A nucleic acid drug delivery system, and preparation and use thereof

Abstract

The present application relates to the technical field of drug delivery, in particular to a nucleic acid drug delivery system and preparation and application thereof. The present application provides a nucleic acid drug delivery system, comprising a lipid nanogel and metal ions, wherein the lipid nanogel comprises a nanogel and a phospholipid layer, the phospholipid layer is wrapped on the outer surface of the nanogel, and the nucleic acid drug and metal ions are placed in the nanogel. The nucleic acid drug delivery system can improve the therapeutic effect of the nucleic acid drug through the biological synergistic effect of the cGAS-STING pathway and the RIG-I / MDA5 pathway, reduce the toxicity generated when the nucleic acid drug system is administered, and has a simple preparation process, easy industrial production and a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of drug delivery technology, specifically to a nucleic acid drug delivery system and its preparation and application. Background Technology

[0002] Cancer is one of the major diseases affecting human health. For tumors that are difficult to cure directly through surgical removal, immune checkpoint inhibitors are a safer treatment option compared to first-line therapies such as radiotherapy and chemotherapy. The mechanism of action of immune checkpoint inhibitors is to block immune checkpoint signaling pathways such as PD-1 / PD-L1 / CTLA4 that cause cytotoxic T cell depletion, thereby continuously activating cytotoxic T cells and exerting an effective tumor-killing effect. However, in clinical practice, due to insufficient tumor-infiltrating T cells in most patients and the inhibitory effect of the tumor immune microenvironment, the activation effect of immune checkpoint inhibitors on T cells is limited, resulting in a low response rate in patients. Therefore, improving the T cell response level in the tumor microenvironment is crucial for improving the efficacy of immune checkpoint inhibitors.

[0003] Dendritic cells (DCs) are an important type of antigen-presenting cells. Activating the type I interferon signaling pathway in DCs can directly and effectively stimulate T cell activation and enable them to exert anti-tumor immune effects. Studies have shown that the artificially synthesized double-stranded RNA analog PolyIC (Polyinosinic-polycytidylic acid) is an effective type I interferon-inducing compound. Its mechanism of action is roughly as follows: On the one hand, PolyIC can be recognized by the double-stranded RNA sensor MDA5 / RIG-I in the DC cytoplasm, recruiting the adaptor protein MAVS in the mitochondria, which in turn recruits TRAF3 to activate TBK1. TBK1 phosphorylates the transcription factor IRF3, thereby inducing the production of type I interferon. On the other hand, PolyIC can also activate the TLR3 signaling pathway in endosomes and recruit TRAF3 to induce the secretion of type I interferon. The former is crucial for activating T cell responses. Currently, many clinical trials have been conducted on the anti-tumor therapeutic effects of PolyIC. Because PolyIC is a double-stranded RNA compound, it is easily degraded by RNases in serum. The widely used formulation in clinical trials is Poly-ICLC. It is a mixture of Poly IC and cationic polymers poly-L-lysine (PLL) and carboxymethyl cellulose (CMC). While improving the plasma stability of Poly IC, it can also increase the endosome escape of Poly IC through the proton sponge effect induced by the cationic polymer in the endosome, thereby more effectively activating MDA5 to produce type I interferon.

[0004] Although Poly-ICLC has shown some therapeutic efficacy in clinical trials, it still faces significant limitations that restrict its widespread clinical use. Firstly, in terms of safety, intravenous injection of high concentrations of Poly-ICLC can cause significant systemic toxicity. This is mainly because the cationic polymers in Poly-ICLC non-specifically adsorb onto negatively charged tissue and cell surfaces, triggering systemic immune activation. Therefore, intratumoral, intramuscular, or subcutaneous injections are more common in clinical practice, but these routes have limitations compared to intravenous administration. Secondly, in terms of efficacy, the therapeutic effect of Poly-ICLC as a single-dose regimen in patients is quite limited. In clinical trials, it is often used as an adjunct therapy in combination with radiotherapy, chemotherapy, and tumor-associated antigens. Therefore, there is an urgent need to develop a new method to improve the anti-tumor therapeutic effect of Poly-ICLC in patients while reducing the toxicity caused by systemic administration. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one objective of this invention is to provide a nucleic acid drug delivery system, its preparation, and its application. The nucleic acid drug delivery system provided by this invention can achieve the co-delivery of metal ions and nucleic acid drugs via intravenous administration. It retains many advantages of intravenous administration, such as avoiding the first-pass effect, rapid arrival at the target tissue, and applicability to cancer types with inaccessible tumor locations. Furthermore, it possesses the high permeability and retention effect (EPR effect) of solid tumors, which is beneficial for enhancing the accumulation of nucleic acid drugs in tumor tissue and achieving better therapeutic effects.

