is not greatly different from that of a virus particle. The liposome comprises a microprecipitated core which is a biodegradable core and can be dissolved in an environment with a pH value lower than the pKa corresponding to the corresponding anion. The liposome is easy to enter cells in an endocytosis mode, and is degraded in a certain acid environment after entering the cells, and the manganese ion micro-precipitation kernel of the liposome is converted into bivalent manganese ions to play an intracellular role.

On one hand, under the condition of not coupling targeting molecules, the liposome is mainly distributed to reticuloendothelial tissues such as liver, spleen and bone, and the like, has the same distribution condition with the virus in vivo, and can be used for treating antiviral immunity or other immunity-related diseases. The liposome can activate cGAS-STING pathway by releasing divalent manganese ions, and improve the sensitivity of cGAS to DNA to realize the function of stimulating innate immunity and/or adaptive immunity. The liposomes can also be used as an immunological adjuvant, including activating T cell activation and/or antibody production.

The treatment of immune-related diseases includes antiviral, antibacterial, and antiparasitic treatments. The virus may be a DNA virus and an RNA virus, among others, such as: herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, hepadnaviridae, flaviviridae, papillomaviraceae, poxviridae and retroviridae, in particular: herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV. The bacteria may be gram-negative and gram-positive bacteria, such as: streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Diplococcus meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumannii. Such as plasmodium, toxoplasma, trypanosoma, schistosoma, filarial worms and leishmania.

The treatment of immune-related diseases also includes the treatment of autoimmune diseases including, but not limited to, type I diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis.

Alternatively, targeting molecules (see fig. 1) can be coupled to the surface of the liposomes of the present invention to specifically distribute them in vivo to tumor tissues and tumor cells for cancer therapy. Such targeting molecules include, but are not limited to, the RGD peptide sequence, folic acid, transferrin, and the like.

Such cancers include, but are not limited to, breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cancer, colorectal cancer, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, and leukemia.

The manganese-containing micro-precipitation liposome is not easy to penetrate through a blood brain barrier, and can reduce the central nervous toxicity of manganese ions to the maximum extent.

The manganese-containing micro-precipitation liposome is mainly prepared by combining the following emulsion mixing method (the first three steps) and a film dispersion method (the last two steps):

1) preparing a compound containing divalent manganese ions into a water solution with a certain concentration, dropwise adding the water solution into an oil solution under the ultrasonic condition, and continuing ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, wherein the volume ratio of a water phase to the oil phase is 1:10-1: 1000;

2) preparing a compound containing anions (phosphate radical, hydrogen phosphate radical, carbonate radical, oxalate radical ions and the like) into an aqueous solution, dropwise adding the aqueous solution into an oil solution under the ultrasonic condition, and continuing ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, wherein the volume ratio of a water phase to the oil phase is 1:10-1: 1000;

3) mixing the two solutions, standing for 10-60 minutes to enable manganese ions to react with corresponding anions to form micro-precipitates, adding a first organic solvent with the volume equivalent to that of the oil phase solution to destroy the oil phase, centrifuging to obtain manganese ion micro-precipitates, and dissolving the manganese ion micro-precipitates in a second organic solvent, wherein the first organic solvent can dissolve the oil phase but not the manganese ion micro-precipitates, and the second organic solvent is a non-polar organic solvent;

4) adding phospholipid material and cholesterol into chloroform solution containing manganese ion micro-precipitate, transferring into an eggplant-shaped bottle, and volatilizing organic solution through a rotary evaporator to form a lipid membrane;

5) adding buffer solution or deionized water, and performing ultrasonic treatment at 40-60 deg.C for 10-60 min to obtain manganese-containing micro-precipitation liposome.

In the step 1), the compound containing divalent manganese ions is soluble in water, and may be selected from one or more of the following compounds: manganese chloride, manganese nitrate, manganese sulfate, and the like. Preferably, the compound containing the divalent manganese ions is prepared into an aqueous solution with the concentration of 10-5000 mmol/L.

In the step 1), the oil solution is a water-incompatible solution, and may be selected from one or more of the following oily solvents: cyclohexane, cyclopentane, petroleum ether, benzene, and the like. A certain amount of nonionic surfactant such as nonoxynol, alkylphenol ethoxylates, octylphenol polyoxyethylene ether, lauryl alcohol polyoxyethylene ether and the like is added into the oil solution to promote emulsification and help the water phase to be uniformly dispersed in the oil phase. Preferably, the volume ratio of the oil solution to the nonionic surfactant is 1.5-7.5.

In the step 2), the anion can form a precipitate with a divalent manganese ion, such as a phosphate ion, a hydrogen phosphate ion, a carbonate ion, an oxalate ion, and the like. The anion-containing compound is soluble in water, typically an alkali metal salt, including but not limited to: sodium hydrogen phosphate, sodium carbonate and sodium oxalate. Preferably, the anion-containing compound is prepared into an aqueous solution with the concentration of 1-500 mmol/L, and the aqueous solution is dropped into the same oil solution as the step 1).

In the step 3), the first organic solvent is preferably an alcohol or ether organic solvent, such as methanol, ethanol, propanol, and diethyl ether, and ethanol is preferably used. The second organic solvent is preferably a volatile nonpolar organic solvent such as chloroform, methylene chloride, carbon tetrachloride, ethyl bromide, benzene, cyclohexane, hexane, diethyl ether, isopropyl ether, ethyl acetate, etc., preferably chloroform. More preferably, the concentration of the manganese ion micro-precipitate dissolved in chloroform is 1-100 mmol/L.

In the step 4), the phospholipid material coupled with the targeting molecules is adopted to prepare the manganese-containing micro-precipitation liposome which has the targeting molecules on the outer surface and can be specifically distributed.

