A STING-activated ionizable lipid, lipid nanoparticles and their applications

Abstract

This invention discloses a STING-activated ionizable lipid, lipid nanoparticles, and their applications. The lipid structure includes a six-membered nitrogen-containing piperazine heterocycle, a dihydroimidazole ring, an alkyl chain connecting the two, and a long alkyl side chain. The alkyl chain connecting the two and the long alkyl side chain linked to the dihydroimidazole ring include, but are not limited to, saturated or unsaturated alkyl chains with one to ten carbons. The auxiliary lipid materials used in the preparation of LNPs include, but are not limited to, myristoylphosphatidylcholine. The nucleic acid drug includes at least one of siRNA and mRNA. The STING-activated ionizable heterocyclic lipid of this invention can be used as a vaccine adjuvant, possessing the ability to load nucleic acid drugs and impart lymphoimmunotropic properties to the prepared LNPs, enhancing their accumulation in immune organs and activating the STING pathway, thereby enhancing the therapeutic effect of tumor vaccines.

Description

Technical Field

[0001] This invention relates to an ionizable lipid, lipid nanoparticles, and applications, specifically to a STING-activated ionizable lipid, lipid nanoparticles, and applications. Background Technology

[0002] mRNA technology can generate specific tumor neoantigens through the protein synthesis system of human cells, thereby inducing a de novo immune response against cancer-specific neoantigens, which can then specifically attack tumor cells and prevent tumor recurrence. This has become one of the important strategies for personalized immunotherapy.

[0003] Although nanomaterials can promote the retention of mRNA vaccines in lymphoid organs, most vaccines fail to effectively reach antigen-presenting cells within these organs, becoming a major rate-limiting step in inducing an immune response. Cancer disease remodels the body's lymphatic system, leading to impaired mobilization of the systemic immune system and immune tolerance. Furthermore, adjuvant and vaccine deposition at the injection site, lymphatic drainage, antigen-presenting barriers, and the amplification of cascade signals between antigen-presenting cells and T cells are all important factors affecting the efficacy of nanovaccines. Therefore, developing lipid nanovaccines that precisely target lymphoid organs, significantly limiting tumor metastasis and promoting the body's immune response while effectively addressing the comprehensive cascade amplification of cellular response signals between mRNA and adjuvant, is crucial for advancing the clinical application of nanovaccines in personalized cancer treatment.

[0004] Based on previous laboratory research, we have discovered that specific proteins can bind to LNP technology to form protein crowns, thereby targeting specific receptors, cells, and organs. Ionizable lipid materials can adsorb nucleic acid drugs through electrostatic interactions; their head groups typically carry a positive charge and can interact with cell membranes to improve lysosomal escape and transfection efficiency of mRNA. Piperazine, as a six-membered nitrogen-containing heterocycle, possesses highly efficient ionization and the ability to penetrate epithelial cells without damage. Therefore, introducing a piperazine ring as a scaffold structure for ionizable lipids can not only transform the "hepatophilicity" of ionizable lipids into "lymphatic tropism," but also enhance the system's penetration ability in the lymphatic system, thereby increasing its potential for biomedical applications.

[0005] Stimulator of interferon genes (STING) signaling agonists, as one of the most promising immune adjuvants, stimulate STING signaling activation in antigen-presenting cells, which can assist mRNA nanovaccines in inducing type I interferon (IFN-I) secretion, thereby promoting tumor antigen cross-presentation, T lymphocyte proliferation and activation, and direct tumor killing. However, free cyclic dinucleotide and aminobenzimidazole dimer-type STING agonists, when used as immune adjuvants in tumor vaccine design, still suffer from poor lymphatic organ delivery efficiency and inability to improve antigen cytoplasmic release, which is one of the key reasons for the current poor clinical efficacy of tumor vaccines. Therefore, developing next-generation STING vaccine adjuvants and effectively combining them with organ-targeted delivery technology of LNP-mRNA to synergistically promote efficient cytoplasmic delivery of tumor antigens in lymphatic organs and STING signaling activation is of great significance for improving tumor vaccine therapy. Summary of the Invention

[0006] Objectives of the Invention: The present invention aims to provide an STING-activated ionizable lipid that combines the functions of STING activator, nucleic acid delivery, and immune system tropism. The second objective of the present invention is to provide an application of the aforementioned STING-activated ionizable lipid in the preparation of lipid nanoparticles with STING activation effect and immune system tropism and in nucleic acid drug delivery. The third objective of the present invention is to provide lipid nanoparticles containing the aforementioned STING-activated ionizable lipid.

