Composite lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses composite lipid nanoparticles as well as a preparation method and application thereof. The composite lipid nanoparticle provided by the invention comprises a cell factor-IgG Fc fusion protein, an Fc receptor protein with a streptavidin tag and a biotinylated lipid nanoparticle, the biotinylated lipid nanoparticles comprise a lipid composition, wherein the lipid composition comprises a biotinylated component with the concentration of 0.375 mol% to 1.5 mol%; the cell factor-IgG Fc fusion protein is connected to the surface of the biotinylated lipid nanoparticle through an Fc receptor protein with a streptavidin tag. The composite lipid nanoparticles have the advantages of low immunogenicity, good biocompatibility, high structural stability, simple preparation process and the like, and have very excellent application prospects in the fields of biological medicine, clinical treatment, vaccine research and development and the like.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, and particularly relates to a composite lipid nanoparticle, its preparation method, and its application. Background Technology

[0002] Lipid nanoparticles (LNPs) are lipid vesicles with a uniform lipid core, widely used as in vivo delivery systems for emerging therapies such as mRNA vaccines, RNA interference, and gene editing. However, in practical applications, LNPs exhibit weak active targeting ability for immune cells such as T cells and monocytes, which have difficulty taking up exogenous particles, resulting in low delivery efficiency and severely limiting their application in CAR-T and immunomodulatory therapies.

[0003] Current research has attempted to enhance the immune cell targeting of LNPs by conjugating antibodies against CD3, CD5, and CD7 to their surface. However, this method involves large molecular weights, complex conformations, and cumbersome conjugation methods, which may lead to reduced LNP particle size stability. Furthermore, antibodies may not effectively mediate rapid and sustained LNP uptake in a dynamic in vivo environment through specific antigen recognition and binding. The effectiveness is also greatly influenced by the expression intensity of the T cell surface antigens targeted by the antibody, resulting in limited improvement in in vivo targeting efficiency. Additionally, it may trigger non-specific immune activation or toxic reactions, thus presenting significant limitations.

[0004] Therefore, obtaining an LNP delivery system that can effectively solve the problems of complex preparation process, strong immunogenicity and low targeted delivery efficiency of existing LNP surface-coupled antibodies is of great significance for realizing the application of LNP in CAR-T, immunomodulation and other fields. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of complex preparation processes, strong immunogenicity, and low targeted delivery efficiency in existing LNP surface-coupled antibodies, and to provide a composite lipid nanoparticle.

[0006] A second objective of this invention is to provide a method for preparing the above-mentioned composite lipid nanoparticles.

[0007] A third objective of this invention is to provide the application of the above-mentioned composite lipid nanoparticles in the preparation of vaccines and / or pharmaceuticals.

[0008] Specifically, the composite lipid nanoparticles provided by the present invention include a cytokine-IgG Fc fusion protein, an Fc receptor protein tagged with streptavidin, and biotinylated lipid nanoparticles; the biotinylated lipid nanoparticles include a lipid composition, the lipid composition including a biotinylated component with a molar concentration of 0.375% to 1.5%; the cytokine-IgG Fc fusion protein is linked to the surface of the biotinylated lipid nanoparticles through the Fc receptor protein tagged with streptavidin.

[0009] Furthermore, the cytokine-IgG Fc fusion protein comprises a cytokine, a linker peptide, and an IgG Fc fragment connected in sequence.

[0010] Furthermore, the cytokines are selected from one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 and TGF-β1.

[0011] Furthermore, the amino acid sequence of the linker peptide is shown in SEQ ID NO:3 or SEQ ID NO:19.

[0012] Furthermore, the amino acid sequence of the IgG Fc fragment is shown in SEQ ID NO:2 or SEQ ID NO:18.

[0013] Furthermore, the Fc receptor protein is Protein A and / or Protein G.

[0014] Furthermore, the amino acid sequence of the Fc receptor protein tagged with streptavidin is shown in SEQ ID NO:13 or SEQ ID NO:15.

[0015] Further, based on the total molar amount of the lipid composition, the lipid composition comprises 40 mol% to 60 mol% of ionizable lipids, 5 mol% to 20 mol% of auxiliary lipids, 30 mol% to 50 mol% of cholesterol, 0 to 5 mol% of polyethylene glycol lipids, and 0.375 mol% to 1.5 mol% of biotinylated polyethylene glycol lipids.

[0016] Further, based on the total molar amount of the lipid composition, the lipid composition comprises 45 mol% to 55 mol% of ionizable lipids, 5 mol% to 15 mol% of auxiliary lipids, 30 mol% to 40 mol% of cholesterol, 0.1 mol% to 1.5 mol% of polyethylene glycol lipids, and 0.375 mol% to 0.75 mol% of biotinylated polyethylene glycol lipids.

[0017] Further, the ionizable lipid is selected from one or more of the following: trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl[2,3-(diolenoyloxy)propyl]ammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl], 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester, 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester, and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol).

[0018] Furthermore, the auxiliary lipid is selected from one or more of dioleoylphosphatidylethanolamine, distearate phosphatidylcholine, and sterols.

[0019] Further, the polyethylene glycol lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, distearate phosphatidylethanolamine-polyethylene glycol, and dioleoylphosphatidylethanolamine-polyethylene glycol.

[0020] Furthermore, the biotinylated polyethylene glycol lipid is distearate phosphatidylethanolamine-polyethylene glycol-biotin and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-biotin.