The liposome containing manganese ion micro-precipitates provided by the invention has the following advantages:

1. the liposome can be used as a nano reservoir of divalent manganese ions, and can be degraded in acidic environment in tissues or cells to release free manganese ions;

2. the liposome is mainly distributed in reticuloendothelial tissues such as liver, spleen, bone and the like after intravenous injection in vivo, is basically consistent with the distribution of viruses, and can be used for antiviral immunity;

3. the liposome can be specifically distributed to target tissues or target cells after surface targeted modification, and is used for anti-tumor treatment;

4. the liposome is not easy to cross blood brain barrier, and can reduce the central nervous toxicity of manganese ions to the maximum extent.

In conclusion, the manganese-containing micro-precipitation liposome can stimulate the innate immune system and/or adaptive immunity or be used as an immunologic adjuvant for treating related diseases such as antiviral, antibacterial, antitumor and autoimmune diseases.

Drawings

FIG. 1 is a schematic structural diagram of a manganese-containing micro-precipitated liposome (NanoMn) according to the present invention.

FIG. 2 is a transmission electron micrograph of the manganese hydrogenphosphate-containing liposome prepared in example 1, with a scale: 50 nm.

FIG. 3 shows the particle size distribution diagram (a) and the surface potential measurement result (b) of the manganese hydrogenphosphate-containing liposome prepared in example 1.

FIG. 4 manganese hydrogenphosphate-containing liposomes Mn in example 7²⁺In vitro leakage versus release profile.

FIG. 5 shows the tissue distribution of the liposomes containing manganese hydrogen phosphate obtained in example 8 in vivo.

FIG. 6. results of experiments on the ability of liposomes containing manganese hydrogenphosphate to activate host cells to resist viral infection. The NanoMn treatment activated host cells to have a peak in their ability to resist viral infection within 12 hours.

FIG. 7 example 10 Mn hydrogen phosphate-containing liposomes with MnCl₂And (3) comparing experimental results of the antiviral infection capacity of the activated host cells. The antiviral infection capacity of the host cell activated by the treatment of the NanoMn is stronger than that of MnCl2, and the effect is obviously reduced after the cGAs knockout.

FIG. 8 shows the liposomes containing manganese hydrogen phosphate and MnCl in example 11₂And (3) analyzing the gene differential expression after treating the dendritic cells. The NanoMn treatment significantly upregulated molecules associated with dendritic cell maturation. .

FIG. 9 manganese hydrogenphosphate-containing liposomes and MnCl in example 11₂T cell stimulation following dendritic cell treatmentExpression of living co-stimulatory molecules and effector factors. NanoMn treatment significantly upregulated T cell activating costimulatory molecules and effectors of dendritic cells.

FIG. 10 shows the liposomes containing manganese hydrogenphosphate in example 11 and MnCl₂The GO functional analysis result of the processed dendritic cells shows that the NanoMn processing obviously up-regulates the antiviral and antitumor biological processes of the dendritic cells.

FIG. 11. liposomes containing manganese hydrogen phosphate in example 11 with MnCl₂After dendritic cells are treated, the analysis result of a Reactome signal channel shows that the antiviral and antitumor signal channel of the dendritic cells is remarkably up-regulated by the treatment of the NanoMn.

FIG. 12 example 12 manganese Hydrogen phosphate-containing liposomes with MnCl₂Inhibition of tumor growth experimental results: tumor growth was inhibited after B16F10 transplantation tumor mice treated with NanoMn.

FIG. 13 example 13 manganese Hydrogen phosphate-containing liposomes with MnCl₂Results of inhibition experiments on tumor metastasis: B16F10 metastatic tumor mice were treated with NanoMn and lung metastatic nodules were inhibited.

FIG. 14 different Mn in example 15²⁺Microscopy and flow cytometry analysis of Hela cells treated against infection with VSV-GFP Virus.

FIG. 15 different Mn in example 15²⁺The rate of viral suppression against infection of HeLa cells by VSV-GFP virus was treated.

FIG. 16 different Mn in example 15²⁺Treatment of HeLa cells induced the expression levels of interferon IFN β and OASL1 genes, a. IFN β mRNA and b. OASL1 mRNA.

FIG. 17 different Mn in example 15²⁺Treatment was performed against infection of Hela cells with different viruses, which expressed the mRNA levels in the host cells.

FIG. 18 different Mn in example 16²⁺After the mice are treated for 36 hours, the clinical index level of acute tissue injury such as ALT, AST and the like in blood is shown.

FIG. 19 GSEA analysis of GO genome for different Mn in example 17²⁺Differences in liver gene expression in treated mice.

FIG. 20 example 17Enrichment analysis of different Mn by combining middle GSEA with GO gene²⁺The expression level of IFN β and IRF7 genes in the liver is detected by an RNA-seq map of the liver gene expression of the treated mice and a qPCR method, namely a.RNA-seq, b.IFN β mRNA expression level in the liver and c.IRF7 mRNA expression level in the liver.

FIG. 21 enrichment analysis of upregulated differentially expressed genes in NanoMn-treated and Vehicle-treated livers by the GO database in example 17.

FIG. 22, example 18C 57BL/6 mice with different Mn²⁺Survival plots of MHV-A59 virus infected with lethal doses after treatment: a. a schematic processing mode; b. mouse survival plots.

FIG. 23, example 18C 57BL/6 mice with different Mn²⁺Graph of body weight change after treatment infected with lethal dose of MHV-A59 virus.

FIG. 24 mice C57BL/6 in example 18 were treated with different Mn²⁺After treatment, MHV-A59 virus with lethal dose is infected, and the qRT-PCR method is used for detecting the expression level of MHV-N genes of livers and spleens: a. expression levels of liver MHV-N mRNA; b. expression levels of spleen MHV-NmRNA.

FIG. 25, different Mn of the C57BL/6 mice in example 19 after infection with a lethal dose of MHV-A59 virus²⁺Survival plots of treatments: a. a schematic processing mode; b. mouse survival plots.