[0007] Technical solution: The STING-activated ionizable lipid of the present invention has the following structural formula:

[0008]

[0009] R1-R6 are selected from saturated or unsaturated alkyl chains with 1 to 10 carbon atoms.

[0010] Preferably, R1, R2, R5, and R6 are selected from saturated or unsaturated straight-chain alkyl groups having 1 to 10 carbon atoms, and R3 and R4 are selected from saturated straight-chain alkyl groups having 1 to 10 carbon atoms. More preferably, R3 and R4 are selected from saturated straight-chain alkyl groups having 1 to 5 carbon atoms.

[0011] The STING-activated ionizable lipids can be used in the preparation of lipid nanoparticles with STING activation effects and immune system tropism, as well as in nucleic acid drug delivery.

[0012] The lipid nanoparticles containing STING-activated ionizable lipids include lipid materials and nucleic acid drugs; the lipid materials include STING-activated ionizable heterocyclic lipids and other lipid materials.

[0013] Preferably, the mass ratio of STING-activated ionizable lipids to other lipid materials is 1:5 to 5:1.

[0014] The other lipid materials are selected from dioleoyl lecithin (DOPC), hydrogenated soybean phosphatidylglycerol (HSPG), lecithinylglycerol (EPG), lecithinositol (EPI), hydrogenated soybean phosphatidylethanolamine (HSPE), phosphatidylethanolamine (EPE), soybean phosphatidylcholine (SPC), soybean phosphatidylglycerol (SPG), soybean phosphatidylserine (SPS), soybean phosphatidylinositol (SPI), hydrogenated soybean phosphatidylserine (HSPS), soybean phosphatidylethanolamine (SPE), soybean phosphatidic acid (SPA), hydrogenated lecithinositol (HEPC), hydrogenated lecithinylglycerol (HEPG), lecithinylserine (EPS), hydrogenated lecithinositol (HEPI), hydrogenated lecithinylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soybean phosphatidylcholine (HSPC), hydrogenated soybean phosphatidic acid (HSPI), and hydrogenated soybean phosphatidic acid (HSPA). Dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylinositol (DSPI), lecithinylcholine (EPC), myristoylphosphatidylcholine (DMPC), myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylcholine (DSPC), distearylphosphatidylglycerol (DSPG), phosphatidic acid (EPA), dioleenoylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PS) PC), dipalmitoylphosphatidylglycerol (DPPA), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, dilauroylethylphosphocholine (DLEP), distearylphosphatidylserine (DSPS), dimyroylethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP) and distearylethylphosphocholine (DSEP), N-(2,3-di-(9-(Z)-octadecenyloxy)-propyl-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-Bis(oleoyloxy)-3-(trimethylammonium)propane (DOTAP), distearylphosphatidylglycerol (DSPG), myristoylphosphatidyl acid (DMPA), distearylphosphatidyl acid (DSPA), myristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), myristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), myristoyl lysophosphatidylcholine (M-LysoPC), palmitoyl lysophosphatidylcholine One or more of the following: (P-LysoPC), stearoyl lysophosphatidylcholine (S-lysoPC), distearyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), phosphatidylcholine-polyethylene glycol (PC-PEG), phosphatidylethanolamine-polyethylene glycol (PE-PEG), distearyl phosphatidylcholine-polyethylene glycol (DSPC-PEG), cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, and other functionalized phospholipids and sterol water-soluble derivatives.

[0015] Preferably, the nucleic acid drug is selected from one or more of siRNA, miRNA, mRNA, ASO, ssRNA, ssDNA, etc.

[0016] Preferably, the method for preparing the above-mentioned lipid nanoparticles is liposome extrusion, thin film hydration, nanoprecipitation, microfluidic process or impingement jet mixing.

[0017] The aforementioned lipid nanoparticles can also be used in the preparation of nucleic acid drugs loaded with nucleic acid, STING activated, and immune system tactical nucleic acid vaccines.