[0021] The preparation method of the above-mentioned composite lipid nanoparticles provided by the present invention includes: S1, mixing a lipid composition with ethanol to obtain an organic phase solution; mixing the organic phase solution with an aqueous phase solution to obtain the biotinylated lipid nanoparticles; S2, incubating a cytokine-IgG Fc fusion protein and an Fc receptor protein with a streptavidin tag for treatment I to obtain a protein complex; S3, incubating the biotinylated lipid nanoparticles and the protein complex for treatment II to obtain the composite lipid nanoparticles.

[0022] Further, in step S1, the concentration of the lipid composition in the organic phase solution is 10 mg / mL to 50 mg / mL.

[0023] Further, in step S1, the aqueous solution includes nucleic acid molecules at a concentration of 0.1 mg / mL to 1 mg / mL.

[0024] Further, in step S1, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is 1:(2~5).

[0025] Further, in step S2, the mass ratio of the added cytokine-IgG Fc fusion protein and the Fc receptor protein with streptavidin tag is 1:(5~10).

[0026] Furthermore, in step S2, the temperature of the incubation treatment I is 1℃~5℃, and the time is 6h~14h.

[0027] Further, in step S3, the mass ratio of the added biotinylated lipid nanoparticles and protein complex is 10:(1~5).

[0028] Furthermore, in step S3, the temperature of the incubation treatment II is 10℃~40℃, and the time is 10min~240min.

[0029] The present invention relates to the application of the above-mentioned composite lipid nanoparticles in the preparation of vaccines and / or drugs, wherein the composite lipid nanoparticles are delivery carriers for nucleic acid molecules and / or drugs.

[0030] Furthermore, the vaccine and / or drug are used in CAR-T therapy and / or immunomodulatory therapy.

[0031] Beneficial effects: The composite lipid nanoparticles provided by this invention utilize a cytokine-IgG Fc fusion protein constructed from the Fc fragments of cytokines and IgG antibodies as functional fragments, serving as a surface modification protein. A specific concentration of biotinylated components is introduced into the lipid nanoparticles, and an Fc receptor protein tagged with streptavidin is used as a linker to modify the surface of the biotinylated lipid nanoparticles with a specific density of the cytokine-IgG Fc fusion protein. The resulting composite lipid nanoparticles simultaneously introduce both cytokines and the Fc receptor protein-IgG Fc complex, two informational substances that work synergistically to form an organic whole that mimics the innate immune signaling mechanism. This improves the binding efficiency and endocytic capacity of the lipid nanoparticles with immune cells, thereby enhancing the targeting and delivery efficiency of immune cells. Furthermore, these composite lipid nanoparticles possess advantages such as low immunogenicity, good biocompatibility, high structural stability, and simple preparation process, showing excellent application prospects in biomedicine, clinical treatment, and vaccine development.

[0032] In some specific embodiments, the cytokine in the cytokine-IgG Fc fusion protein is preferably IL-2. In this case, the introduced IL-2 and Fc receptor protein-IgG Fc complex can significantly enhance the targeted delivery effect of the composite lipid nanoparticles to T cells, especially CD25-overexpressing T cells, and at the same time endow the obtained composite lipid nanoparticles with targeting properties for T cell-rich tissues such as the spleen and lymph nodes. Attached Figure Description

[0033] Figure 1 This is one of the experimental results of testing the mRNA delivery efficiency of the composite lipid nanoparticles provided in Example 2 of the present invention on mouse T cells under in vitro conditions (by changing the density of IL-2-Fc fusion protein and CD8 in the composite lipid nanoparticles). + T cells); Figure 2 Figure 2 shows the experimental results of the composite lipid nanoparticles provided in Example 2 of this invention, testing the mRNA delivery efficiency of mouse T cells under in vitro conditions (by changing the density of IL-2-Fc fusion protein and CD4 in the composite lipid nanoparticles). + T cells); Figure 3 Figure 3 shows the experimental results of the composite lipid nanoparticles provided in Example 2 of this invention, testing the mRNA delivery efficiency of mouse T cells under in vitro conditions (by altering the cytokine linked in the cytokine-Fc fusion protein, CD8). + T cells); Figure 4 Figure 4 shows the experimental results of the composite lipid nanoparticles provided in Example 2 of this invention on the mRNA delivery efficiency of mouse T cells under in vitro conditions (altering the cytokine linked in the cytokine-Fc fusion protein, CD4). + T cells); Figure 5 This is one of the experimental results of testing the mRNA delivery efficiency of the composite lipid nanoparticles provided in Example 4 of this invention to mouse T cells under in vivo conditions (distribution of LNP-mediated delivered mRNA in different organs). Figure 6 Figure 2 shows the experimental results of the composite lipid nanoparticles provided in Example 4 of this invention in vivo to test the mRNA delivery efficiency of mouse T cells (distribution of LNP-mediated delivered mRNA in different organs). Figure 7 Figure 3 shows the experimental results of the composite lipid nanoparticles provided in Example 4 of this invention in vivo to test the mRNA delivery efficiency of mouse T cells (distribution of LNP-mediated delivered mRNA in different organs). Figure 8 Figure 4 shows the experimental results of the composite lipid nanoparticles provided in Example 4 of this invention in vivo to test the mRNA delivery efficiency of mouse T cells (distribution of LNP-mediated delivered mRNA in immune cells of different organs). Figure 9Figure 5 shows the experimental results of the composite lipid nanoparticles provided in Example 4 of this invention in vivo to test the mRNA delivery efficiency of mouse T cells (distribution of LNP-mediated delivered mRNA in immune cells of different organs). Figure 10 Figure 6 shows the experimental results of the composite lipid nanoparticles provided in Example 4 of this invention in vivo to test the mRNA delivery efficiency of mouse T cells (distribution of LNP-mediated delivered mRNA in immune cells of different organs). Figure 11 This is one of the experimental results (CD8) of the composite lipid nanoparticles provided in Example 6 of this invention, which tested the mRNA delivery efficiency of human primary T cells under in vitro conditions. + T cells); Figure 12 Figure 2 shows the experimental results of the composite lipid nanoparticles provided in Example 6 of this invention, testing their mRNA delivery efficiency to human primary T cells under in vitro conditions (CD4). + T cells).