FIG. 26, different Mn of the C57BL/6 mice in example 19 after infection with a lethal dose of MHV-A59 virus²⁺And (3) processing, detecting the expression level of liver and spleen MHV-N gene by a qRT-PCR method: a. expression levels of liver MHV-N mRNA; b. expression levels of spleen MHV-NmRNA.

FIG. 27A graph of the enrichment analysis of differentially expressed genes with upregulated expression in NanoMn and Vehicle group mice using GO database in example 19 and uninfected or different Mn²⁺Heat map of the metabolic-related gene expression levels of treated infected mice: go database enrichment analysis map; b. heat map of mouse metabolism-related gene expression levels under different treatments.

FIG. 28. example 19C 57BL/6 mice infected with lethal doses of MHV-A59 virus at different Mn²⁺Treatment, spleen weightSpleen lymphocyte number and spleen parenchymal map: a. spleen weight graph; b. spleen lymphocyte count; c. spleen in picture.

FIG. 29 percentage and cell count of Lymphocytes (LY) and Monocytes (MO) following infection of C57BL/6 mice with a lethal dose of MHV-A59 virus in example 19: a. percentage and cell count of Lymphocytes (LY); b. percentage of Monocytes (MO) and cell count.

FIG. 30. C57BL/6 mice in example 20 infected with non-lethal doses of MHV-A59 virus at different Mn²⁺Survival profiles of mice treated, reinfected with lethal doses of MHV-A59 virus on day 14 post-infection: a. a schematic processing mode; b. mouse survival plots.

FIG. 31. C57BL/6 mice in example 20 infected with non-lethal doses of MHV-A59 virus at different Mn²⁺Treatment, reinfection with a lethal dose of MHV-A59 virus on day 14 post-infection, mouse liver MHV-N mRNA expression levels.

FIG. 32. C57BL/6 mice in example 20 infected with non-lethal doses of MHV-A59 virus at different Mn²⁺Treatment, flow cytometry detection of mouse liver and spleen CD8 memory T cells (CD 8)⁺CD44^hiCD62L⁺) Percentage of (A): a. a flow cytogram; b. mouse liver and spleen CD8 memory T cell (CD 8)⁺CD44^hiCD62L⁺) Percentage of (c).

FIG. 33. infection of C57BL/6 mice in example 20 with a non-lethal dose of MHV-A59 virus at different Mn²⁺Treating, infecting again with lethal dose of MHV-A59 virus, and detecting mouse liver CD8 by flow cytometry⁺CD25⁺And CD4⁺CD25⁺Percentage of cells: a. a flow cytogram; b. mouse liver CD8⁺CD25⁺And CD4⁺CD25⁺Percentage of cells.

FIG. 34. C57BL/6 mice in example 20 infected with non-lethal doses of MHV-A59 virus at different Mn²⁺Treating, reinfection with a lethal dose of MHV-A59 virus, mouse liver and spleen CD8⁺、CD4⁺、B220⁺Percentage of cells and spleen CD8⁺CD44^hiAnd CD4⁺CD44^hiCD25 in cells⁺Percentage of cells: a. mouse liver and spleen CD8⁺、CD4⁺、B220⁺Flow cytograms and percentile statistics; b. mouse spleen CD8⁺CD44^hiAnd CD4⁺CD44^hiCD25 in cells⁺Flow cytograms and percentage statistics.

FIG. 35 infection of C57BL/6 mice in example 20 with non-lethal doses of MHV-A59 virus at different Mn²⁺Treating, reinfecting with lethal dose of MHV-A59 virus, treating mouse liver and spleen lymphocytes with 100ng/mL PMA and 500ng/mL ionomycin for 5h, and detecting CD8 by flow cytometry⁺And CD4⁺Percentage of cells producing tumor necrosis factor TNF α and interferon IFN γ a. mouse liver CD8⁺And CD4⁺Flow cytogram and percentage statistical chart of tumor necrosis factor TNF α and interferon IFN gamma generated by cell, and mouse spleen CD8⁺And CD4⁺Cells produce flow cytograms and percentage statistics of tumor necrosis factor TNF α and interferon IFN γ.

FIG. 36. C57BL/6 mice in example 20 were inoculated subcutaneously with non-lethal doses of MHV-A59 virus, varying in Mn²⁺Treatment, mice infected with a lethal dose of MHV-a59 virus were re-i.p. on day 14 post-infection for weight change and serum IgG concentration: a. a schematic processing mode; b. body weight change of mice; c. mouse serum IgG concentration.

Detailed Description

The invention is further illustrated and explained by the following examples, which are not to be construed as limiting the invention.

Example 1 preparation of liposomes containing manganese hydrogen phosphate

Preparing manganese chloride into 500mM aqueous solution, dropwise adding 300 mu L of the solution into 15mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the ultrasonic condition, and continuing ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase;

preparing sodium hydrogen phosphate into 25mM aqueous solution, dropwise adding 300 mu L of the solution into 15mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the condition of ultrasonic treatment, continuing the ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, and then adding 200 mu L of 20mg/mL DOPA chloroform solution;

mixing the two solutions, standing for 30 minutes to enable manganese ions to react with hydrogen phosphate ions to form micro-precipitates, adding ethanol with the volume equivalent to that of the oil phase solution to destroy the oil phase, centrifuging to obtain the manganese hydrogen phosphate micro-precipitates, washing with ethanol for 2-3 times, and dissolving in 1mL of chloroform;

adding 100 μ L DOTAP (10mM), cholesterol (10mM) and DSPE-PEG (3mM) to the chloroform solution containing the manganese hydrogen phosphate micro-precipitate (7.5mM) obtained above, transferring to an eggplant-shaped bottle, and volatilizing the organic solution by a rotary evaporator to form a lipid film; finally, 800 mu L of deionized water is added, and the mixture is subjected to ultrasonic treatment for 30 minutes at the temperature of 50 ℃ to obtain the ultrasonic wave-absorbing material.