[0006] Therefore, in a first aspect, the present invention provides a nucleic acid drug delivery system comprising a lipid nanogel (LNG) and metal ions;

[0007] The lipid nanogel comprises a nanogel and a phospholipid layer, wherein the phospholipid layer coats the outer surface of the nanogel;

[0008] The nucleic acid drug and metal ions are placed in the nanogel;

[0009] The metal ions include at least one selected from manganese ions, zinc ions, cobalt ions, nickel ions, iron ions, ferrous ions, magnesium ions, copper ions, chromium ions, aluminum ions, silver ions, barium ions, and gadolinium ions.

[0010] The inventors discovered that nucleic acid drugs can be encapsulated in a nanogel core by utilizing the interaction of ionizable phospholipids with negatively charged nucleic acid drugs under acidic conditions. Simultaneously, metal ions, acting as STING pathway agonists, are adsorbed into the nanogel core through heating and incubation, forming stable chelates with the nucleic acid drugs. This enhances the therapeutic efficacy of the nucleic acid drugs and reduces toxicity during systemic administration. Compared to conventional drug delivery carriers (LNPs), this invention, using LNG, achieves both high nucleic acid drug encapsulation efficiency and significantly enhanced acid-responsive release capability and biological activity.

[0011] According to an embodiment of the present invention, the nucleic acid drug comprises DNA or RNA.

[0012] According to an embodiment of the present invention, the nucleic acid drug includes at least one selected from mRNA, siRNA, tRNA, snRNA, miRNA, piRNA, and circRNA.

[0013] According to an embodiment of the present invention, the nucleic acid drug includes those selected from TLR agonists.

[0014] According to an embodiment of the present invention, the nucleic acid drug includes at least one selected from Poly IC, CpG, Poly I:U and their derivatives.

[0015] According to a preferred embodiment of the present invention, the nucleic acid drug comprises a selection from Poly IC.

[0016] According to an embodiment of the present invention, the Poly IC includes a selection from LMW Poly IC or HMW Poly IC.

[0017] According to an embodiment of the present invention, the nanogel is prepared from polysaccharides.

[0018] According to an embodiment of the present invention, the polysaccharide includes at least one selected from hyaluronic acid, sodium alginate, dextran and its derivatives.

[0019] According to a preferred embodiment of the present invention, the polysaccharide comprises a component selected from hyaluronic acid.

[0020] According to an embodiment of the present invention, the phospholipid layer comprises at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids.

[0021] According to an embodiment of the present invention, the phospholipid layer comprises cationic lipids or polyethylene glycol lipids.

[0022] According to an embodiment of the present invention, the cationic lipid includes at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, and DC-Chol.

[0023] According to an embodiment of the present invention, the polyethylene glycol lipid includes at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG.

[0024] According to an embodiment of the present invention, the phospholipid layer further comprises accessory lipids or steroidal lipids.

[0025] According to an embodiment of the present invention, the auxiliary lipid includes at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, and ALC-0159.

[0026] According to an embodiment of the present invention, the steroidal lipids include cholesterol.

[0027] According to an embodiment of the present invention, the phospholipid layer comprises cationic lipids, polyethylene glycol lipids, auxiliary lipids, and steroidal lipids.

[0028] According to an embodiment of the present invention, the molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid and steroidal lipid is (2-6):(0.2-1.5):(0-5):(0-10).

[0029] According to a preferred embodiment of the present invention, the molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid and steroidal lipid is 4:0.5:1:5.

[0030] A second aspect of the present invention provides a method for preparing the nucleic acid drug delivery system described in the first aspect, comprising:

[0031] (1) Prepare an aqueous solution of nanogel and an aqueous solution of nucleic acid drug by mixing the aqueous solution of nanogel and the aqueous solution of nucleic acid drug to obtain an aqueous phase component;

[0032] (2) Prepare the phospholipid layer to obtain the alcohol phase component;

[0033] (3) The aqueous phase component and the alcohol phase component are mixed to obtain a lipid nanogel encapsulating nucleic acid drugs;

[0034] (4) Add a metal ion solution to the lipid nanogel and heat to incubate to obtain the nucleic acid drug delivery system.