[0034] Note: **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0035] To improve the targeted delivery efficiency of LNPs to immune cells such as T cells and monocytes that have difficulty uptakeing exogenous particles, the inventors of this invention, through extensive and in-depth research, discovered that cytokines with immunomodulatory functions have excellent application prospects in improving the targeted delivery efficiency of LNPs to immune cells. However, the inventors found in experiments that the physicochemical properties of cytokines are unstable, and their modification on the LNP surface suffers from problems such as unstable linkage, uncontrollable linkage direction and linkage density. This leads to unsatisfactory effects on improving the immune cell targeting and delivery efficiency of LNPs, and also results in reduced controllability of LNP particle size and increased immunogenicity.

[0036] Based on the problems encountered in the application of cytokines to LNP surface modification, the inventors, through further in-depth research and extensive experiments, creatively discovered that cytokine-IgG can be obtained by coupling the Fc fragment of an IgG antibody with a cytokine via a specific linker peptide. The Fc fusion protein not only maintains the targeting affinity of cytokines for immune cells but also exhibits excellent stability, enabling it to function continuously in the dynamic in vivo environment. Furthermore, the connection strategy of "introducing biotinylated components into LNPs and using Fc receptor proteins tagged with streptavidin as linkers" achieves stable binding of cytokines to the LNP surface and enhances the controllability of cytokine binding direction and density. The introduction of heterologous Fc receptor proteins tagged with streptavidin does not induce excessive activation of non-specific T cells or cytotoxic responses. Moreover, the antigen-antibody complex formed by its binding with Fc fragments, modified at a specific density on the LNP surface, can synergistically cooperate with cytokines to mimic the innate immune signaling mechanism, thereby improving the binding efficiency of LNPs to immune cells and better mediating endocytosis. This enhances the targeting and delivery efficiency of immune cells. The resulting composite lipid nanoparticles possess advantages such as low immunogenicity, good immune cell targeting, and excellent delivery efficiency. Based on this, the technical solution of this invention is obtained.

[0037] The composite lipid nanoparticles provided by the present invention specifically include: a cytokine-IgG Fc fusion protein, an Fc receptor protein tagged with streptavidin, and biotinylated lipid nanoparticles, wherein the cytokine-IgG Fc fusion protein is linked to the surface of the biotinylated lipid nanoparticles via the Fc receptor protein tagged with streptavidin.

[0038] In this invention, the cytokine-IgG Fc fusion protein refers to a fusion protein obtained by coupling a cytokine and an IgG antibody Fc fragment, and its structure specifically includes a cytokine, a linker peptide, and an IgG Fc fragment connected in sequence.

[0039] In the cytokine-IgG Fc fusion protein, the cytokine refers to a class of polypeptides with biological activity that transmits signals to regulate the physiological activities and immune responses of cells. The cytokines can be selected and combined from existing known cytokines according to the cells to be targeted by the composite lipid nanoparticles and the actual application scenario. Specific examples include, but are not limited to, one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 and TGF-β1.

[0040] In some specific embodiments, the cytokine is preferably IL-2. In this case, the composite lipid nanoparticles exhibit excellent targeted delivery to T cells, especially CD25-overexpressing T cells.

[0041] In the cytokine-IgG Fc fusion protein, the linker peptide refers to a short peptide sequence that can achieve the linking between the Fc fragments of cytokines and IgG antibodies through its own flexible characteristics.

[0042] In some specific embodiments, the amino acid sequence of the linker peptide is preferably as shown in SEQ ID NO:3. In this case, the linker peptide, while achieving the linking of the Fc fragments of the cytokine and IgG antibody, can effectively maintain their respective native conformations and biological activities, thereby endowing the resulting cytokine-IgG Fc fusion protein with excellent targeting binding capability.

[0043] In the cytokine-IgG Fc fusion protein, the IgG Fc fragment refers to the fragment composed of heavy chain CH2-CH3 in the IgG antibody molecule. It can be selected from existing IgG antibodies according to the target of the composite lipid nanoparticles. The present invention does not particularly limit it.

[0044] In some specific embodiments, the amino acid sequence of the IgG Fc fragment is as shown in SEQ ID NO:2 or SEQ ID NO:18.

[0045] In this invention, the Fc receptor protein with a streptavidin tag refers to a fusion protein obtained by coupling streptavidin and Fc receptor protein, the structure of which specifically includes streptavidin and Fc receptor protein.

[0046] In the Fc receptor proteins tagged with streptavidin, the Fc receptor protein refers to a class of proteins capable of specifically recognizing and binding to the Fc fragment of IgG antibodies, specific examples including, but not limited to, Protein A and / or Protein G. Protein A is produced by Staphylococcus aureus (…). Staphylococcus aureus Protein G is a cell wall protein secreted by Streptococcus (streptococcus), capable of specifically binding to the Fc fragment of IgG antibodies from various mammals, including humans, rabbits, and pigs. Streptococcus The cell wall protein secreted by the IgG can specifically bind to the Fc fragment of IgG antibodies from various mammals, including humans, rabbits, and pigs.

[0047] In some specific embodiments, the amino acid sequence of the Fc receptor protein tagged with streptavidin is preferably as shown in SEQ ID NO:13 or SEQ ID NO:15.

