Metal element lipid composition with atypical crystalline structure and preparation method and use thereof

Abstract

The present invention discloses a metal element lipid composition with an atypical crystalline structure, a preparation method thereof and a use thereof. The calcium metal element in the lipid composition of the present invention has a specific structural morphology, which can overcome the problem that free metal ions are difficult to directly enter cells, successfully deliver the metal element into cells, and overcome the deficiency that traditional calcium phosphate cannot activate IFNβ. Functional experiments confirm that it can significantly induce the production of IFNβ, thereby playing an important role in immune responses such as anti-viral infection and anti-tumor.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a metal element lipid composition having an atypical crystal structure and capable of activating IFNβ, and a preparation method and application thereof. Background Art

[0002] Type I interferon IFNβ is one of the most important core molecules in the innate immune response, and plays an important role in anti-viral infection and anti-tumor. Studies have found that manganese can induce the production of IFNβ by activating the cytoplasmic DNA pattern recognition receptor cGAS to exert anti-viral and anti-tumor effects. However, there are no reports on other metal elements inducing the production of IFNβ.

[0003] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0004] In view of at least some of the technical problems in the prior art, the present invention provides a metal element lipid composition with a specific structure, a preparation method and use thereof. The calcium salt in the lipid composition of the present invention has an atypical crystalline structure, which can overcome the problem that free metal ions are difficult to directly enter cells, successfully deliver metal elements into cells, and at the same time, the atypical crystalline structure can activate or greatly enhance the production of IFNβ and / or IFNλ1 in an organism. The present invention is completed at least in part based on the above findings. Specifically, the present invention includes the following contents.

[0005] In a first aspect of the present invention, a metal element lipid composition having a special crystal structure is provided, which comprises a lipid membrane shell layer and a loose calcium salt core wrapped by the lipid membrane shell layer.

[0006] In certain embodiments, according to the metal element lipid composition of the present invention, the loose calcium salt is an atypical crystalline calcium phosphate.

[0007] In certain embodiments, according to the metal element lipid composition of the present invention, the electron density of the atypical crystalline calcium phosphate is lower than the electron density of the stable calcium phosphate when detected by small angle X-ray scattering, preferably, it has Figure 6 Figure shown.

[0008] In certain embodiments, according to the metal element lipid composition of the present invention, the lipid membrane comprises phospholipids and optionally cholesterol.

[0009] In certain embodiments, according to the metal element lipid composition of the present invention, the lipid membrane comprises at least one of phospholipids, sterols, sphingolipids and glycerolipids; preferably, phosphatidic acid selected from cholesterol, sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol and C5-C25 saturated or unsaturated fatty acids.

[0010] The second aspect of the present invention provides a method for preparing a metal element lipid composition, which comprises encapsulating metal element particles with lipids to obtain the metal element lipid composition.

[0011] In certain embodiments, the method for preparing the metal element lipid composition according to the present invention further comprises the step of obtaining metal element core particles.

[0012] In certain embodiments, the method for preparing the metal element lipid composition according to the present invention comprises obtaining the metal element core particles by the following steps:

[0013] (1) dispersing the metal salt solution into the oil phase to obtain a first microemulsion;

[0014] (2) dispersing the anionic compound into the oil phase to obtain a second microemulsion; and

[0015] (3) The first microemulsion and the second microemulsion are fused, and the oil phase is removed to obtain metal element core particles.

[0016] In certain embodiments, according to the method for preparing the metal element lipid composition of the present invention, step (2) further comprises dispersing the anionic compound into the oil phase and then adding the lipid solution for ultrasonic treatment to obtain a second microemulsion.

[0017] The third aspect of the present invention provides a pharmaceutical composition or immune adjuvant, which comprises the metal element lipid composition described in the first aspect and an optional pharmaceutically acceptable carrier or excipient.

[0018] The fourth aspect of the present invention provides the use of a metal element lipid composition in the preparation of a drug for activating IFNβ, IFNλ1 or inducing the production of IFNβ or IFNλ1, a drug for immune-related diseases or conditions, a drug for increasing the number of immune cells, or an antimicrobial or antitumor drug.

[0019] The fifth aspect of the present invention provides a method for activating interferon in an organism in vitro or inducing its production, which includes the step of contacting an organism with the metal element lipid composition described in the first aspect of the present invention in vitro; preferably, the organism includes cells or tissues; preferably, the interferon includes interferon IFNβ and / or interferon IFNλ1.

[0020] The present invention aims to solve the problem that free metal ions are difficult to directly enter cells. In order to successfully deliver metal elements into cells, a series of metal liposome nanoparticles are prepared by thin film dispersion method and reverse microemulsion precipitation method. Functional experimental screening finds that a specific metal element lipid composition can significantly induce the production of IFNβ. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flow chart of the preparation of exemplary metal nanoparticles (i.e., NanoCa metal nanoparticles).

[0022] Figure 2 Characterization of exemplary NanoCa metal nanoparticles. Figure 2 A is a physical picture of NanoCa; Figure 2 B is a transmission electron microscopy image of NanoCa; Figure 2 C is the particle size of NanoCa; Figure 2 D is the membrane potential diagram of NanoCa.

[0023] Figure 3 : is an energy dispersive X-ray spectroscopy elemental analysis diagram of exemplary NanoCa metal nanoparticles.

[0024] Figure 4 The figure below is an exemplary diagram of the physical and chemical properties of NanoCa metal nanoparticles. The left figure shows the CCK8 detection of cell activity after different concentrations of NanoCa were used to treat HepG2, HT29, RAW264.7 and HT1080 cells for 24 hours. In each group of bar graphs, from left to right are HepG2, HT29, RAW264.7 and HT1080; the right figure shows the release efficiency of NanoCa in PBS buffers with different pH values ​​detected by inductively coupled plasma.

[0025] Figure 5 The left picture is a crystal form picture of CaCO3, and the right picture is a crystal form picture of NanoCa.

[0026] Figure 6 The left picture is the small angle X-ray scattering result of CaP, the middle picture is the small angle X-ray scattering result of NanoCa, and the right picture is the Ca of NanoCa, CaP and CaCO3. 2+ Signal diagram.