[0035] The preparation method of the nucleic acid drug delivery system provided by this invention can improve the encapsulation rate of nucleic acid drugs, greatly simplify the preparation process, facilitate industrial production scale-up, and use safer production excipients, which is conducive to the clinical application and transformation of this nucleic acid drug delivery system.

[0036] According to an embodiment of the present invention, step (1) further includes:

[0037] The aqueous solution of the nanogel, the aqueous solution of the nucleic acid drug, and the buffer solution are mixed to obtain the aqueous phase component.

[0038] According to an embodiment of the present invention, the concentration of the nanogel aqueous solution is 0.001-10 mg / ml.

[0039] According to an embodiment of the present invention, the concentration of the nucleic acid drug aqueous solution is 0.001-5 mg / ml.

[0040] According to an embodiment of the present invention, the buffer solution includes at least one selected from citrate-sodium citrate buffer, acetic acid-sodium acetate buffer, MES buffer, and glycine-hydrochloric acid buffer.

[0041] According to an embodiment of the present invention, the buffer solution has a pH of 3-7 and a concentration of 0.001-1M.

[0042] According to an embodiment of the present invention, step (3) further includes:

[0043] The obtained aqueous and alcoholic components are mixed using a microfluidic chip.

[0044] According to an embodiment of the present invention, the flow rate during the mixing process via the microfluidic chip is 5-15 ml / min.

[0045] According to an embodiment of the present invention, the volume ratio of the aqueous phase component to the alcohol phase component is 3:1-9:1.

[0046] According to an embodiment of the present invention, the metal ion solution in step (4) includes at least one selected from soluble manganese ion solution, soluble zinc ion solution, soluble cobalt ion solution, soluble nickel ion solution, soluble iron ion solution, soluble ferrous ion solution, soluble magnesium ion solution, soluble copper ion solution, soluble chromium ion solution, soluble aluminum ion solution, soluble silver ion solution, soluble barium ion solution, and soluble gadolinium ion solution.

[0047] According to an embodiment of the present invention, the soluble manganese ion solution includes at least one selected from manganese sulfate solution, manganese nitrate solution, manganese acetate solution, and manganese citrate solution.

[0048] According to an embodiment of the present invention, the concentration of the soluble manganese ion solution is 0.5-4M.

[0049] According to an embodiment of the present invention, the heating and incubation temperature in step (4) is 37.5-60°C, and the incubation time is 1-6h.

[0050] According to an embodiment of the present invention, the preparation method further includes:

[0051] The obtained nucleic acid drug delivery system was subjected to dialysis, concentration, filtration, and isotonic adjustment.

[0052] According to an embodiment of the present invention, a third aspect of the present invention provides a pharmaceutical composition comprising the nucleic acid drug delivery system described in the first aspect.

[0053] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0054] The fourth aspect of the present invention provides the use of the nucleic acid drug delivery system of the first aspect or the pharmaceutical composition of the third aspect in the preparation of a medicament, wherein the medicament is used for antitumor immunotherapy.

[0055] According to an embodiment of the present invention, the drug is used for antitumor immunotherapy or anti-infection therapy.

[0056] According to an embodiment of the present invention, the method of administration of the drug includes at least one selected from intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection.

[0057] A fifth aspect of the present invention provides a vaccine comprising an antigen and the nucleic acid drug delivery system described in the first aspect.

[0058] The sixth aspect of the present invention provides the use of the vaccine described in the fifth aspect in the prevention or treatment of tumors or infectious diseases.

[0059] The present invention has the following advantages over the prior art:

[0060] (1) Higher safety: This invention develops a novel nucleic acid drug delivery system with good safety. All excipients used have been approved by the FDA. At the same time, compared with poly-ICLC containing cationic polymers, the nucleic acid drug delivery system provided by this invention has a negatively charged surface and is masked by polyethylene glycol, making it less likely to be non-specifically adsorbed with cells or proteins in plasma, which greatly reduces the risk of drug systemic toxicity.

[0061] (2) The preparation process is simple and the obtained nucleic acid drug delivery system has a uniform and stable particle size, which is easy to scale up for industrial production: Compared with the traditional microemulsion method, the present invention adopts microfluidic technology to greatly simplify the preparation process of existing similar preparations, avoids the use of a variety of toxic reagents, and is conducive to the application of this process to large-scale industrial production; at the same time, the nucleic acid drug delivery system prepared by microfluidic technology has a smaller particle size, is more uniform and stable, which is conducive to the accumulation of drugs at the tumor site;

[0062] (3) Effective biological activity: The nucleic acid drug delivery system prepared in this invention can exhibit good biological activity in vitro and in vivo. For example, LNG-Mn-pIC (i.e., a nucleic acid drug delivery system loaded with divalent manganese ions and nucleic acid drugs as Poly ICs) has a stronger type I interferon induction ability in mouse BMDC cells compared with single-component LNG-Mn and LNG-pIC; it also has a stronger type I interferon induction ability compared with LNP-Mn-pIC without nanogel; at the same time, LNG-Mn-pIC also showed a more significant anti-tumor growth effect than single-component delivery systems in the MC38 mouse tumor model, and is expected to be used as an effective method for tumor immunotherapy.