[0048] In this invention, the biotinylated lipid nanoparticles refer to a type of functionalized nanocarrier formed by introducing biotinylated components into the structure of lipid nanoparticles. The lipid composition used to prepare these biotinylated lipid nanoparticles specifically includes a biotinylated component at a concentration of 0.375 mol% to 1.5 mol%, specifically 0.375 mol%, 0.4 mol%, 0.45 mol%, 0.6 mol%, 0.7 mol%, 1.125 mol%, 1.5 mol%, or any value between these concentrations. In this case, the density of the cytokine-IgG Fc fusion protein linked to the surface of the composite lipid nanoparticles is ideal, exhibiting advantages such as good particle size uniformity, low immunogenicity, and high cell-targeting binding efficiency.

[0049] In the biotinylated lipid nanoparticles, the lipid composition specifically includes ionizable lipids, auxiliary lipids, cholesterol, polyethylene glycol lipids, and biotinylated polyethylene glycol lipids. The ionizable lipids refer to a class of lipids with ionization properties, capable of freely crossing cell membranes. This is a commonly used raw material in the preparation of existing lipid nanoparticles, and the present invention does not specifically limit it. Specific examples include, but are not limited to: trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl[2,3-(dioleoyloxy)propyl]ammonium chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl] (DC-Chol), 8-[(2-hydroxyethyl)[6-oxo-6-] One or more of the following: -(undecyloxy)hexyl]amino]octanoic acid-octylnonyl ester (SM-102), 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl ester (Dlin-MC3-DMA), and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200). The auxiliary lipids refer to a class of neutral lipids that improve stability and in vivo circulation, and are a class of raw materials commonly used in the preparation of existing lipid nanoparticles. This invention does not specifically limit them, but specific examples include, but are not limited to, one or more of dioleoylphosphatidylethanolamine (OEA), distearate phosphatidylcholine (DSPC), and sterols. The polyethylene glycol lipids refer to amphiphilic polymer molecules composed of hydrophilic polyethylene glycol chains and hydrophobic alkyl chains and / or dialkyl chains. They are a type of raw material commonly used in the preparation of existing lipid nanoparticles. This invention does not specifically limit their use, but specific examples include, but are not limited to, one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG), distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG). The biotinylated polyethylene glycol lipids refer to a type of chemically modified polyethylene glycol lipid molecule with biotin linked in its structure. Specific examples include, but are not limited to, distearate phosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-Biotin) and / or 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-biotin (DMG-PEG-Biotin).

[0050] In some specific embodiments, based on the total molar amount of the lipid composition, the concentration of the ionizable lipids is preferably 40 mol% to 60 mol%, specifically 40 mol%, 40.5 mol%, 41 mol%, 43 mol%, 45 mol%, 48 mol%, 50 mol%, 51.5 mol%, 53 mol%, 55 mol%, 58 mol%, 60 mol%, or any value between them; the concentration of the auxiliary lipids is preferably 5 mol% to 20 mol%, specifically 5 mol%, 5.1 mol%, 5.3 mol%, 5.8 mol%, 6 mol%, 6.5 mol%, 7 mol%, 9 mol%, 10 mol%, 15 mol%, 18 mol%, 20 mol%, or any value between them; the concentration of cholesterol is preferably 30 mol% to 5 mol%. The concentration of the polyethylene glycol lipid is preferably 0-5 mol%, specifically 0 mol%, 0.01 mol%, 0.375 mol%, 0.5 mol%, 0.75 mol%, 1 mol%, 1.125 mol%, 1.5 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, or any value between them; the concentration of the biotinylated polyethylene glycol lipid is preferably 0.375 mol% to 1.5 mol%, specifically 0.375 mol%, 0.75 mol%, 1 mol%, 1.125 mol%, 1.5 mol%, or any value between them.

[0051] In some preferred embodiments, based on the total molar amount of the lipid composition, the concentration of the ionizable lipid is more preferably 45 mol% to 55 mol%; the concentration of the auxiliary lipid is more preferably 5 mol% to 15 mol%; the concentration of cholesterol is more preferably 30 mol% to 40 mol%; the concentration of polyethylene glycol lipid is more preferably 0.1 mol% to 1.5 mol%; and the concentration of biotinylated polyethylene glycol lipid is more preferably 0.375 mol% to 0.75 mol%.

[0052] Based on obtaining the above-mentioned composite lipid nanoparticles, the present invention also provides a method for preparing the above-mentioned composite lipid nanoparticles. The preparation method specifically includes: S1, mixing a lipid composition with ethanol to obtain an organic phase solution; mixing the organic phase solution with an aqueous phase solution to obtain the biotinylated lipid nanoparticles; S2, incubating a cytokine-IgG Fc fusion protein and an Fc receptor protein tagged with streptavidin for treatment I to obtain a protein complex; S3, incubating the biotinylated lipid nanoparticles and the protein complex for treatment II to obtain the composite lipid nanoparticles.

[0053] In this invention, in step S1, the concentration of the lipid composition in the organic phase solution is preferably 10 mg / mL to 50 mg / mL, specifically 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL or any value between them.

[0054] In this invention, the aqueous solution in step S1 refers to a solution system that uses water as a solvent and includes a buffer system, encapsulated substances, and other solutes. It is a type of raw material commonly used in the preparation of existing lipid nanoparticles, and this invention does not impose any particular limitations on it.

[0055] In this invention, specific examples of the encapsulated substances in the aqueous solution in step S1 include, but are not limited to, nucleic acid molecules such as mRNA, siRNA, and DNA; and / or, small molecule drugs.

[0056] In some specific embodiments, in step S1, when the encapsulated substance is specifically a nucleic acid molecule, the concentration of the nucleic acid molecule in the aqueous solution is preferably 0.1 mg / mL to 1 mg / mL, specifically 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 1 mg / mL or any value between them.