[0018] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The STING-activated ionizable heterocyclic lipids and lipid nanoparticles possess, on the one hand, highly efficient nucleic acid drug loading capacity and protective effects; on the other hand, they have STING signaling activation effects, thereby inducing the expression of type I IFNs, initiating interferon immune responses, and IFNs stimulate the proliferation of anti-tumor T cells, their penetration into tumor tissues, and their direct killing. Furthermore, downstream signal transduction of STING leads to the activation of antigen-presenting cells (APCs) and the production of inflammatory cytokines, thereby promoting the initiation and recruitment of T cells. On the other hand, enhancing the tropism of the immune system can not only reduce the off-target effects of nucleic acid vaccines and cause side effects such as liver damage, but also increase the accumulation of adjuvants and vaccines in lymph nodes, enhance the translation and presentation of intracellular antigens, and enable the immune system to form a positive feedback loop of antigen presentation / interferon signaling / CD8+ T cells, thereby changing the tumor immune pattern, triggering the immune infiltration of inflammatory macrophages, neutrophils, and natural killer (NK) populations, and improving the persistence of the immune response. Attached Figure Description

[0019] Figure 1This is the synthetic route for the STING-activated ionizable heterocyclic lipids in Example 1. Figure 2 Its proton nuclear magnetic resonance spectrum;

[0020] Figure 3 This is the synthetic route for the STING-activated ionizable heterocyclic lipids in Example 2;

[0021] Figure 4 The results of molecular docking between lipids and STING protein in Example 1;

[0022] Figure 5 The results of isothermal titration calorimetry detection of lipids and STING protein in Example 1 are shown.

[0023] Figure 6 The particle size distribution is shown in the characterization diagram of the formulation in Example 3.

[0024] Figure 7 Here is a scanning electron microscope image of the formulation in Example 3;

[0025] Figure 8 The nuclease stability and serum stability of the formulation in Example 3;

[0026] Figure 9 , Figure 10 and Figure 11 This study investigates the effects of the formulation in Example 4 on BMDC activation, antigen presentation enhancement, and OVA-specific T cell proliferation.

[0027] Figure 12 The BrdU method was used to detect the proliferative effect of the preparation on T cells in Example 3;

[0028] Figure 13 and Figure 14 To observe the distribution of the formulation prepared according to the method of Example 1 in living and isolated mouse tissues using a small animal in vivo imaging system. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0030] Example 1

[0031] The preparation process of a STING-activated ionizable heterocyclic lipid is as follows (synthetic route as shown). Figure 1 (as shown)

[0032] Accurately measure the prescribed amounts of 1,4-bis(3-aminopropyl)piperazine (0.25 mmol), 8-15-alkylone (1 mmol), and ethyl isocyanate (0.75 mmol) into a volumetric flask containing 5 mL of chloroform. The entire reaction system was reacted in a 60°C oil bath with stirring, protected from light, for 48 h. After the reaction, the target product was obtained by silica gel column chromatography and analyzed by… Figure 2 The 1H NMR spectrum shown is used to determine the structure of the product.

[0033] Example 2

[0034] The preparation process of another STING-activated ionizable heterocyclic lipid is as follows (synthetic route). Figure 3 (as shown)

[0035] Accurately measure the prescribed amounts of 1,4-piperazine dibutylamine (0.25 mmol), 2,13-pentadecanediene-8-one (1 mmol), and ethyl isocyanate (0.75 mmol) into a volumetric flask containing 5 mL of chloroform. The entire reaction system was reacted in the dark under oil bath heating and stirring at 60 °C for 48 h. After the reaction was completed, the target product was obtained by silica gel column chromatography.

[0036] Example 3

[0037] Prescription composition:

[0038]

[0039] Preparation Process: Lipid nanoparticles were prepared using microfluidic technology. Precise amounts of the ionizable heterocyclic lipids prepared in Example 1 were weighed and added to 300 μL of methanol:dioxane (1:1) solution, followed by sonication until completely dissolved. Precise amounts of cholesterol were weighed and added to 100 μL of ethanol, followed by sonication until completely dissolved. Precise amounts of DSPE-PEG6000 and DMPC were weighed and added to 100 μL of methanol, followed by sonication until completely dissolved. The solutions were mixed to obtain the organic phase. Approximately 8 OD of siRNA was dissolved in 255 μL of DEPC-containing water, and then 1.245 μL of pH 4.0 HEPES buffer was added as the aqueous phase. The corresponding solutions were added to the two microfluidic inlets, and the aqueous and organic phases were uniformly mixed using a program. The resulting formulation was diluted 40-fold with PBS, and the organic phase was removed by ultrafiltration.