[0063] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0064] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0065] Figure 1 This diagram illustrates how the LNG-Mn-pIC dual pathway synergistically activates anti-tumor immunity and enhances anti-tumor efficacy.

[0066] Figure 2The preparation, characterization, and preliminary activity verification of the LNG-Mn-pIC nucleic acid drug delivery system in Examples 1 and 2 of this invention are shown. Figure A shows the microfluidic preparation process of LNG-Mn-pIC; Figure B shows the particle size comparison between LNG-pIC and LNG-Mn-pIC; Figure C shows the zeta potential comparison between LNG-pIC and LNG-Mn-pIC; Figures DF show the particle size distribution, zeta potential, and manganese ion loading comparison between LNG-Mn-pIC and LNP-Mn-pIC; Figure G shows the manganese ion acid response release experiment of LNG-Mn-pIC (labeled as LNG in the figure) and LNP-Mn-pIC (labeled as LNP in the figure); Figure H shows the activity comparison of LNG-Mn-pIC and LNP-Mn-pIC in in vitro activation of BMDC (***P<0.001); Figure I shows a schematic diagram of the quantification of pIC in LNG-Mn-pIC (labeled as LNG in the figure) and LNP-Mn-pIC (labeled as LNP in the figure) by agarose gel electrophoresis;

[0067] Figure 3 This paper illustrates the optimization of the LNG-Mn-pIC preparation process in Example 3 of the present invention. Specifically, Figures A and C show the manganese ion loading (A), average particle size (B), and zeta potential (C) of LNG-Mn-pIC obtained at different incubation temperatures when DSPC is used as the auxiliary phospholipid; Figures DF and DF show the manganese ion loading (D), average particle size (E), and zeta potential (F) of LNG-Mn-pIC obtained at different incubation temperatures when DOPE is used as the auxiliary phospholipid; Figures GI and GI show the manganese ion loading (G), average particle size (H), and zeta potential (I) of LNG-Mn-pIC obtained at different incubation times when DSPC is used as the auxiliary phospholipid; Figures J and L show the manganese ion loading (J), average particle size (K), and zeta potential (L) of LNG-Mn-pIC obtained at different incubation times when DOPE is used as the auxiliary phospholipid; and Figures MN show the pH-responsive release comparison of LNG-Mn-pIC when DSPC and DOPE are used as auxiliary phospholipids.