[0057] In this invention, the method of mixing the organic phase solution and the aqueous phase solution to form nanoparticles in step S1 is a conventional technique used in the preparation of existing lipid nanoparticles, and this invention does not impose any particular limitations on it.

[0058] In this invention, in step S1, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is preferably 1:(2~5), specifically 1:2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:4, 1:5, or any value between them. More specifically, the flow rate of the organic phase solution is preferably 2 mL / min to 5 mL / min, specifically 2 mL / min, 2.3 mL / min, 2.5 mL / min, 2.8 mL / min, 3 mL / min, 4 mL / min, 4.5 mL / min, 5 mL / min, or any value between them; the flow rate of the aqueous phase solution is preferably 4 mL / min to 25 mL / min, specifically 4 mL / min, 5 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 12 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, or any value between them.

[0059] In this invention, in step S2, the preferred mass ratio of the added cytokine-IgG Fc fusion protein and the Fc receptor protein with streptavidin tag is 1:(5~10), specifically it can be 1:5, 1:5.3, 1:5.5, 1:6, 1:7.5, 1:8, 1:9, 1:10 or any value between them.

[0060] In this invention, in step S2, the conditions for the incubation treatment I specifically include a temperature preferably of 1℃ to 5℃, specifically 1℃, 1.2℃, 1.5℃, 1.8℃, 2℃, 2.5℃, 3℃, 4℃, 5℃ or any value between them; and a time preferably of 6h to 14h, specifically 6h, 6.5h, 7h, 8h, 9h, 10h, 12h, 14h or any value between them.

[0061] In this invention, in step S3, the mass ratio of the added biotinylated lipid nanoparticles and protein complex is 10:(1~5), specifically 10:1, 10:1.9, 10:3, 10:4.3, 10:5 or any value between them.

[0062] In this invention, in step S3, the conditions for the incubation treatment II include a temperature preferably of 10°C to 40°C, specifically 10°C, 15°C, 18°C, 20°C, 25°C, 30°C, 33°C, 38°C, 40°C or any value between them; and a time preferably of 10 min to 240 min, specifically 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 80 min, 100 min, 120 min, 150 min, 180 min, 200 min, 240 min or any value between them.

[0063] Based on the advantages of the aforementioned composite lipid nanoparticles, such as low immunogenicity, good biocompatibility, high structural stability, and simple preparation process, this invention also provides the application of the aforementioned composite lipid nanoparticles in the preparation of vaccines and / or drugs. Specifically, the composite lipid nanoparticles are used as delivery carriers for nucleic acid molecules and / or drugs.

[0064] In this invention, the specific application areas of the vaccine and / or drug may be, but are not limited to: CAR-T therapy and / or immunomodulatory therapy.

[0065] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0066] Preparation Example 1 This preparation example illustrates the construction of an IL-2-Fc fusion protein, specifically including: 1. Structural design of IL-2-Fc fusion protein: Using the mouse IL-2 fragment with the sequence shown in SEQ ID NO:1 and the IgG Fc fragment-I with the sequence shown in SEQ ID NO:2 as functional fragments, and using the linker peptide with the sequence shown in SEQ ID NO:3, the IL-2-Fc fusion protein with the sequence shown in SEQ ID NO:4 was constructed.

[0067] 2. Construction of IL-2-Fc fusion protein expression vector: Based on the amino acid sequence of IL-2-Fc fusion protein and the codon preference of HEK293 cells, the encoding gene of IL-2-Fc fusion protein (sequence shown in SEQ ID NO:5) was obtained, sent to a biosynthesis company for synthesis, and after verifying the correct sequence, it was carried into the pcDNA3.1(+) plasmid vector to construct the IL-2-Fc fusion protein expression vector.

[0068] 3. Expression and purification of IL-2-Fc fusion protein: (1) Take the IL-2-Fc fusion protein expression vector and use PEI 40000 HEK293 cells were transfected with the transfection reagent (Polysciences, catalog number #24765, hereinafter the same) according to the instructions to obtain recombinant cells. The cells were cultured at 37°C, 120 rpm and 5% CO2 for 72 h, and then centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain crude protein solution. (2) The crude protein solution was purified by using a Ni-NTA column (Tiandi Renhe, catalog number SA101100, the same below) and according to the instructions to obtain the IL-2-Fc fusion protein, which was stored at 4°C for later use.

[0069] The sequence information of the IL-2-Fc fusion protein and its related fragments involved in this preparation example is shown in Table 1.

[0070] Table 1.

[0071] Preparation Example 2 This preparation example uses the method provided in Preparation Example 1 to construct a cytokine-Fc fusion protein. The difference is that the sequence information of the constructed cytokine-Fc fusion protein is shown in Table 2. Under the same conditions, the cytokine-Fc fusion protein was constructed.

[0072] Table 2.

[0073]

[0074] Preparation Example 3 This preparation example uses the method provided in Preparation Example 1 to construct an Fc receptor protein tagged with streptavidin. The difference is that the sequence information of the constructed Fc receptor protein tagged with streptavidin and its related fragments is shown in Table 3. Under the same conditions, the streptavidin-Protein A fusion protein was constructed.

[0075] Table 3.

[0076] Example 1 This embodiment illustrates the preparation of a composite lipid nanoparticle, specifically including: S1, (1) Mix SM-102 (Aveto, catalog number O02010), DSPC (Aveto, catalog number DSPC), cholesterol (Sigma, catalog number C8667), DMG-PEG2000 (Aveto, catalog number O02005) and DSPE-PEG2000-biotin (Xi'an Ruixi Biotechnology, catalog number R-1304-2k) to obtain a lipid composition; dissolve the lipid composition in anhydrous ethanol according to the addition amount of 25 mg / mL to obtain an organic phase solution.