[0027] Figure 7 RT-qPCR was used to detect the expression of type I interferon IFNβ1 in HepG2 cells after they were treated with different metal nanoparticles for 24 hours.

[0028] Figure 8 Screening of interferon activated by NanoCa in different cells. Figure 8 A is the RT-qPCR detection of IFNβ1 and Figure 8 B, expression of IFNλ1; Figure 8 C is the RT-qPCR detection of IFNβ1 and Figure 8 D is the expression of IFNλ1; Figure 8 E is the expression of IFNβ1 detected by RT-qPCR after Caco2 cells were treated for 24 h. Figure 8 F is the expression of IFNλ1; Figure 8 G is the expression of IFNβ1 detected by RT-qPCR after treating RAW264.7 cells for 24 h; Figure 8 H is the expression of IFNβ1 detected by RT-qPCR after EL4 cells were treated for 24 h; Figure 8 I is the RT-qPCR detection of IFNβ1 and Figure 8 J is the expression of IFNλ1; Figure 8 K is the final concentration of 100 μM / L NanoCa treatment of HepG2 cells at different time points after RT-qPCR detection of IFNβ1 and Figure 8 L is the expression of IFNλ1.

[0029] Fig. 9 This is the differential gene enrichment analysis of HT29 cell RNA-Seq. Fig. 9 A is the RNA-Seq differential gene heat map of HT29 cells treated with 100 μM / L NanoCa, MnCl2 and Nanovector for 24 hours; Fig. 9 B is the volcano plot of differentially expressed genes in HT29 cell RNA-Seq; Fig. 9 C is the GO pathway enrichment analysis of differentially expressed genes in RNA-Seq of HT29 cells and Fig. 9 D is KEGG pathway enrichment analysis (n=2).

[0030] Fig.10 This is the differential gene enrichment analysis of RAW264.7 cells RNA-Seq. Among them, Fig.10 A is the heat map of RNA-Seq differentially expressed genes after RAW264.7 cells were treated with NanoCa, MnCl2 and Nanovector at a final concentration of 100 μM / L for 24 h; Fig.10 B is the GO pathway enrichment analysis of differentially expressed genes in RAW264.7 cell RNA-Seq and Fig.10 C is KEGG pathway enrichment analysis (n=2).

[0031] Fig.11NanoCa activates IFNβ in C57BL / 6 mice. Fig.11 A shows the expression of IFNβ1 and IL-6 in the spleen of wild-type C57BL / 6 mice after tail vein injection of 100 μL of 8 mM / L NanoCa. Fig.11 B is ELISA to detect IFNβ1 in blood.

[0032] Fig.12 Flow chart of animal experiments. Each mouse was injected subcutaneously with 5×10 5 B16 cells, equivalent to a tumor volume of about 500mm 3 100 μl of NanoCa and Nanovector with an initial concentration of 8 mM / L were injected into the tail vein at 1 pm. The above results were obtained by three independent repeated experiments.

[0033] Fig.13 The left picture is a picture of subcutaneous melanoma analysis (5 animals in each group); the middle picture is a quantitative picture of tumor volume; the right picture is a curve of mouse weight. The above results were obtained by three independent repeated experiments.

[0034] Fig.14 The following are the physical pictures of the main organs: heart, liver, lung and kidney. Each result was independently repeated three times.

[0035] Fig.15 HE staining of the main organs: heart, liver, spleen, lung and kidney. Each result was independently repeated three times.

[0036] Fig.16 Figure 1 shows the actual lung of a melanoma lung metastasis model (3 animals in each group). Each mouse was injected with 5×10 5 The above results were performed with three independent replicates.

[0037] Fig.17 The figure is a quantification graph of lung tissue weight. The above results were obtained by three independent repeated experiments.

[0038] Fig.18 This is the result of the synergistic effect between NanoCa and PD1 antibody. In order to study whether NanoCa has the effect of synergizing with PD1 antibody, BALB / c mice were subcutaneously injected with CT26 to establish the model. After the tumor grew to a certain volume, NanoCa, PD1, and NanoCa+PD1 were injected respectively. The tumor volume was regularly tested. The results showed that the tumor volume and weight of the NanoCa+PD1 group were the smallest, indicating that NanoCa can synergize with PD1 to exert an anti-tumor effect.

[0039] Fig.19The results of NanoCa's anti-tumor effect in pancreatic cancer. In order to study whether NanoCa has an anti-tumor effect in pancreatic cancer, we injected PANO2 cells subcutaneously into C57BL / 6 mice to create a model. After the tumor grew to a certain size, we injected NanoCa and Nanovector respectively. The results showed that the tumor size in the NanoCa group was smaller, indicating that NanoCa has an anti-pancreatic cancer effect.

[0040] Fig. 20 NanoCa exerts anti-tumor effects by increasing the number of immune cells. Fig. 20 A is cell flow cytometry detection CD 3+ CD 8+ T lymphocyte flow cytometry and quantification result bar graph; Fig. 20 B is the flow cytometry graph and quantitative result bar graph of NK cells detected by flow cytometry; Fig. 20 C is the CD4 T cells secreting IFNγ detected by flow cytometry 8+ Flow cytometry and quantification results bar graph of T cells; Fig. 20 D is the proportion of CD8+T cells in tumors of NanoCa group, PD1 group, NanoCa+PD1 group, Nanovector group and mock group detected by cell flow cytometry. Fig. 20 E is the proportion of CD8+T cells secreting IFNγ in the tumors of NanoCa group, PD1 group, NanoCa+PD1 group, Nanovector group and mock group detected by flow cytometry. The above results were independently repeated three times.

[0041] Fig.21 Calcium deficiency can reduce the immunity of mice. Fig.21 A is a real picture of the tumor (4 mice in each group); Fig.21 B is the quantification of tumor volume; Fig.21 C is the weight curve of mice; Fig.21 D is RT-qPCR detection of mouse spleen FNβ1 and Fig.21 E is the expression of IFNγ; Fig.21 F is the expression of ISG15 in mouse spleen detected by RT-qPCR; Fig.21 G is the flow cytometry graph and quantitative result bar graph of B lymphocytes detected by flow cytometry; Fig.21 H is cell flow cytometry detection CD 3+ CD 8+ T lymphocyte flow cytometry and quantification results bar graph.