The prepared manganese hydrogen phosphate-containing liposome (NanoMn) is shown in figure 2 by a transmission electron microscope, and the particle diameter and the surface potential measured by a laser particle diameter instrument are shown in figure 3.

Example 2 preparation of liposomes containing manganese phosphate

Manganese chloride is prepared into 1M aqueous solution, 600 mu L of the solution is dropwise added into 30mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the ultrasonic condition, and the ultrasonic treatment is continued, so that water drops are cracked and uniformly dispersed in an oil phase;

preparing sodium phosphate into 50mM aqueous solution, dropwise adding 600 mu L of the solution into 30mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the condition of ultrasonic treatment, continuing ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, and then adding 200 mu L of 20mg/mL DOPA chloroform solution;

mixing the two solutions, standing for 30 minutes to enable manganese ions to react with phosphate ions to form micro-precipitates, adding ethanol with the volume equivalent to that of the oil phase solution to destroy the oil phase, centrifuging to obtain manganese phosphate micro-precipitates, washing with ethanol for 2-3 times, and dissolving in 2mL of chloroform;

adding 200 μ L DOTAP (10mM), cholesterol (10mM) and DSPE-PEG (3mM) to the chloroform solution containing the manganese phosphate micro-precipitate (15mM), transferring to an eggplant-shaped bottle, and volatilizing the organic solution by a rotary evaporator to form a lipid film; finally, 1mL of deionized water is added, and the mixture is subjected to ultrasonic treatment at 45 ℃ for 40 minutes to obtain the ultrasonic probe.

Example 3 preparation of liposomes containing manganese carbonate

preparing sodium carbonate into a 25mM aqueous solution, dropwise adding 300 mu L of the solution into 15mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the condition of ultrasonic treatment, continuing the ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, and then adding 200 mu L of 20mg/mL DOPA chloroform solution;

mixing the two solutions, standing for 30 minutes to enable manganese ions to react with carbonate ions to form micro-precipitates, adding ethanol with the volume equivalent to that of the oil phase solution to destroy the oil phase, centrifuging to obtain the manganese carbonate micro-precipitates, washing with ethanol for 2-3 times, and dissolving in 1mL of chloroform;

adding 100 μ L DOTAP (10mM), cholesterol (10mM) and DSPE-PEG (3mM) to the chloroform solution containing the manganese carbonate micro-precipitate (7.5mM) obtained above, transferring to an eggplant-shaped bottle, and volatilizing the organic solution by a rotary evaporator to form a lipid membrane; finally, 1mL of deionized water is added, and the mixture is subjected to ultrasonic treatment at 60 ℃ for 20 minutes to obtain the ultrasonic wave-absorbing material.

Example 4 preparation of liposomes containing manganese oxalate

Preparing manganese chloride into a 300mM aqueous solution, dropwise adding 200 mu L of the solution into 10mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the ultrasonic condition, and continuing ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase;

preparing sodium oxalate into a 20mM aqueous solution, dropwise adding 200 mu L of the solution into 10mL of cyclohexane/nonoxynol mixed oil solution (70/30, v/v) under the condition of ultrasonic treatment, continuing the ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, and then adding 200 mu L of 20mg/mL DOPA chloroform solution;

mixing the two solutions, standing for 30 minutes to enable manganese ions to react with oxalate ions to form micro-precipitates, adding ethanol with the volume equivalent to that of the oil phase solution to destroy the oil phase, centrifuging to obtain the manganese oxalate micro-precipitates, washing with ethanol for 2-3 times, and dissolving in 1mL of chloroform;

adding 100. mu.L of DOTAP (10mM), cholesterol (10mM) and DSPE-PEG (3mM) to the chloroform solution containing the manganese oxalate micro-precipitate (7.5mM) obtained above, transferring to an eggplant-shaped bottle, and evaporating the organic solution by a rotary evaporator to form a lipid film; finally, 800 mu L of deionized water is added, and the mixture is subjected to ultrasonic treatment at 50 ℃ for 50 minutes to obtain the product.

Example 5 TEM image of liposomes containing manganese Hydrogen phosphate

The manganese hydrogenphosphate-containing liposome prepared in example 1 was diluted 10-fold with deionized water, and the structure thereof was observed by a transmission electron microscope (JEM-200X, JEOL, Japan). And (2) keeping the temperature of the diluted sample at room temperature for 30min, dropwise adding the diluted sample on a copper mesh coated with a carbon film, directly loading the sample into a JEM-200X transmission electron microscope after water is volatilized, and observing the particle size and the shape at an accelerating voltage of 80kv, wherein the manganese hydrogen phosphate-containing liposome is spherical and has the particle size of about 10-40 nm as shown in figure 2.

Example 6 measurement of particle size and surface potential of liposomes containing manganese Hydrogen phosphate

The liposome containing manganese hydrogen phosphate prepared in example 1 was taken, and the particle size and potential at 25 ℃ were measured using a particle size cup and a potential cup, respectively, using a laser particle sizer (Zetasizer, us PSS). Equilibrate for 1 minute before measurement after each loading into the cuvette. As shown in FIG. 3, the particle size distribution was mostly 30 to 50nm, and the potential was about-2.3 mV.

Example 7 Liposome Mn containing manganese Hydrogen phosphate Microprecipitates²⁺In vitro release of

To study the pH sensitive release of NanoMn, we performed typical experiments under different pH conditions. 2mL of NanoMn suspension (8mM) was added to a dialysis bag (molecular weight cut-off: 8000-14000). Different dialysis bags containing NanoMn were immersed in 10mL of Phosphate Buffered Saline (PBS) with different pH values (4.5, 6.5, 7.4), respectively, and shaken at 40rpm at 37 ℃. At predetermined time points (5min, 15min, 1h, 2h, 4h and 24h), 200. mu.L of sample was taken each time and replaced with the same volume of fresh medium. Further determination of Mn for each sample²⁺The concentration of the active ingredients in the mixture is,drawing Mn²⁺The release profile is accumulated.