[0077] (2) Take EGFP mRNA (including the EGFP encoding gene) at a final concentration of 0.1 mg / mL and suspend it in citrate buffer (10 mM, pH=3.0) to obtain an aqueous solution.

[0078] (3) The organic phase solution and the aqueous phase solution were mixed in a microfluidic mixer at a flow rate of 3 mL / min and 9 mL / min, respectively, to obtain a suspension containing LNP. The suspension was dialyzed overnight using a Pur-A-Lyzer™ Midi dialysis kit (Merck, catalog number PURD35030-1KT) according to the instructions to obtain biotinylated lipid nanoparticles. Then, the mRNA in the biotinylated lipid nanoparticles was quantified using Quant-iT RiboGreen RNA reagent (Thermo Fisher Scientific, catalog number R11491) according to the instructions.

[0079] S2. Take the cytokine-Fc fusion protein provided in Preparation Example 1 or 2 and the Fc receptor protein with streptavidin tag provided in Preparation Example 3 at a mass ratio of 1:8, mix them, and incubate them overnight at 4°C to obtain protein complexes.

[0080] S3. Biotinylated lipid nanoparticles and protein complexes were mixed at a mass ratio of 10:3 and incubated at room temperature for 30 min. Then, the composite lipid nanoparticles were purified using a size exclusion chromatography column packed with Sepharose CL-2B (Sigma-Aldrich, catalog number CL2B300, the same below). The purified composite lipid nanoparticles were then concentrated using an Amicon Ultra centrifugal filter to obtain each composite lipid nanoparticle.

[0081] The specific raw materials and their proportions used in each composite lipid nanoparticle are shown in Table 4.

[0082] Table 4.

[0083] Note: X1:X2:X3:X4:X5 refers to the molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000 and DSPE-PEG2000-biotin added to the lipid composition.

[0084] Example 2 This embodiment illustrates the delivery efficiency of the composite lipid nanoparticles provided in Example 1 to mouse T cells under in vitro conditions. The specific tests include: 1. Effect of LNP surface IL-2-Fc fusion protein density on delivery efficiency: (1) According to 1×10 6 The seeding amount of cells / well was determined by adding primary T cells derived from the lymph nodes of C57BL / 6 mice into a 12-well plate and culturing them at 37°C for 48 hours to obtain the cell solution to be tested. (2) Add the composite lipid nanoparticles LNP-1~LNP-4 to the cell culture medium at a dosage of 200 ng / well, incubate at 37°C for 24 h, and then detect CD8 using flow cytometry. + and CD4 + The EGFP positivity rate in T cells was measured, with composite lipid nanoparticles LNP-1'~LNP-4' used as a control. The results are as follows: Figure 1 and 2 As shown.

[0085] Depend on Figure 1 and 2 The results show that the density of the surface-modified IL-2-Fc fusion protein (achieved by changing the molar percentage of DSPE-PEG2000-biotin in the lipid composition) has a significant impact on the delivery efficiency of the composite lipid nanoparticles. When the concentration of DSPE-PEG2000-biotin in the lipid composition is 0.375 mol%~0.75 mol%, the resulting composite lipid nanoparticles have a high delivery efficiency for CD8. + and CD4 + T cells have excellent targeted delivery efficiency.

[0086] 2. Effect of LNP surface-modified cytokine type on delivery efficiency: (1) Add anti-CD3ε antibody solution (Biolegend, catalog number 100340) to a 12-well plate at a final concentration of 5 μg / mL, incubate at 37℃ for 2 h, then remove the solution and add 1×10 6 Primary T cells derived from C57BL / 6 mouse lymph nodes were added to 12-well plates. Anti-CD28 antibody solution (Biolegend, catalog number 102116) and recombinant mouse IL-2 solution (Yisheng Biotechnology, catalog number 90142ES08) were added to a final concentration of 1 μg / mL and cultured at 37℃ for 48 h to obtain the cell solution to be tested. (2) Add composite lipid nanoparticles LNP-2, LNP-5~LNP-10 at a dosage of 200 ng / well to the cell culture medium to be tested. After incubation at 37℃ for 24 h, CD8 were detected by flow cytometry. + and CD4 + The EGFP positivity rate in T cells was measured, with composite lipid nanoparticles LNP-0 and LNP-2' used as controls. The results are as follows: Figure 3 and 4 As shown.

[0087] Depend on Figure 3 and 4The results show that, compared with cytokines such as IL-7, IL-10, IL-15, and TGF-β, the introduction of the IL-2-Fc fusion protein, constructed from IL-2 and IgG Fc fragment-I, into composite lipid nanoparticles significantly improves the targeted delivery efficiency of the composite lipid nanoparticles to T cells—CD8. + The EGFP positivity rate in T cells was greater than 80%, a 4-fold increase compared to LNP-0; CD4 + The EGFP positivity rate in T cells was greater than 80%, which was more than twice that of LNP-0.

[0088] Depend on Figures 1-4 The results show that, compared to unactivated primary T cells, delivery of CD25-overexpressing T cells (activated by dual signals of anti-CD3ε antibody and anti-CD28 antibody) using composite lipid nanoparticles significantly improved CD8 expression. + T cells and CD4 + The EGFP positivity rate in T cells is increased by 2 to 4 times, resulting in superior targeted delivery efficiency.

[0089] Example 3 This embodiment uses the method provided in Example 1 to prepare composite lipid nanoparticles. The difference is that an equal amount of Firefly luciferase mRNA (including the firefly luciferase encoding gene, labeled with Cy5) is added instead of EGFP mRNA, while other conditions are the same, to obtain composite lipid nanoparticles.