[0042] Fig. 22 NanoCa has antiviral effects. Fig. 22A is the detection of HSV-1 in HT29 cells infected with 0.1 MOI of HSV-1 and treated with 100 μM / L NanoCa and Nanovector for 24 h; Fig. 22 B is the detection of VSV after HT29 cells infected with 0.1 MOI of VSV were treated with 100 μM / L NanoCa and Nanovector for 24 h; Fig. 22 C is the detection of MHV after RAW264.7 cells infected with 0.1 MOI MHV were treated with 100 μM / L NanoCa and Nanovector for 24 h; Fig. 22 D is a 6-8 week old female mouse that was injected intravenously with 5×10 7 Survival curve of PFU HSV-1 (10 mice per group); Fig. 22 E is the HSV-1 virus titer in the blood; Fig. 22 F is the detection of IFNβ1 and (G) ISG15 in blood; Fig. 22 H was injected into the tail vein of 6-8 week-old female mice with 1×10 7 Survival curve of PFU VSV (10 mice in each group); Fig. 22 I is the VSV virus titer in blood; Fig. 22 J is the level of IFNβ1 and Fig. 22 K is the detection of ISG15. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] [Metallic element lipid composition]

[0047] In a first aspect of the present invention, a metal element lipid composition is provided, which is sometimes referred to herein as "the lipid composition of the present invention" or "the metal element lipid composition of the present invention", comprising a lipid membrane and a calcium salt. Preferably, the lipid membrane encapsulates the calcium salt to form a core-shell structure.

[0048] In the present invention, calcium salt refers to a salt as an active ingredient for activating IFNβ, and optionally further includes other metal elements, which can be used to enhance or improve the activation function of IFNβ, and can also have other functions, such as a function of supplementing nutrition, examples of which include but are not limited to iron, manganese, zinc, etc. In addition, the lipid composition of the present invention may further contain other metal elements as impurities. Such other metal elements are generally non-essential elements introduced during the preparation process, such as sodium, potassium, etc.

[0049] In the present invention, the calcium salt is preferably present in the form of a slightly soluble loose solid powder, and its illustrative examples include calcium salts that are slightly soluble in water, such as calcium phosphate. The present invention finds that the activity of activating interferon can only be produced when the calcium salt exists in a specific form. The calcium salt of the present invention is not a traditional salt form, but exists in a special form or an atypical crystalline form. When the calcium salt in this form is detected by small-angle X-ray scattering, the electron density of the atypical crystalline calcium phosphate is lower than the electron density of stable calcium phosphate. Herein, stable calcium phosphate is a traditional calcium phosphate, which has a higher electron density.

[0050] In the present invention, the lipid components constituting the lipid membrane are not limited, and components known in the art can be used, including but not limited to phospholipids, neutral lipids and amphoteric lipids. As phospholipids, examples include but are not limited to natural phospholipids, sphingolipids, glycosphingolipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and sphingomyelin, dipalmitoylphosphatidylcholine, serine or ethanolamine glycerol or the corresponding oleic acid esters of these compounds, phosphorylcholine as an intermediate in the synthesis of phosphatidylcholine, natural phospholipid derivatives, egg phosphorylcholine, soybean phosphorylcholine, hydrogenated soybean phosphorylcholine, phosphorylcholine (didecanoyl-L-α-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine) and phospholipids. choline [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoylphosphatidylcholine [DPPC], distearoylphosphatidylcholine [DSPC], dioleoylphosphatidylcholine [DOPC], 1-palmitoyl, 2-oleoylphosphatidylcholine [POPC], di-re-oleoylphosphatidylcholine [DEPC]), phosphoglycerol (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], 1,2-dipalmitoyl-sn-glycero- 3-phosphoglycerol [DPPG], 1,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG]), phosphatidic acids (1,2-dimyristoyl-sn-glycero-3-phosphoglyceric acid [DMPA], dipalmitoylphosphatidic acid [DPPA], distearoyl-phosphatidic acid [DSPA]), phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [ DMPE], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [DSPE], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]), phosphoserine, polyethylene glycol [PEG] phospholipids (mPEG-phospholipids, polyglycerol-phospholipids, functionalized phospholipids, terminal activated phospholipids), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP). As neutral lipids, examples thereof include, but are not limited to, dioleoylphosphatidylethanolamine DOPE, cholesterol, methoxy (polyethylene glycol)-distearoyl mPEG-DS. In the present invention, the lipid film may further include other components. For example, cholesterol. In certain embodiments, the lipids of the present invention include modified lipids, such as polyethylene glycol-modified lipids, examples of which include, but are not limited to, DSPE-PEG. In certain embodiments, the lipids of the present invention include at least one of DOPA, DOTAP and DSPE-PEG.

[0051] In the present invention, the metal element lipid composition is preferably present in the form of particles. The particle size is not limited, preferably nanoparticles, and the average particle size is generally 1-900nm, such as 5-800nm, 6-600nm, 8-500nm, 10-200nm, etc., such as 20, 50, 100, 150nm, etc.

[0052] [Preparation method]

[0053] The second aspect of the present invention provides a method for preparing a metal element lipid composition. As long as the lipid composition of the composition and structure described in the first aspect can be obtained, the specific preparation method is not particularly limited. Generally speaking, it includes the step of making lipid-coated metal element particles obtain a metal element lipid composition. The formation of liposomes by lipid-coated particles can adopt any technology known in the art. The lipids here are generally composed of multiple components. Exemplarily, including but not limited to DOPA, cholesterol and DSPE-PEG, the dosage ratio is not limited and can be freely set by those skilled in the art as needed. The exemplary dosage ratio of DOPA and cholesterol is 0.5:2 to 2:0.5. The dosage of DOPA and DSPE-PEG is based on a molar ratio of 5:1 to 5:6, etc.

[0054] In certain embodiments, step (4) of the present invention further comprises the step of evaporating the solvent to form a film on the surface of the metal element core particles. The evaporation conditions are not limited, for example, it can be carried out by rotary evaporation at room temperature.