[0040] Example 4

[0041] Prescription composition:

[0042]

[0043] Preparation process: Lipid nanoparticles were prepared using a nanoprecipitation method. Accurately weigh the prescribed amounts of the ionizable heterocyclic lipids, DSPE-PEG6000, cholesterol, and DMPC prepared in Example 1. Add 100 μL of methanol:tetrahydrofuran (1:1) and sonicate until completely dissolved to form the organic phase. Add 1 mL of pH 3.0 HEPES Buffer to a 10 mL volumetric flask as the aqueous phase. Stir vigorously for 3-5 min, then add 0.05 mg of OVA mRNA and continue stirring for 3-5 min. Then add another 100 μL of the organic phase and continue stirring for 5 min. Place the formulation in a dialysis bag (MW3500) and dialyze with enzyme-free HEPES Buffer (pH 7.4) for 2 h to remove the organic phase and adjust the pH of the formulation to 7.4.

[0044] Example 5

[0045] Prescription composition:

[0046]

[0047] Preparation process: Lipid nanoparticles were prepared using a nanoprecipitation method. Precise amounts of the ionizable heterocyclic lipids, DSPE-PEG6000, cholesterol, and DMPC prepared in Example 1 were weighed and added to 100 μL of methanol:tetrahydrofuran (1:1), and sonicated until completely dissolved to form the organic phase. 1 mL of pH 3.0 HEPES Buffer was added to a 10 mL volumetric flask as the aqueous phase. The mixture was stirred vigorously with a magnetic stirrer for 3-5 min, followed by the addition of 0.05 mg of miRNA and continued stirring for another 3-5 min. Then, 100 μL of the organic phase was added and the mixture was stirred for another 5 min. A rotary evaporator was used to remove any residual organic phase from the formulation, and the complete removal of the organic phase was confirmed by weighing the volumetric flask before and after rotary evaporation.

[0048] The STING-activated ionizable heterocyclic lipids prepared in Example 1 were used to simulate the binding of small lipid molecules and STING proteins via molecular docking, such as... Figure 4 The diagram shows simulated binding and simulated binding forces with different crystalline forms of STING protein. The binding affinity between small lipid molecules and human STING protein was detected using isothermal calorimetric titration (ITC) with a MicroCal PEAQ-ITC instrument, such as... Figure 5 The titration curve and the obtained binding parameters are shown. It can be seen that the binding force between the lipid molecule and the STING protein is in the range of μM to nM, and the binding energy ΔG and enthalpy change ΔH are negative, indicating that the reaction system is an exothermic reaction that can proceed spontaneously, and the affinity between the lipid and the STING protein mainly comes from specific binding abilities such as hydrogen bonds and van der Waals forces.

[0049] The formulation prepared in Example 3 was used to measure the particle size using a Malven zetasizer. Figure 6 As shown, lipid nanoparticles with small particle size and uniform PDI were prepared, with a particle size of about 162 nm and a PDI of 0.18. Figure 7 TEM scanning electron microscope image of the prepared formulation.

[0050] The NC siRNA-containing formulation prepared in Example 3 was incubated with nuclease and serum for a certain period of time, and then SDS and heparin were added for nucleic acid drug extraction. The stability of the formulation was detected by nucleic acid gel electrophoresis.

[0051] Figure 8 The results of nucleic acid gel electrophoresis are shown. It is evident that the formulation exhibits good stability after co-incubation with nucleases for 0-4 hours and co-incubation with serum for 0-24 hours, demonstrating strong protection for the loaded nucleic acid drug.

[0052] The OVA mRNA-containing formulation prepared in Example 4 was used to evaluate its effects on BMDC activation and antigen presentation enhancement. Bone marrow cells were extracted from the femur of C57BL / 6J mice and cultured in RPMI 1640 medium containing GM-CSF. On day 7, suspended and loosely adherent cells were collected, and the CD11c positivity rate was measured. After grouping and administration, cells were collected 24 hours later. Flow cytometry staining and detection were performed using monoclonal antibodies against CD11c, CD80, CD86, MHC II, and MHC I-OVA257-264 (SIINFEKL). The activation and antigen presentation enhancement of BMDCs were evaluated by the proportion of positive cells and the mean fluorescence intensity. Spleens were harvested from C57BL / 6J mice, and splenic cells were extracted. After grouping and administration, cells were collected 24 hours later. Flow cytometry staining and detection were performed using monoclonal antibodies against CD3, CD4, CD8, and MHC I-OVA257-264 (SIINFEKL). The activation effect of OVA-specific T cells was evaluated by the proportion of positive cells.