[0090] The specific raw materials and their proportions used in each composite lipid nanoparticle are shown in Table 5.

[0091] Table 5.

[0092] Note: X1:X2:X3:X4:X5 refers to the molar ratio of SM-102, DSPC, cholesterol, DMG-PEG2000 and DSPE-PEG2000-biotin added to the lipid composition.

[0093] Example 4 This embodiment illustrates the delivery efficiency of the composite lipid nanoparticles provided in Example 3 to mouse T cells under in vivo conditions. The specific tests include: 1. Distribution of LNP-mediated delivered mRNA in different organs: Six 8-week-old female C57BL / 6 mice were randomly divided into two groups of 3 mice each (n=3), and the following operations were performed: (1) LNP-IgG group: Each mouse was injected with 5 μg of LNP-11' via the tail vein. Six hours after the injection, D-Luciferin (APExBIO, catalog number C3654, the same below) was injected into the mice via intraperitoneal injection at a dose of 150 mg / kg. (2) LNP-IL2-Fc group: Each mouse was injected with 5 μg of LNP-11 via the tail vein. Six hours after the injection, D-Luciferin was injected into the mice via intraperitoneal injection at a dose of 150 mg / kg. Ten minutes after D-Luciferin injection, mice were euthanized by cervical dislocation, and their organs were dissected. The luminescence signals of each organ were quantitatively analyzed using the IVIS Spectrum imaging platform and Living IMAGE software. The results are as follows: Figures 5-7 As shown.

[0094] Depend on Figures 5-7 The results show that, compared with the composite lipid nanoparticle LNP-11', the mRNA delivered by LNP-11 mediated by the surface modified with IL-2-Fc fusion protein is mainly enriched in tissues rich in T cells, such as the spleen and lymph nodes, while the distribution of hepatocytes in non-target tissues is significantly reduced. This indicates that the modification with IL-2-Fc fusion protein significantly improves the targeting of composite lipid nanoparticles to T cells and tissues under in vivo conditions.

[0095] 2. Distribution of LNP-mediated delivered mRNA in immune cells of different organs: Nine 8-week-old female C57BL / 6 mice were randomly divided into three groups of 3 mice each (n=3), and the following operations were performed: (1) PBS group: Each mouse was injected with 5 μg of PBS buffer (10 mM, pH=7.4) via the tail vein. (2) LNP-IgG group: Each mouse was injected with 5 μg of LNP-11' via the tail vein; (3) LNP-IL2-Fc group: Each mouse was injected with 5 μg of LNP-11 via the tail vein; Four hours after injection, blood was collected from the retro-orbital vascular plexus of mice. The blood samples were processed with erythrocyte lysis buffer (Solepro, catalog number R1010, the same below) and resuspended in PBS buffer (10mM, pH=7.4) to obtain a single-cell suspension of blood. Mice were euthanized by cervical dislocation, and their spleens and lymph nodes were collected and homogenized. The spleen and lymph node samples were processed using erythrocyte lysis buffer and resuspended in PBS buffer (10 mM, pH 7.4) to obtain single-cell suspensions of the spleen and lymph nodes. Flow cytometry was used for analysis, and the results are as follows: Figures 8-10 As shown.

[0096] Depend on Figures 8-10 The results show that, compared with the composite lipid nanoparticles LNP-11', the mRNA delivered by LNP-11 mediated by the surface modified with IL-2-Fc fusion protein is mainly enriched in the spleen, and has strong in vivo distribution specificity.

[0097] Preparation Example 4 This preparation example uses the method provided in Preparation Example 1 to construct the IL-2-Fc fusion protein. The difference is that the sequence information of the constructed IL-2-Fc fusion protein and its related fragments is shown in Table 6. Under the same conditions, the IL-2-Fc fusion protein was constructed.

[0098] Table 6.

[0099] Comparative preparation example This preparation example uses the method provided in Preparation Example 1 to construct a fusion protein. The difference is that the sequence information of the constructed fusion protein and its related fragments is shown in Table 7. Under the same conditions, the fusion protein was constructed.

[0100] Table 7.

[0101] Example 5 This embodiment uses the method provided in Example 1 to prepare composite lipid nanoparticles. The difference is that the fusion protein provided in Preparation Example 4 or the comparative preparation example is used instead of the cytokine-Fc fusion protein provided in Preparation Example 1 or 2. All other conditions are the same, and composite lipid nanoparticles are obtained.

[0102] The specific raw materials and their proportions used in each composite lipid nanoparticle are shown in Table 8.

[0103] Table 8.

[0104] Example 6 This embodiment illustrates the delivery efficiency of the composite lipid nanoparticles provided in Example 5 to human cells under in vitro conditions. The specific tests include: (1) According to 1×10 6 For cell / well seeding, human primary T cells were added to a 12-well plate and cultured at 37°C for 48 h to obtain the cell solution to be tested. (2) Add anti-CD3ε antibody solution (Biolegend, catalog number 317301) to a 12-well plate at a final concentration of 10 μg / mL. Incubate at 37°C for 2 hours, then remove the solution. Add 1×10 6 For cell / well seeding, human primary T cells were added to 12-well plates. Anti-CD28 antibody solution (Biolegend, catalog number 302901) was added at a final concentration of 5 μg / mL, and recombinant human IL-2 solution (Yisheng Biotechnology, catalog number 90103ES76) was added at a final concentration of 50 IU / mL. The cells were cultured at 37℃ for 48 h to obtain the test cell solution-II. (3) Add 200 ng / well of composite lipid nanoparticles LNP-12~LNP-14 to the cell solution I or cell solution II to be tested, incubate at 37°C for 24 h, and then detect CD8 by flow cytometry. + and CD4 + EGFP positivity rate in T cells, results as follows Figure 11 and Figure 12 As shown.