[0055] In certain embodiments, step (4) of the present invention further comprises adding water or buffer to the film-formed core particles for dispersion.

[0056] In the present invention, the metal element particles can be prepared by any known method or purchased, for example, a method of preparing calcium phosphate nanostructured powder by microwave-assisted hydrothermal reaction using water-soluble calcium salt as a calcium source and phosphorus-containing biomolecules as a phosphorus source.

[0057] Exemplarily, the present invention uses a thin film dispersion combined with a reverse microemulsion precipitation method to illustrate the preparation method of the metal element lipid composition of the present invention, which specifically includes the following steps:

[0058] (1) dispersing the metal salt solution into the oil phase to obtain a first microemulsion;

[0059] (2) dispersing the anionic compound into the oil phase to obtain a second microemulsion;

[0060] (3) dissolving the first microemulsion and the second microemulsion, and removing the oil phase to obtain metal element core particles.

[0061] It will be understood by those skilled in the art that the numbers (1), (2), etc. representing the steps are only for the purpose of distinguishing different steps, and do not mean the order of the steps. As long as the purpose of the present invention can be achieved, the order of the above steps is not particularly limited. In addition, without affecting the purpose of the present invention, two or more of the above steps can be combined and performed simultaneously. In addition, it will be understood by those skilled in the art that other steps or operations may be included before and after the above steps (1)-(4), or between these arbitrary steps, such as further optimizing and / or improving the method described in the present invention.

[0062] Step (1)

[0063] Step (1) of the present invention is a preparation step of the first microemulsion, i.e., a metal salt microemulsion. Specifically, a solution of a soluble metal salt is dispersed into the oil phase. Wherein the soluble metal salt is generally different from the metal salt in the lipid composition. The metal salt in the lipid composition is generally an insoluble or slightly soluble or precipitated metal salt, while the soluble metal salt refers to a metal salt soluble in a solvent, especially an aqueous solvent, examples of which include but are not limited to calcium chloride, calcium nitrate, calcium fluoride, etc. In the solution of the soluble metal salt, the concentration of the metal salt is not limited, and is generally related to the solubility of the metal salt. Exemplary concentrations are generally 0.01-9000 μM / L, preferably 0.1-5000 μM / L, more preferably 1-1000 μM / L, for example 10 μM / L, 20 μM / L, 50 μM / L, 100 μM / L, 200 μM / L, 300 μM / L, 400 μM / L. The oil phase herein refers to the commonly used oily substance in emulsion, including oil, fat, wax of natural plants and animals, including fatty acid glyceride, lanolin, lecithin, squalane, etc. Mineral oil substances include stearic acid (octadecanoic acid), cetyl alcohol (hexadecanol), cholesterol, stearyl alcohol (octadecyl alcohol), isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl linoleate, benzoic acid lauryl ester, isostearyl isostearate, fatty acid lactate, decyl oleate, octyl palmitate and silicone oil, etc. The present invention can use the oil phase of single composition, or the oil phase of two or more compositions. When two or more oily substances are used in combination, the ratio of each oily substance is not limited, and can be set by those skilled in the art as required.

[0064] In the present invention, the amount of the soluble metal salt solution to the oil phase is not limited, and is generally 1:2000, preferably 1:1000, such as 1:800, 1:500, 1:200, 1:100, etc., based on volume. In order to improve the dispersion effect of the soluble metal salt solution in the oil phase, the soluble metal salt solution is preferably added dropwise to the oil phase under ultrasonic conditions. In order to accelerate dispersion, preferably, a stirring or vibration step is further included.

[0065] Step (2)

[0066] In the present invention, step (2) is a step of obtaining a second microemulsion, specifically, comprising the step of dispersing the anionic compound into the oil phase. The anionic compound can be added to the oil phase in the form of a solution or a solid form of the anionic compound when dispersed. In order to improve the effect during dispersion, it is preferably dispersed into the oil phase in the form of an anionic compound solution. The anionic compound solution here can be an aqueous solution, that is, the anionic compound is dissolved in an aqueous solvent to obtain an anionic compound solution. The anionic compound is not limited, and preferably an anionic compound that can react with a metal salt solution to obtain a metal salt in the lipid composition, examples of which include soluble hydrogen phosphates, phosphates, dihydrogen phosphates, etc., especially alkali metal salts, such as potassium salts, sodium salts, etc.

[0067] In the present invention, the concentration of anions in the anion solution is not limited, and can be, for example, 0.01-200mM / L, such as 0.1mM / L, 0.5mM / L, 1mM / L, 5mM / L, 10mM / L, 15mM / L, 20mM / L, 25mM / L, 30mM / L, 35mM / L, 40mM / L, 45mM / L, 50mM / L, 100mM / L, 150mM / L, etc.

[0068] The oil phase in step (2) of the present invention refers to an oily substance commonly used in an emulsion, and for details, please refer to the oil phase in step (1). The oil phase in step (2) may be the same as or different from the oil phase in step (1).

[0069] In step (2) of the present invention, the amount of the anion solution to the oil phase is not limited, and is generally 1:2000, preferably 1:1000, such as 1:800, 1:500, 1:200, 1:100, etc., based on volume. In order to improve the dispersion effect of the anion solution in the oil phase, it is preferred to drop a solution of a soluble metal salt into the oil phase under ultrasonic conditions. In order to accelerate dispersion, preferably, a stirring or vibration step is further included.

[0070] In step (2) of the present invention, preferably, after dispersing the anionic solution in the oil phase, a lipid solution is further added to promote the fusion of the first microemulsion and the second microemulsion. The lipid added here is preferably 2,3-dioleoyl-propyl-trimethylamine, which can greatly enhance the fusion of the first microemulsion and the second microemulsion compared with other lipids. The concentration of the lipid solution in the lipid solution here is generally 0.1-200 mg / ml. If the lipid concentration is too low, the effect of promoting fusion tends to deteriorate. On the other hand, if the lipid concentration is too high, the stability of the second microemulsion tends to deteriorate. The preferred concentration is 1-150 mg / ml, more preferably 5-100 mg / ml, such as 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 50 mg / ml, etc. The amount of the lipid solution used here is based on the amount of the anionic aqueous solution. The ratio is 1:0.5 to 1:5, preferably 1:1 to 1:3. Preferably, a further sonication step is performed after the addition of the lipid solution.