[0053] Figure 9 The figure shows the proportion of CD80 and CD86 positive cells, indicating that the formulation group can stimulate DC cell maturation more effectively than the PBS group; Figure 10 The average fluorescence intensity of CD80, MHC II, and MHC I-OVA257-264 (SIINFEKL) is shown in the figure. Figure 11 The image shows the OVA-specific CD3 levels in different groups. + CD8 + T cells and OVA-specific CD3 + CD4 +The study showed that, compared to the control group, the formulation group could promote antigen cross-presentation by stimulating the activation of STING signaling in DC cells, and further promote the proliferation of OVA-specific T cells after continuous use with siSTAT3.

[0054] STAT3 siRNA was selected as the model siRNA. A formulation containing STAT3 siRNA was prepared according to the preparation protocol in Example 3. T cell proliferation was detected using the BrdU method. Spleens were harvested from C57BL / 6J mice, and splenic cells were extracted. After 1 hour of BrdU labeling, cells were grouped, plated, and administered the drug. Cells were collected 36 hours later, and flow cytometry staining with CD3, CD8, and BrdU antibodies was used to detect T cell proliferation levels. Figure 12 The study showed that, compared to the PBS group, the formulation group significantly increased cytotoxic T lymphocytes (CD3+). + CD8 + ), Th1 cells (CD3) + CD183 + ) and NK cells (CD49b + The proliferation level of ) was significantly improved when used in combination with siSTAT3.

[0055] Cy5-siRNA was selected as the model siRNA. The preparation containing Cy5-siRNA, prepared according to the preparation scheme in Example 3, was evaluated for in vivo distribution in mice. ICR mice were selected as the model mice. The formulation ratio was kept unchanged, and the ionizable lipid was replaced with the cationic lipid DOTAP as a control. Both the LNPs-Cy5-siRNA and DOTAP-LNPs-Cy5-siRNA groups of mice were injected intravenously with the corresponding formulations. The distribution of the fluorescent formulation in vivo was observed using a small animal in vivo imaging system at 1h, 2h, 3h, 4h, 6h, and 24h after administration. The mice were then euthanized, and the heart, liver, spleen, lung, kidney, and lymph nodes were harvested to observe the distribution of the fluorescent formulation in isolated tissues.

[0056] Figure 13 The figure shows the distribution of each fluorescent formulation in mice at different time points. From 1 h to 6 h, compared with the DOTAP-LNPs control group, the formulation in the LNPs group showed more significant uptake and accumulation in mice, and a small amount was still present at 24 h, indicating that the formulation has the ability to circulate in vivo for a long time. Figure 14The distribution of each fluorescent formulation in isolated mouse tissues (heart, liver, spleen, lung, kidney, and lymph nodes) at different time points was shown. At each time point, the LNPs group showed significantly higher accumulation in the spleen and lymph nodes than the DOTAP-LNPs group, indicating that the synthesized ionizable lipids endowed the lipid nanoparticles with immune system tropism, increasing their accumulation in the immune organs, spleen and lymph nodes.

Claims

1. A STING-activated ionizable lipid, characterized in that: The structural formula is as follows: ， Among them, R1, R2, R5, and R6 are selected from saturated straight-chain alkyl groups with 1 to 10 carbon atoms, and R3 and R4 are selected from saturated straight-chain alkyl groups with 1 to 5 carbon atoms.

2. The use of the STING-activated ionizable lipid of claim 1 in the preparation of lipid nanoparticles with STING activation effect, wherein the lipid nanoparticles are used for nucleic acid drug delivery.

3. A lipid nanoparticle, characterized in that: It includes lipid materials and nucleic acid drugs, wherein the lipid materials are the STING-activated ionizable lipids as described in claim 1 and other lipid materials.

4. The lipid nanoparticles according to claim 3, characterized in that: The mass ratio of the STING-activated ionizable lipid to other lipid materials is 1:5 to 5:

1.

5. The lipid nanoparticles according to claim 4, characterized in that: The other lipid materials are selected from one or more of cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol.

6. The lipid nanoparticles according to claim 3, characterized in that: The nucleic acid drug is selected from one or more of siRNA, miRNA, mRNA, ASO, and ssRNA.

7. The lipid nanoparticles according to claim 3, characterized in that: Methods for preparing the lipid nanoparticles include liposome extrusion, thin film hydration, nanoprecipitation, microfluidic or impingement jet mixing.

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