Mn²⁺The in vitro leakage and release curves are shown in FIG. 4, indicating that NanoMn releases Mn continuously in a pH-dependent manner² ⁺. Mn of NanoMn at pH values of 4.5 and 6.5²⁺The cumulative release rate is much higher than Mn at pH 7.4²⁺Cumulative release rate (greater than 90% at pH 4.5 and less than 10% at pH 7.4).

Example 8 tissue distribution of liposomes containing manganese hydrogen phosphate microprecipitates in vivo

In order to examine the distribution of liposomes containing manganese hydrogen phosphate micro-precipitates in vivo after administration, DiR near-infrared fluorescent dye-labeled liposomes were prepared by the same method as in example 1, wherein DiR dye (50 μ L of DiR dye at a concentration of 0.1mg/mL per mL of micro-precipitated chloroform solution) was added together with the final addition of the lipid material, and the remaining steps were completely identical to example 1.

A Balb/c mouse with the duration of 6-8 weeks is taken, 200 mu L/40 mu L/8 mu L of near-infrared dye DiR fluorescence labeled manganese hydrogen phosphate liposome solution is injected into the tail vein of the mouse respectively, the mouse is killed by using a cervical dislocation method 24 hours after administration, main organs of heart, liver, spleen, lung, kidney, brain and thymus are dissected and collected, the main organs are placed under a living body imaging instrument of the mouse for imaging observation, the wavelength of exciting light is set to be 720nm, and the wavelength of emitting light is set to be 780 nm. The tissue distribution of the prepared manganese hydrogen phosphate-containing micro-precipitated liposomes in mice is shown in fig. 5, and the liposomes are mainly distributed in liver and spleen, and are distributed in lung in a small amount (color image is changed into black and white image, and the conclusion is drawn according to the original image).

Example 9 time gradient experiment of IFN beta production in Liposome-activated host cells containing manganese phosphate Microprecipitates

To study the time-dependent changes in the production of IFN β by NanoMn-stimulated host cells, NanoMn-treated host cells were collected for different time periods and assayed for IFN β expression.

Laying iBMDM cells on a 24-well plate, adding 200 mu M of NanoMn when the iBMDM cells are 70-80% of confluence, treating the iBMDM cells for 0h,6h,12h,24h,36h and 48h respectively, then collecting the cells, extracting RNA by a TRIzol method, carrying out reverse transcription, then carrying out qRT-PCR, and detecting the expression level of IFN beta mRNA. As shown in FIG. 6, the results showed that IFN β production by the host cells peaked at 12 hours.

Example 10 comparison of liposomes containing manganese hydrogen phosphate Microprecipitates and MnCl₂Host cell activation IFN β production assay

To compare NanoMn with MnCl₂Activating IFN β production level difference of host cells, and taking mouse primary dendritic cells, NanoMn and MnCl₂Respectively treating at 200 mu M for 12 hours, collecting cells, extracting RNA, performing qRT-PCR, and detecting the expression level of IFN β.

Taking the bone marrow cells of the femur and the tibia of a 6-8 week C57BL/6 mouse, culturing in a 1640 culture medium, and adding a colony stimulating factor GM-CSF and interleukin IL-4 to induce the bone marrow cells to differentiate into Bone Marrow Dendritic Cells (BMDC). The medium was changed and supplemented with induction factors on day 4. Lipopolysaccharide was added overnight on day 7 to stimulate dendritic cell maturation. After 12h the mature dendritic cells were blown down from the culture dish (the dendritic cells were semi-adherent). Followed by NanoMn and MnCl₂The treatment was carried out as in example 9, with a treatment time of 12 h. qRT-PCR for IFN β mRNA expression levels, as shown in FIG. 7, the results showed that Vehicle and NanoCa, NanoMn and MnCl were present in comparison with the control group₂Can obviously stimulate dendritic cells to express IFN β, and the NanoMn is stronger than MnCl₂Approximately 2 times.

To verify NanoMn and MnCl₂Whether all produced IFN β through the cGAS-STING pathway and exerted biological effects, dendritic cells, NanoMn and MnCl, were also induced in cGAS knockout mice₂IFN β mRNA expression levels were measured after treatment, as shown in FIG. 7, the results indicate that either NanoMn or MnCl is present₂The ability of dendritic cells to be stimulated to produce IFN β was significantly down-regulated, indicating that both are dependent on the cGAS-STING pathway to produce IFN β.

Example 11 RNA-Seq comparison of liposomes containing manganese hydrogen phosphate Microprecipitates with MnCl₂Stimulation of host cell differential Gene expression experiments

To analyze NanoMn and MnCl₂The reason for the difference in the ability to activate IFN β in the host cells was the use of mouse primary dendritic cells, NanoMn and MnCl₂Respectively 200 muM treatment for 12 hours. Cells were harvested and RNA-Seq was performed.

C57BL/6 mouse primary dendritic cell induction and treatment were the same as in example 10. After cell collection, the cells were vortexed in TRIzol until completely lysed, frozen at-80 ℃ and transcriptome sequencing was performed. The results are shown in FIG. 8, and the gene differential expression analysis shows that compared with the dendritic cell maturation associated molecules in the control group, the dendritic cell maturation associated molecules are NanoMn and MnCl₂After treatment, the protein is obviously up-regulated. Shows that the NanoMn and the MnCl₂Can promote the maturation of dendritic cells, further enhance the antigen presentation of the dendritic cells, finally induce T-cells and play the anti-infection and anti-tumor roles. In addition, compared to MnCl₂The effect of the NanoMn on most genes is more obvious, which shows that the NanoMn has stronger advantages. Furthermore, T cell activating costimulatory molecules, effectors, were significantly elevated after NanoMn treatment and higher than MnCl₂Groups (fig. 9). The nano Mn has the capability of more strongly activating effector T cells to play anti-infection and anti-tumor roles.