[0071] Step (3)

[0072] Step (3) of the present invention is a step for preparing metal element core particles, which includes fusing the first microemulsion and the second microemulsion, and removing the oil phase to obtain the metal element core particles. In order to promote the fusion of the first microemulsion and the second microemulsion, a solvent is preferably added to the mixed solution of the first microemulsion and the second microemulsion to dissolve the oil phase. The solvent here can be any solvent as long as it is mutually soluble in oil, and examples thereof include alcohols, ethers, alkanes, etc.

[0073] The step (3) of the present invention may further include the step of removing the mixed solvent of the oil phase and the solvent. The step of dispersing the obtained metal element core particles in the solvent is not limited and may be a low molecular weight alkane or a halogenated alkane.

[0074] [Pharmaceutical composition or adjuvant]

[0075] The third aspect of the present invention provides a pharmaceutical composition or immune adjuvant, which comprises the metal element lipid composition described in the first aspect and an optional pharmaceutically acceptable carrier or excipient.

[0076] The pharmaceutical composition of the present invention comprises at least a metal element lipid composition as an active ingredient. Optionally, it further comprises a pharmaceutically acceptable carrier or excipient. In the present invention, pharmaceutically acceptable carriers or excipients are well known in the art, and a person of ordinary skill in the art can determine that they meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients.

[0077] In the present invention, examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg / ml to 25 mg / ml human serum albumin; (2) 0.9% saline (0.9% w / v sodium chloride), and (3) 5% (w / v) glucose; antioxidants such as tryptamine and stabilizers such as Tween 20 may also be included.

[0078] The pharmaceutical composition of the present invention can be any suitable dosage form. For example, injection, suspension, emulsifier, etc. The pharmaceutical composition of the present invention can be applied to the body by known means. For example, delivered to the tissue of interest by intramuscular injection, optionally administered via intravenous, percutaneous, intranasal, oral, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered herein can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0079] The pharmaceutical composition of the present invention can activate type I interferon IFNβ as an adjuvant, thereby activating T cell activation and / or antibody production. Preferably, the pharmaceutical composition of the present invention further comprises an immunogenic component, such as a vaccine, an antigen, etc. An immunogenic component refers to a substance that can be recognized by the immune system and can cause an antigen-specific immune response by forming antibodies and / or antigen-specific T cells. Generally, an antigen can be a protein or polypeptide that contains at least one antigenic epitope and can be presented to the surface of a T cell by a major histocompatibility complex (MHC).

[0080] In certain embodiments, the immunogenic component of the present invention is a microbial antigen, such as an immunogenic peptide segment of bacteria, viruses, fungi, or other pathogens. The pathogens include, but are not limited to, human hepatitis viruses including HAV, HBV, HCV, cytomegalovirus CMV, human immunodeficiency virus HIV, Epstein-Barr virus, dengue virus, human papillomavirus (HPV), respiratory syncytial virus, rhinovirus, human T-lymphotropic virus type I (HTLV-1), influenza, bovine leukemia virus (BLV), pertussis, poliomyelitis, measles, mumps, rubella, smallpox, herpes zoster, anthrax, tetanus, rotavirus, rabies virus, fowlpox, meningococcus, anthrax, encephalitis, pneumococcus, streptococcus, staphylococcus, Neisseria, Escherichia coli, Shigella, Leishmania, respiratory syncytial virus, parainfluenza, adenovirus, varicella, flavivirus, Mycobacterium tuberculosis, and malaria.

[0081] In certain embodiments, the immunogenic component of the present invention is a tumor antigen, that is, a protein that is overexpressed in tumor cells compared to normal cells. Tumor antigens can be further divided into tumor-associated antigens (TAA) and tumor-specific antigens (TSA). TAA is a class of antigen molecules that exist in both tumor cells and normal cells, examples of which include: embryonic proteins, glycoprotein antigens, squamous cell antigens, etc. TAA is not unique to tumor cells, and normal cells can also synthesize it in trace amounts, and it is highly expressed when tumor cells proliferate. TSA refers to a new antigen that is only expressed on the surface of tumor cells and not in normal cells. This type of antigen may be present in tumors of the same tissue type in different individuals, such as melanoma-specific antigens encoded by human malignant melanoma genes, which may be present in melanoma cells of different individuals, but not expressed in normal melanocytes. TSA can also be shared by tumors of different histological types, such as mutated Ras gene products that can be commonly found in lung cancer, digestive tract tumors, etc., but because its amino acid sequence is inconsistent with the expression product of the normal proto-oncogene Ras, it can be recognized by the body's immune system and stimulate the body's immune response. Usually, this type of antigen produced by mutation is called a neoantigen. These antigens can be recognized by cytotoxic T lymphocytes, and cells presenting the antigens can be killed by T lymphocytes.

[0082] [use]

[0083] The fourth aspect of the present invention provides the use of a metal element lipid composition in the preparation of a drug for activating IFNβ or inducing IFNβ production or a drug for immune-related diseases or conditions, preferably an antiviral or anti-tumor drug.

[0084] In certain embodiments, the drug of the present invention is a drug for antimicrobial infection, such as antiviral, antibacterial and antiparasitic drugs. Exemplary viruses are selected from DNA viruses and RNA viruses, such as Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Hepadnaviridae, Flaviviridae, Papillomaviridae, Poxviridae and Retroviridae, more preferably from: Herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV. Wherein, bacteria are selected from Gram-negative bacteria and Gram-positive bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa and Acinetobacter baumannii. Wherein, parasites are selected from Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filarial worms and Leishmania, etc.

[0085] In certain embodiments, the drug of the present invention is an anti-tumor or anti-cancer drug. The tumor or cancer is not limited, and illustratively, cancer includes but is not limited to breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colorectal cancer, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer and leukemia.