[0105] Depend on Figure 11 and Figure 12 The results show that, compared with LNP-13 and LNP-14, LNP-12, which is modified with a specific density of IL-2-Fc fusion protein, has excellent targeted delivery efficiency for CD25-overexpressing T cells (activated by dual signals of anti-CD3ε antibody and anti-CD28 antibody).

[0106] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0107] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A complex lipid nanoparticle characterized in that, The complex lipid nanoparticle comprises a cytokine-IgG Fc fusion protein, an Fc receptor protein with a streptavidin tag, and a biotinylated lipid nanoparticle; the biotinylated lipid nanoparticle comprises a lipid composition comprising a biotinylated component at a concentration of 0.375 mol% to 1.5 mol%; the cytokine-IgG Fc fusion protein is connected to the surface of the biotinylated lipid nanoparticle through the Fc receptor protein with a streptavidin tag.

2. The complexed lipid nanoparticle of claim 1, wherein, The cytokine-IgG Fc fusion protein comprises, in sequence, a cytokine, a connecting peptide segment, and an IgG Fc fragment; Optionally, the cytokine is selected from one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and TGF-β1; Optionally, the amino acid sequence of the connecting peptide segment is as shown in SEQ ID NO: 3; Optionally, the amino acid sequence of the IgG Fc fragment is as shown in SEQ ID NO: 2 or SEQ ID NO:

18.

3. The complexed lipid nanoparticle of claim 1, wherein, The Fc receptor protein is Protein A and / or Protein G; Optionally, the amino acid sequence of the Fc receptor protein with a streptavidin tag is as shown in SEQ ID NO: 13 or SEQ ID NO:

15.

4. The complexed lipid nanoparticle of claim 1, wherein, The lipid composition comprises, based on the total molar amount of the lipid composition, an ionizable lipid at a concentration of 40 mol% to 60 mol%, a helper lipid at a concentration of 5 mol% to 20 mol%, cholesterol at a concentration of 30 mol% to 50 mol%, a polyethylene glycol lipid at a concentration of 0 to 5 mol%, and a biotinylated polyethylene glycol lipid at a concentration of 0.375 mol% to 1.5 mol%; Optionally, the lipid composition comprises an ionizable lipid at a concentration of 45 mol% to 55 mol%, a helper lipid at a concentration of 5 mol% to 15 mol%, cholesterol at a concentration of 30 mol% to 40 mol%, a polyethylene glycol lipid at a concentration of 0.1 mol% to 1.5 mol%, and a biotinylated polyethylene glycol lipid at a concentration of 0.375 mol% to 0.75 mol%; Optionally, the ionizable lipid is selected from one or more of trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl [2,3-(dioleyloxy)propyl] ammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl] 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino] octanoic acid-1-octyl nonyl ester, 4-(N,N-dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester, and 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl) bis(dodecan-2-ol). Optionally, the helper lipid is selected from one or more of dioleoylphosphatidyl ethanolamine, distearoylphosphatidyl choline and sterol; Optionally, the polyethylene glycol lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, distearic acid phosphatidyl ethanolamine-polyethylene glycol and dioleoylphosphatidyl ethanolamine-polyethylene glycol; Optionally, the biotinylated polyethylene glycol lipid is distearoylphosphatidyl ethanolamine-polyethylene glycol-biotin and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-biotin.

5. The method of producing the complex lipid nanoparticle according to any one of claims 1 to 4, characterized in that, The preparation method comprises: S1, mixing a lipid composition and ethanol to obtain an organic phase solution; mixing the organic phase solution and an aqueous phase solution to obtain the biotinylated lipid nanoparticle; S2, incubating a cytokine-IgG Fc fusion protein and a streptavidin-labeled Fc receptor protein to obtain a protein complex; S3, incubating the biotinylated lipid nanoparticle and the protein complex to obtain the complex lipid nanoparticle.

6. The method of claim 5, wherein the complex lipid nanoparticle is prepared by a method comprising: In step S1, the concentration of the lipid composition in the organic phase solution is 10 mg / mL to 50 mg / mL; Optionally, the aqueous phase solution comprises a nucleic acid molecule at a concentration of 0.1 mg / mL to 1 mg / mL; Optionally, the mixing flow rate ratio of the organic phase solution and the aqueous phase solution is 1:(2 to 5).

7. The method of claim 5, wherein the complex lipid nanoparticle is prepared by a method comprising: In step S2, the mass ratio of the cytokine-IgG Fc fusion protein and the streptavidin-labeled Fc receptor protein is 1:(5 to 10); Optionally, the temperature of the incubation treatment I is 1℃ to 5℃, and the time is 6h to 14h.

8. The method of claim 4, wherein the composite lipid nanoparticle is prepared by a method comprising: In step S3, the mass ratio of the biotinylated lipid nanoparticle and the protein complex is 10:(1 to 5); Optionally, the temperature of the incubation treatment II is 10℃ to 40℃, and the time is 10min to 240min.

9. Use of the complex lipid nanoparticle according to any one of claims 1 to 4 for the manufacture of a vaccine and / or a medicament, characterized in that, The complex lipid nanoparticle is a delivery carrier for nucleic acid molecules and / or drugs.

10. Use of the complexed lipid nanoparticle according to claim 9 for the manufacture of a vaccine and / or a medicament, characterized in that, The vaccine and / or drug are applied in CAR-T treatment and / or immunoregulatory treatment.

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