GO functional analysis shows that compared with MnCl₂The biological processes of the NanoMn antiviral and antitumor are more enriched; reactome signal pathway analysis found that compared to MnCl₂The NanoMn antiviral and antitumor signaling pathway was more enriched (fig. 10, fig. 11). These results show that compared to MnCl₂The NanoMn has stronger effect in the antiviral and antitumor processes.

Example 12, B16F10 melanoma mice transplantation tumor experiment

To verify the comparison to MnCl₂The NanoMn has stronger effect in the anti-tumor process. Mouse transplantation tumor models were prepared in C57BL/6 mice, and NanoMn and MnCl were performed, respectively₂And (4) processing and observing the growth condition of the tumor.

Taking C57BL/6 mice 6-8 weeks later, inoculating 5 × 10 subcutaneously on the back⁵B16F10 melanoma cells. 3 mug/g NanoMn or MnCl is injected into the abdominal cavity on the 3 rd, 7 th and 11 th days respectively₂. Tumor growth was observed and it was found that the NanoMn group had tumor growth relative to MnCl₂The group is slow, indicating that NanoMn may have stronger tumor inhibition effect. The results are shown in FIG. 12.

Example 13, B16F10 melanoma mice metastasis experiment

To verify the comparison to MnCl₂The NanoMn has stronger effect in the anti-tumor process. Mouse metastatic tumor model was made in C57BL/6 mice, and NanoMn and MnCl were performed separately₂And (5) processing, and observing the growth condition of metastatic nodules in the lung of the mouse.

Injecting C57BL/6 mice 6-8 weeks later into tail vein of 1 × 10⁶B16F10 melanoma cells. Injecting 20 mu g/g of NanoMn or MnCl into the abdominal cavity on the 5 th day and the 10 th day respectively₂. Mice were dissected 21 days later and observed for metastatic nodules of lung tumors. Found to compare to MnCl₂Group, NanoMn group, had few metastatic nodules. The results are shown in fig. 13, which indicates that NanoMn may have a stronger tumor metastasis inhibition effect.

Example 15 inhibition of viral infection by NanoMn

To investigate the inhibitory effect of NanoMn on viral infection, HeLa cells were treated with NanoMn at various concentrations, and MnCl was used₂And Vehicle as a control, Hela cells were infected with vesicular stomatitis virus (VSV-GFP) expressing Green Fluorescent Protein (GFP) at 0.01MOI for 16h, the cells were analyzed by microscopy and flow cytometry (fig. 14), and EC50 was calculated as a percentage of GFP-positive cells relative to untreated controls (fig. 15) to observe the efficacy of NanoMn in viral infection. With free Mn²⁺(EC50 ═ 71.67 μ M) the inhibition of viral replication by NanoMn treatment was greater (EC50 ═ 71.67 μ M)₅₀＝3.897μM)。

With 10. mu.M MnCl₂Or treating HeLa cells with NanoMn for specified hours, detecting the expression level of interferon IFN β and OASL1 genes by quantitative reverse transcription polymerase chain reaction, and treating with NanoMn to MnCl similarly to the antiviral effect₂Treatment induced a stronger type I interferon response (figure 16).

With 10. mu.M MnCl₂HeLa cells were treated with NanoMn or PBS. At the same time, the virus was shown to infect cells for 24h at 0.01 MOI. The expression level of the viral RNA was detected by the qRT-PCR method. As shown in FIG. 17, NanoMn showed broad-spectrum antiviral action and inhibited replication of the coronavirus MHV-A59 and other viruses in host cells. The data indicate that NanoMn triggers the host interferon response, limiting viral transmission.

Example 16 acute toxicity of NanoMn to organs

To investigate the acute toxic effect of NanoMn on organs, we analyzed NanoMn and MnCl₂And clinical indexes of acute tissue injury such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and the like in the blood of mice after the mice are treated by Vehicle for 36 h. C57BL/6 mice were injected intravenously with 1.8mM/kg MnCl₂NanoMn or PBS. Mice were sacrificed after 36 hours and blood was collected for biochemical blood testing. As shown in FIG. 18, with MnCl₂Compared with mild hepatotoxicity and cardiotoxicity caused by the treatment, the treatment of the NanoMn has little influence on the levels of functional parameters of the liver, kidney and heart, and shows that the NanoMn has no obvious acute damage to main organs of a host.

Example 17 NanoMn triggers an Interferon response in the reticuloendothelial System

To test whether NanoMn could elicit a type I interferon response in vivo, we analyzed the transcriptome of NanoMn, manganese chloride and Vehicle-treated mouse liver. C57BL/6 mice were injected intraperitoneally with 1.8mM/kg MnCl₂NanoMn or PbS. After 18 hours, mouse livers were taken for RNA-seq assay. As shown in FIG. 19, GSEA analysis of MnCl with GO genome₂And differences in liver gene expression in NanoMn-treated mice. ES is an abbreviation for enrichment fraction, NES denotes normalized enrichment fraction. NanoMn treated mice to MnCl₂The treated mice detected higher mRNA levels of the interferon signal-related gene. Further, unlike NanoMn, MnCl₂These results were further confirmed by subsequent qPCR methods examining the expression levels of IFN β and IRF7 genes in the liver (fig. 20).

In addition to activating the interferon signaling pathway, we noted that NanoMn treatment also enhanced signaling associated with antigen processing and presentation (fig. 21). In conclusion, our findings indicate that the NanoMn prepared in example 1 is non-toxic and triggers an interferon response of the reticuloendothelial system in vivo.