[0086] Example

[0087] 1. Preparation of Metal Liposome Nanoparticles

[0088] 1. Preparation of liposome core

[0089] (1) Preparation of oil phase: a mixed solvent of cyclohexane / nonoxynol ether (Cyclohexane / Igepal CO-520) was prepared in a volume ratio of 71:29 as the oil phase for later use;

[0090] (2) Ca 2+ Preparation of microemulsion: add 300 μl of 500 μM / L CaCl2 aqueous solution to 15 ml of oil phase under ultrasound, vibrate while adding, to ensure that all water droplets at the bottom of the tube are evenly dispersed into the oil phase; let stand for 5 min;

[0091] (3)HPO4 2- Preparation of microemulsion: 300 μl of Na2HPO4 aqueous solution (25 mM / L, pH=9) was added dropwise to 15 ml of oil phase under ultrasonic conditions, and the mixture was shaken while being added to ensure that all water droplets at the bottom of the tube were evenly dispersed into the oil phase; then 200 μl of 2,3-dioleoyl-propyl-trimethylamine solution (20 mg / ml) was added, ultrasonicated for 5-10 seconds, and allowed to stand for 5 minutes;

[0092] (4) Preparation of CaP core: 2+ Microemulsion Pour HPO4 2- In microemulsion, let stand for 20 minutes after mixing; divide into two special Invitrogen centrifuge tubes, add 15ml of anhydrous ethanol to each tube, and fully dissolve for 5 minutes; centrifuge at 15000g for 20 minutes, discard the supernatant oil phase / ethanol mixed solvent, and then add ethanol to the Invitrogen centrifuge tube to wash twice to completely wash away the oil phase solvent. After slightly drying with a hair dryer, add 2-4ml of chloroform to the centrifuge tube, blow the tube wall thoroughly, and transfer to a 10ml eggplant-shaped bottle.

[0093] 2. Preparation of liposome outer core

[0094] (5) Add 100 μl of DOPA / cholesterol (10 mM / L, volume ratio of 1:1) and 100 μl of DSPE-PEG (3 mM / L) chloroform solution into the eggplant-shaped flask respectively;

[0095] (6) Rotate thoroughly at 37°C to evaporate chloroform to form a film;

[0096] (7) Add 800 μl of double distilled water or 5 ml of Tris-HCl buffer (pH = 7.4), sonicate at 50°C for 5 min, and ensure that all membranes are sonicated into the solution. The prepared solution is NanoCa liposomes.

[0097] The preparation process of other metal ion liposomes is consistent with this, and the cationic and anionic reactants can be changed according to the characteristics of the generated metal ions.

[0098] 3. Preparation of stable CaP

[0099] Prepare 62.27 mM CaCl2 solution: weigh 6.9113 g of CaCl2 solid and dilute to 1 L with ddH2O;

[0100] Prepare 37.36mM Na2HPO4 12H2O solution: weigh 13.3801g Na2HPO4 12H2O solid and dilute to 1L with ddH2O for later use;

[0101] The pH of the Na2HPO4 12H2O solution was adjusted to 12.20 with 10 M NaOH (8 g dissolved in 20 ml).

[0102] Add 30 ml of Na2HPO4 12H2O solution dropwise into the CaCl2 solution and stir while mixing until it is fully mixed. The result is a CaP solution.

[0103] 2. Physicochemical properties and cytotoxicity experiments of calcium phosphate liposomes

[0104] Inductively coupled plasma method for measuring intracellular calcium concentration

[0105] About 5 mg of NanoCa dry powder was dissolved in 5 ml of buffer solution with different pH values ​​(4.5, 6.5 and 7.5), and then the buffer solution was shaken at 37°C at a rate of 40 rpm / min. At the predetermined time points (0, 1, 3, 6, 9, 12 h), 200 μL of the sample was extracted each time and replaced with the same volume of fresh buffer. The Ca content of each sample was determined by inductively coupled plasma emission spectrometry. 2+ concentration, plotting Ca 2+ Cumulative release profile.

[0106] Transmission electron microscopy to measure the size of nanoparticles

[0107] (1) To prevent contamination, wash the glassware and instruments required for the experiment thoroughly with deionized water and dry them.

[0108] (2) Drop 10 μl of NanoCa onto a copper mesh with a carbon film as a sample mesh.

[0109] (3) Cover the sample net with a petri dish and leave it at room temperature for 10-15 minutes until the sample is naturally dry.

[0110] (4) Turn on the transmission electron microscope for measurement and select an appropriate magnification so that a large number of nanoparticles appear in the field of view, while ensuring that the resolution at this magnification is much smaller than the size of the nanoparticles (e.g., less than 1 / 100).

[0111] (5) Data analysis and measurement parameters.

[0112] Enhanced CCK-8 cytotoxicity assay

[0113] (1) HepG2, HT29, RAW264.7 and HT1080 cells were plated in 96-well plates, with 5,000 cells per well in 100 μl.

[0114] (2) According to the experimental requirements, HepG2, HT29, RAW264.7 and HT1080 cells were treated with different concentrations of NanoCa for 24 h, and the wells containing cell culture medium, NanoCa and enhanced CCK-8 solution but no cells were added served as blank controls.

[0115] (3) After adding 10 μl of enhanced CCK-8 solution to each well, the cells were incubated in a 37°C cell culture incubator for 1 h.

[0116] (4) Measure the absorbance at 450 nm.

[0117] The results showed that the prepared NanoCa had a transparent and uniform appearance ( Figure 2 A). Through transmission electron microscopy, it was found that the particle size was between 20-30nm, the shape and size were consistent, and the distribution was uniform ( Figure 2 BC); the overall potential is positive ( Figure 2 D); Energy dispersive X-ray spectrometer further analysis showed that Ca and P elements were successfully encapsulated, indicating that NanoCa was successfully prepared ( Figure 3 ).

[0118] Small angle X-ray scattering was used to detect the structure, particles and properties of the prepared NanoCa, CaP and CaCO3. The results showed that the NanoCa with a special crystal form was not a typical crystal form, while CaCO3 was a classic calcite structure. The electron density of NanoCa was lower than that of calcium phosphate, indicating that the atomic density of nano calcium was lower than that of calcium phosphate, and its internal arrangement was not as tight as that of ordinary calcium phosphate, which was more conducive to the release of Ca after nano calcium entered the cell.2+ , and the flow cytometry results showed that the Ca released after NanoCa entered the cells 2+ Ca release ratio between CaP and CaCO3 2+ many.