Example 18 NanoMn treatment improves treatment of coronavirus infection

To determine whether an enhanced interferon response could improve treatment of coronavirus infection, C57BL/6 mice were intraperitoneally injected with 1.8mM/kg of NanoMn, MnCl, respectively₂Or Vehicle.24 hours later, the mice were injected with a lethal dose of 3 × 10⁵MHV-A59 of PFU. As shown in fig. 22, although MnCl₂Treatment extended the survival of virus-infected mice to some extent, but either Vehicle or MnCl₂The treated mice all died within 8 days after viral infection. Notably, more than half of the NanoMn pretreated mice survived this process.

Furthermore, in the use of Vehicle or MnCl₂But not the NanoMn-pretreated mice, severe weight loss was detected (fig. 23). Additionally, mice were sacrificed on day 4 post infection, spleens and livers were removed. The expression level of MHV-N gene was detected by qRT-PCR method. qRT-PCR analysis showed that the NanoMn treatment significantly reduced viral titers in the liver and spleen (fig. 24). Thus, our data indicate that NanoMn treatment can improve treatment of coronavirus infection.

Example 19 NanoMn amelioration of coronavirus induced tissue damage

To verify the therapeutic effect of NanoMn on coronavirus infection, we used 3 × 10⁵PFU MHV-A59 was injected intraperitoneally into C57BL/6 mice. After 24 hours, 1.8mM/kg of MnCl was intraperitoneally injected, respectively₂NanoMn or PBS. As shown in FIG. 25, similar to the NanoMn pretreatment, the survival of coronavirus infected mice was also significantly prolonged by post-infection injection of NanoMn, while either Vehicle or MnCl was used₂Other mice treated died within 7 days after viral infection.

On the 4 th day after infection, organs of the infected mice were collected, and the expression level of the MHV-N gene in each organ was examined by the qRT-PCR method. As shown in FIG. 26, with Vehicle or MnCl₂In contrast to treated mice, the NanoMn treatment limited viral propagation in the liver and spleen.

In order to fully analyze the protective effect of NanoMn on coronavirus infection, differential expression genes of liver tissues in the virus infection process are analyzed by an RNA-seq method. Abdominal cavity injection of C57BL/6 miceMHV-A59 was infected by the injection, and mouse livers were taken on day 4 post infection for RNA-seq analysis. Enrichment analysis of differentially expressed genes with upregulated expression in NanoMn and Vehicle group mice was performed using the GO database (a in fig. 27). The heatmap (b in fig. 27) shows the expression levels of genes associated with metabolism in untreated or infected mice. With Vehicle or MnCl₂In contrast to treated mice, NanoMn rescued gene expression from metabolic process-related pathways affected by coronavirus infection.

In addition, C57BL/6 mice were injected intraperitoneally with 3 × 10⁵PFU MHV-A59, intraperitoneal injection of 1.8mM/kg MnCl 24h after infection₂NanoMn or PBS. On day 4 post-infection, mice spleens were photographed and weighed, and splenic lymphocytes were isolated and counted for statistical analysis. In addition to the liver, we also noted that NanoMn treatment resulted in mild enlargement of the spleen and an increase in the number of splenocytes (fig. 28).

Day 0, C57BL/6 mice were infected intraperitoneally with 3 × 10⁵MHV-A59 of PFU. After 24 hours, mice were injected intraperitoneally with 1.8mm/kg MnCl₂NanoMn or PBS. Blood from the inner canthus of the mouse is collected on the designated days and routine blood detection is carried out. Statistical analysis was performed using the percentage and cell count of Lymphocytes (LY) and Monocytes (MO). By analyzing the percentage of immune cells in blood, we found that during coronavirus infection, NanoMn increased the number of lymphocytes and monocytes in blood (fig. 29). Thus, the data indicate that, in addition to the innate immune response, host acquired immunity may also be involved in the protective effect of NanoMn on coronavirus infection.

Example 20 NanoMn as vaccine adjuvant enhances host adaptive immunity

To determine whether host acquired immunity was also involved in coronavirus infection, we injected C57BL/6 mice intraperitoneally with an infecting non-lethal dose of MHV-A59(3 × 10)⁴PFU), intraperitoneally injected with 1.8mM/kg NanoMn or PBS 24 hours after infection, and intraperitoneally infected with a lethal dose of MHV-A59(3 × 10) on day 14 after infection⁵PFU). As shown in figure 30, we re-challenged mice with a lethal dose of MHV-a59 2 weeks after the primary viral infection, and all mice that were re-challenged with virus survived.

In addition, mice were sacrificed 3 days after the second challenge, liver and spleen lymphocytes were collected for flow cytometry analysis, and qRT-PCR analysis showed a significant reduction in virus titer in virus re-challenged mice compared to mice initially infected with virus (fig. 31). Thus, our data suggest that coronavirus infection can provoke the development of an adaptive immune response and immune memory in the host.

To investigate the role of NanoMn in adaptive immune regulation, we evaluated T cell development at day 7 after non-lethal viral infection. Mice were sacrificed on day 7 post infection with a non-lethal dose of MHV-A59 and organs were collected. Organ lymphocytes are separated, and CD8 memory T cells (CD 8) are detected by a flow cytometer⁺CD44^hiCD62L⁺) Percentage of (c). As shown in FIG. 32, NanoMn treatment promoted CD8 in the liver and spleen⁺Memory T cell (CD 44)^hiCD62L⁺) Polarization of (1).

To further confirm the stimulatory effect of NanoMn on memory T cell function, we re-challenged virus-infected mice with a lethal dose of MHV-a59 with or without NanoMn treatment. On day 3 after reinfection with MHV-A59, the mice were sacrificed, and the livers of the infected mice were removed and lymphocytes were isolated. Flow cytometry for detecting CD8⁺CD25⁺And CD4⁺CD25⁺Percentage of cells, statistical analysis was performed. As shown in FIG. 33, CD8 in the liver of NanoMn-treated mice⁺CD25⁺(activated) T cell proliferation, CD4⁺CD25⁺(regulatory) T cell depletion.