[0119] In order to detect whether the successfully prepared NanoCa has cytotoxicity, HepG2, HT29, RAW264.7 and HT1080 cells were treated with different concentrations of NanoCa. After 24 hours, it was found that the concentration was not cytotoxic in the range of 0-200 μm / L, and the cell survival rate began to decrease at 300 μm / L ( Figure 4 left picture).

[0120] In order to understand its release efficiency in different buffers, NanoCa was placed in PBS buffers with pH values ​​of 4.5, 6.5 and 7.5, respectively. The free calcium ion concentration in the buffer was detected by inductively coupled plasma method. It was found that NanoCa had the highest release efficiency under acidic conditions and the release efficiency was lower under neutral and alkaline conditions, indicating that NanoCa was more easily released in an acidic environment ( Figure 4 (right picture).

[0121] The above results indicate that uniform and stable pH-responsive NanoCa was successfully prepared.

[0122] 3. Screening of metal liposome nanoparticles capable of activating IFNβ

[0123] IFNβ is a core molecule that plays an effective role in natural immunity. In order to screen out metal nanoparticle liposomes that can activate IFNβ, a series of metal liposome nanoparticles were prepared, including nano-iron, nano-ferrous iron, nano-magnesium, nano-calcium, nano-manganese, nano-calcium oxalate, nano-calcium carbonate, nano-calcium citrate, nano-zinc, nano-silver, nano-cobalt and nano-copper. HepG2 cells were treated with these nanoparticles for 24 hours, and MnCl2 was used as a control. The results showed that MnCl2, NanoMn and NanoCa can all activate type I interferon IFNβ ( Figure 7 ). When HT29, HT1080 and CaCO2 cells were treated at the same time, it was found that NanoCa activated type I interferon IFNβ and type III interferon IFNλ1 ( Figure 8 AF). It was found that NanoCa can also activate type I interferon IFNβ ( Figure 8GH). Studies have shown that MnCl2 and NanoMn activate type I interferon IFNβ through the cGAS-STING signaling pathway and have good antiviral and anti-tumor effects. However, it is still unclear how NanoCa activates IFNβ and what applications it has, so NanoCa will be the subject of subsequent research.

[0124] In order to study the optimal activation concentration and strongest activation time corresponding to NanoCa, different concentrations of NanoCa were used for different time periods. It was found that the strongest activation occurred when the final concentration was 100 μM / L for 24 h. Therefore, the final concentration of 100 μM / L was used for 24 h in subsequent experiments. Figure 3 ).

[0125] In addition to activating IFNβ and IFNλ1, can NanoCa also activate other cytokines? To this end, we treated HT29 cells with NanoCa, used MnCl2 and Nanovector as controls, and analyzed through RNA-seq sequencing that NanoCa can activate a series of immune positive regulatory factors, including Cxcl15, Il-2, Irf7, Isg15, Oasl, Gzmb, Ifnβ, Ifnλ1, Ifit2, Ifit3 and Isg20 ( Fig. 9 AB). GO pathway analysis results showed that the differentially expressed genes were mainly enriched in pathways such as type I interferon, antiviral response, interleukin 1, and tumor necrosis factor alpha ( Fig. 9 C); KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in signaling pathways such as tumor necrosis factor alpha, type I interferon, and cytokine regulation of immune response ( Fig. 9 D).

[0126] Then, RAW264.7 cells were treated with NanoCa, and MnCl2 and Nanovector were used as controls. RNA-seq sequencing and analysis showed that NanoCa can also activate a series of positive immune regulatory factors, including Ccl4, Cd86, Il2, Il6, Tbx21, Cxcl2, and Cxcl3 ( Fig.10 AB). The results of GO pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in the positive regulation of chemokine-mediated signaling pathways, natural killer cell pathways, and negative regulation of viral transcription ( Fig.10 B); KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in pathways such as pattern recognition receptor activity, chemokine-mediated signal transduction, and positive regulation of type I interferon response ( Fig.10 C). This indicates that NanoCa may exert antiviral and anti-tumor effects by stimulating cells to produce cytokines that positively regulate immune responses.

[0127] 4. Anti-tumor effect of NanoCa

[0128] The above results have confirmed that NanoCa can activate IFNβ, IFNλ1 and a series of immune-related cytokines at the cellular level. The next step is to verify whether it can activate IFNβ and other cytokines in vivo. Wild-type C57BL / 6 mice were injected with 100μL of NanoCa and Nanovector with an initial concentration of 8mM / L through the tail vein. After 24 hours, the spleen and blood of the mice were obtained, and the spleen RNA was extracted and reverse transcribed into cDNA. RT-qPCR was used to detect the RNA level of IFNβ; ELISA was used to detect IFNβ in the blood. The results showed that IFNβ could be detected in the blood and spleen ( Fig.11 A and B), indicating that NanoCa can also activate IFNβ in vivo.

[0129] IFNβ can activate innate immunity and adaptive immunity, and is also a positive regulator of class I major histocompatibility complex (MHC). In addition to its direct anti-tumor effect, it can also act on the body's immune system and participate in tumor killing by mobilizing innate immunity and adaptive immunity, such as stimulating DCs and macrophages to promote their maturation to enhance the antigen presentation effect, increase the production of granzymes and perforins of CTLs and NK cells, and increase the proportion of memory T cells to exert anti-tumor effects.

[0130] To investigate whether NanoCa has an anti-tumor effect, melanoma cells induced by B16 cells were injected subcutaneously. Fig.12 )、4T1 breast cancer( Fig.17 )、CT26 Colorectal cancer( Fig.18 ) and PANO2 pancreatic cancer ( Fig.19 ) models, and when the tumor volume reaches about 500mm 3 NanoCa was injected into the tail vein, and the tumor volume and mouse body weight were regularly monitored. Fig.13 , 18 and 19), the results showed that compared with the Nanovector and con groups, there was no significant difference in the body weight of mice in the NanoCa group, and the tumor volume was significantly reduced ( Fig.13 ); there was no significant difference in the shape and size of the main organs, heart, liver, lung and kidney ( Fig.14 ), and HE staining results showed that there were no abnormalities in the NanoCa group, Nanovector group, and con group ( Fig.15), indicating that NanoCa has an anti-subcutaneous tumor effect and has no toxic side effects on the main organs of mice. In order to explore whether NanoCa can cooperate with anti-PD-1 antibodies to exert a synergistic effect, NanoCa and PD-1 antibodies were used to treat CT26 subcutaneous tumors at the same time. The results showed that NanoCa can cooperate with PD-1 antibodies to exert anti-tumor effects ( Fig.18 ).