Using CD8⁺、CD4⁺、B220⁺The cell percentage was statistically analyzed, and spleen CD8 was statistically analyzed⁺CD44^hiAnd CD4⁺CD44^hiCD25 in cells⁺Percentage of cells, we found that unlike the primary viral infection, which elicited a systemic immune response, the immunostimulatory effect of NanoMn was mainly concentrated in the liver rather than the spleen during the secondary viral infection (fig. 34).

On day 3 after the second challenge, lymphocytes were isolated from the liver of infected mice and the mice were treated with 100ng/mL PMA and 500ng/mL ionomycin5h, detecting CD8 by using flow cytometry⁺And CD4⁺Cells produced tumor necrosis factor TNF α and interferon IFN γ As shown in FIG. 35, NanoMn treatment resulted in T cells producing more effector cytokines in the liver rather than the spleen as a result of T cell polarization (TNF α)⁺IFNγ⁺). Thus, our data indicate that NanoMn enhances the cellular immune response to coronaviruses.

To determine whether NanoMn affects humoral immunity, we subcutaneously inoculated C57BL/6 mice with 3 × 10⁴PFUMHV-A59 and 1.8mM/kg NanoMn or PBS 14 days after primary viral infection, mice were injected intraperitoneally with a lethal dose of 3 × 10⁵MHV-A59 of PFU. As shown in fig. 36, all immunized mice survived similar to the i.p. injection. By body weight analysis, the NanoMn-treated group significantly reduced viral toxicity compared to the control group. More importantly, the NanoMn treatment increased serum IgG concentrations relative to control mice (fig. 36). Taken together, our data indicate that NanoMn can be used as a vaccine adjuvant to enhance adaptive immunity and protect a host from coronavirus infection.

Claims

1. A manganese-containing micro-precipitation liposome is a biodegradable liposome coated with manganese ion precipitates, and consists of a micro-precipitation inner core containing manganese ions and a phospholipid bilayer shell.

2. The liposome of claim 1, wherein the liposome has a particle size of 20-200nm, and a phospholipid bilayer shell with a positive charge, wherein the liposome slowly degrades and releases manganese ions in a more acidic environment after entering cells.

3. The liposome of claim 1, wherein the manganese ion is a divalent manganese ion and the manganese ion-containing microprecipitated core is selected from one or more of the following compounds: manganese phosphate, manganese hydrogen phosphate, manganese carbonate and manganese oxalate.

4. The liposome of claim 1, wherein the phospholipid bilayer enclosure is comprised of a phospholipid material and cholesterol, wherein the phospholipid material is selected from one or more of the following materials: DOPA, DOTAP, DSPE-PEG.

5. The liposome of claim 4, wherein the liposome comprises 20% to 80% DOPA, 8% to 40% DOTAP, 8% to 40% cholesterol, and 2% to 20% DSPE-PEG by mole percent in the phospholipid bilayer.

6. The liposome of claim 1, wherein a targeting molecule is coupled to the surface of the liposome.

7. Use of the manganese-containing microprecipitated liposome of any one of claims 1-6 for the preparation of a medicament for the treatment of immune-related diseases.

8. The use of claim 7, wherein said manganese-containing microprecipitated liposomes are used as a component for stimulating innate and/or adaptive immunity or as an immunoadjuvant in said medicament.

9. The use as claimed in claim 7, wherein the medicament is an antiviral, antibacterial, antiparasitic, or autoimmune disease treating medicament, or cancer treating medicament.

10. Use according to claim 9, wherein the virus is a DNA virus or an RNA virus, preferably selected from the group consisting of herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, hepadnaviridae, flaviviridae, papilloma viridae, poxviridae and retroviridae; the bacteria are gram-negative bacteria or gram-positive bacteria, preferably selected from streptococcus pneumoniae, haemophilus influenzae, salmonella, meningococcus, staphylococcus epidermidis, staphylococcus aureus, escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, enterobacter cloacae, citrobacter freundii, pseudomonas aeruginosa and acinetobacter baumannii; the parasite is selected from the group consisting of plasmodium, toxoplasma, trypanosoma, schistosoma, filarial worms and leishmania; the autoimmune diseases include, but are not limited to, type I diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis; such cancers include, but are not limited to, breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cancer, colorectal cancer, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, and leukemia.

11. The method for preparing a manganese-containing microprecipitated liposome of any one of claims 1 to 6, comprising the steps of:

1) preparing a compound containing divalent manganese ions into a water solution with a certain concentration, dropwise adding the water solution into an oil solution under the ultrasonic condition, continuing to perform ultrasonic treatment to crack water drops and uniformly disperse the water drops in an oil phase, wherein the volume ratio of a water phase to the oil phase is 1:10-1: 1000;

2) preparing a compound containing anions in the micro-precipitate into an aqueous solution, dropwise adding the aqueous solution into an oil solution under the ultrasonic condition, and continuing to perform ultrasonic treatment to crack water droplets and uniformly disperse the water droplets in an oil phase, wherein the volume ratio of the water phase to the oil phase is 1:10-1: 1000;

12. The method according to claim 11, wherein the compound containing divalent manganese ions in step 1) is selected from one or more of the following compounds: manganese chloride, manganese nitrate, manganese sulfate; the compound containing anions in the micro-precipitate in the step 2) is selected from one or more of the following compounds: sodium hydrogen phosphate, sodium carbonate and sodium oxalate.

13. The method of claim 11, wherein the oil solution in steps 1) and 2) is selected from one or more of the following oily solvents: cyclohexane, cyclopentane, petroleum ether, benzene; a nonionic surfactant is added to the oil solution.

14. The method according to claim 11, wherein the first organic solvent in step 3) is an alcohol or ether organic solvent, preferably selected from methanol, ethanol, propanol and diethyl ether; the second organic solvent is a volatile non-polar organic solvent, preferably selected from chloroform, dichloromethane, carbon tetrachloride, bromoethane, benzene, cyclohexane, hexane, diethyl ether, isopropyl ether and ethyl acetate.