[0131] To further investigate whether NanoCa has anti-metastatic effects, a lung metastasis model was established by injecting B16F10 cells into the tail vein of WT mice. At the same time, 100ul of NanoCa and Nanovector were injected into the tail vein respectively. The results showed that the number of lung metastases in the NanoCa group was significantly reduced and the weight of lung tissue was significantly lighter than that in the Nanovector group ( Figure 16-17 ), indicating that NanoCa has an anti-metastatic effect. This result shows that NanoCa has an anti-tumor effect.

[0132] Studies have shown that in resting T lymphocytes, the CD3ε / ζ cytoplasmic segment binds to the plasma membrane, allowing ITAM tyrosine to be inserted into the hydrophobic end of the plasma membrane, preventing spontaneous ITAM phosphorylation in unstimulated T cells. After the TCR of T cells is stimulated by antigens, the calcium ion channels on the cell surface open automatically, and calcium ions enter the cells, rapidly increasing the intracellular calcium ion concentration and distributing it unevenly in T cells. The increased calcium ion-mediated signal transduction initiates the activation of immune cells, prompting immune cells to differentiate, grow, and secrete a series of immune molecules to exert effector functions. In order to verify whether NanoCa can activate T cell immunity and exert anti-tumor effects after entering the body, CD3 in tumor tissues was detected by (Flow Cytometry, FCM). 3+ CD 8+ T cells, NK cells, and IFNγ-secreting CD4 T cells 8+ The results showed that compared with the Nanovector and con groups, the NanoCa group had a higher CD4 T cell ratio. 8+ T cells ( Fig. 20 A) NK cells ( Fig. 20 B) and CD4+ secretion of IFNγ 8+ T cells ( Fig. 20 C) ratio increased significantly. This indicates that NanoCa can increase CD 8+ T cells, NK cells, and IFNγ-secreting CD4 T cells 8+ The ratio of T cells exerts an anti-tumor effect. 8+ T cells and IFNγ-secreting CD4 T cells 8+ The proportion of T cells increased significantly ( Fig. 20D and E), indicating that NanoCa combined with PD1 can increase CD 8+ T cells and IFNγ-secreting CD4 T cells 8+ The proportion of T cells exerts an anti-tumor effect.

[0133] 5. Ca deficiency reduces the anti-tumor ability of mice

[0134] The above results show that intravenous injection of NanoCa can activate immune response by increasing calcium ion concentration. In order to verify how long-term calcium deficiency will affect the immune system, wild-type C57BL / 6J mice were fed with calcium-free feed for 3 months and then injected with B16 cells subcutaneously. Compared with wild-type mice fed normally, there was no difference in body weight between the two groups of mice, but the tumor growth rate of mice in the calcium-deficient group was significantly faster than that of wild-type mice ( Fig.21 AC), and compared with wild-type mice, the expression levels of IFNβ, IFNγ and ISG15 in the spleen of tumor-injected calcium-deficient mice were significantly reduced ( Fig.21 DF); at the same time, the results of flow cytometry showed that there was no significant difference in the number of B lymphocytes in the spleen of the two groups of mice ( Fig.21 G) while the calcium deficiency group CD 8+ The proportion of T cells was significantly lower than that of the wild-type group ( Fig.21 H). It shows that long-term calcium deficiency will affect CD 8+ The number of T cells affects immunity.

[0135] VI. Antiviral Effect of NanoCa

[0136] In order to explore the antiviral effect of NanoCa, HT29 cells were infected with HSV-1 and VSV at an MOI of 0.1, and RAW264.7 cells were infected with MHV at an MOI of 0.1. The results showed that compared with the control group, the viral loads of HSV-1, VSV and MHV in the NanoCa-treated group were significantly decreased, while the viral load of the Nanovector group was increased ( Fig. 22 AC), indicating that NanoCa has an antiviral effect at the cellular level.

[0137] In order to verify whether NanoCa can play an effective antiviral effect in vivo, WT mice were infected with lethal doses of HSV-1 and VSV viruses, and blood was taken to extract RNA and measure viral load and cytokines before and 24 hours after NanoCa injection. The results showed that mice in the NanoCa group were resistant to HSV-1 and VSV and had a higher survival rate; 24 hours after NanoCa injection, the virus titer in the blood was lower, and the expression of IFNβ and ISG15 increased significantly ( Fig. 22 DK). This indicates that NanoCa can exert antiviral effects by increasing the expression of IFNβ and ISG15.

[0138] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A metal element lipid composition, characterized in that: The metal element lipid composition is composed of a lipid membrane shell layer and a calcium phosphate core wrapped by the lipid membrane shell layer, and the metal element lipid composition is obtained by a method comprising the following steps: Dispersing a metal salt solution into an oil phase to obtain a first microemulsion, wherein the metal salt is calcium chloride; dispersing an anionic compound into the oil phase to obtain a second microemulsion, wherein the anionic compound is a soluble hydrogen phosphate; fusing the first microemulsion and the second microemulsion, and removing the oil phase to obtain calcium phosphate core particles; and The calcium phosphate core particles are wrapped with lipids, the solvent is evaporated, and then water or buffer is added for dispersion to obtain the metal element lipid composition, wherein the lipids are DOPA, cholesterol and DSPE-PEG.

2. A pharmaceutical composition or immune adjuvant, characterized in that: It comprises the metal element lipid composition according to claim 1 and a pharmaceutically acceptable carrier or excipient.

3. Use of the metal element lipid composition according to claim 1 in the preparation of antimicrobial or antitumor drugs, characterized in that: The microorganism is HSV-1, VSV or MHV, and the tumor is melanoma, breast cancer, colorectal cancer or pancreatic cancer.

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