[0068] Figure 4This invention demonstrates the bioactivity of LNG-Mn-pIC in in vitro activation of BMDC cells and killing of tumor cells in Example 4 of this invention. Figure A shows the synergistic effect of LNG-pIC with LNG-Mn or free manganese ions in activating BMDC to produce IFN-β; Figure B shows the activity comparison of LNG-Mn-pIC, LNG-pIC, and LNG-Mn in activating BMDC to produce IFN-β; Figure C shows the synergistic effect of free manganese ions and free pIC at different concentrations and ratios in activating BMDC to produce IFN-β (presented as a heatmap with average values ​​of n=3); Figure D shows the synergistic effect of LNG-Mn-pIC at different concentrations and ratios of manganese ions and pIC in activating BMDC to produce IFN-β (presented as a heatmap with average values ​​of n=3); Figure E shows the toxicity of free manganese ions and pIC at different concentrations and ratios in MC38 cells (presented as a heatmap with average values ​​of n=3); Figure F shows the synergistic effect of free manganese ions and pIC at different concentrations and ratios in activating BMDC to produce IFN-β (n=3, quantitative results). Each pIC in the figure... The manganese ion concentrations at each pIC concentration, from left to right, are 0 μM, 100 μM, 200 μM, 300 μM, and 400 μM, respectively. Figure G shows the synergistic effect of LNG-Mn-pIC in activating BMDC to generate IFN-β at different manganese ion and pIC concentrations and ratios (n=3, quantitative results). The manganese ion concentrations at each pIC concentration in the figure, from left to right, are 0 μM, 100 μM, 200 μM, 300 μM, and 400 μM, respectively. Figure H shows the synergistic effect of different manganese ion and pIC concentrations and ratios. The toxicity of LNG-Mn-pIC at different concentrations and ratios in MC38 cells (n=3, quantitative results). The manganese ion concentrations at each pIC concentration, from left to right, are 0 μM, 100 μM, 200 μM, 300 μM, and 400 μM, respectively. Figure I shows the IFN-β activation levels of LNG-Mn-pIC in BMDCs of wild-type mice (WT), STING knockout mice (STING-KO), and MAVS knockout mice (MAVS-KO). No significant difference was observed (ns). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0069] Figure 5This study demonstrates the in vivo tissue distribution and antitumor activity of LNG-Mn-pIC in Example 5 of the present invention. Figure A shows the distribution of LNG-Mn-pIC-IR780 in various tissues of MC38 subcutaneous tumor-bearing mice; Figure BD shows the antitumor dose escalation experiment of LNG-Mn-pIC in the MC38 subcutaneous tumor model (manganese ion and pIC doses of 25ug and 5ug, 25ug and 10ug, 25ug and 20ug, n=3), the corresponding dosing regimen diagram (B), tumor growth curve (C), and mouse weight change (D); Figure EH shows the component-dependent experiment of LNG-Mn-pIC in the MC38 subcutaneous tumor model (manganese ion and pIC doses of 25ug and 10ug, n=5), the corresponding dosing regimen diagram (E), tumor growth curve (F), mouse weight change (G), and mouse survival monitoring results (H); Figure IL shows the independent growth curves of mice in the NT group (I), LNG-pIC group (J), LNG-Mn group (K), and LNG-Mn-pIC group (L) in the LNG-Mn-pIC component-dependent experiment. Detailed Implementation

[0070] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0071] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0072] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0073] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0074] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0075] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0076] Terminology Definitions and Explanations

[0077] In this document, the term "LMW Poly IC" should be understood as low molecular weight Poly IC with an average molecular weight range of 0.2-1 kb; the term "HMW Poly IC" should be understood as high molecular weight Poly IC with an average molecular weight range of 1.5-8 kb.

[0078] The term "pharmaceutically acceptable" indicates that a pharmaceutical composition can be administered to a subject without producing adverse physiological reactions that would impede the administration of the pharmaceutical composition. For example, "pharmaceuticalally acceptable excipients" refers to excipients useful in the preparation of generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of such excipients or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous mediators, such as non-volatile oils, may also be used.

[0079] The term "pharmaceutically acceptable carrier" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to extend the shelf life or potency of antibodies.

[0080] According to embodiments of the present invention, a first aspect of the present invention provides a nucleic acid drug delivery system, the nucleic acid drug delivery system comprising lipid nanogels and metal ions;

[0081] The lipid nanogel comprises a nanogel and a phospholipid layer, wherein the phospholipid layer coats the outer surface of the nanogel;

[0082] The nucleic acid drug and metal ions are placed in the nanogel;

[0083] The metal ions include at least one selected from manganese ions, zinc ions, cobalt ions, nickel ions, iron ions, ferrous ions, magnesium ions, copper ions, chromium ions, aluminum ions, silver ions, barium ions, and gadolinium ions.

[0084] The aforementioned metal ions can act as STING pathway agonists to activate the cGAS-STING signaling pathway responsible for sensing cytoplasmic DNA, thereby effectively inducing DC cells to produce type I interferon and activating CD8. + T cells, and experiments have demonstrated a multi-layered biological synergistic effect between the cGAS-STING signaling pathway and the RIG-I / MDA5 signaling pathway activated by nucleic acid drugs, such as Poly IC. Figure 1 Specifically, firstly, activation of the STING signaling pathway can also recruit TBK1 and phosphorylate IRF3, thereby inducing the production of type I interferon. Both activate the same transcription factors and effector molecules in downstream signal transduction. Secondly, activation of the two pathways can upregulate the expression levels of key proteins in each pathway. For example, ssDNA can upregulate the transcription level of RIG-I mRNA, while PolyIC can upregulate the transcription level of STING mRNA, thus achieving positive feedback regulation in their respective signal transduction processes. In addition, double-stranded RNA can promote the assembly of the RIG-I / MAVS / STING ternary protein complex, further amplifying the activation level of downstream signals. Furthermore, compared to expensive synthetic cyclic dinucleotide small molecule STING agonists that are easily degraded and metabolized in vivo, the metal ions used in this invention are inexpensive, readily available, and stable in plasma. They can also be used as MRI contrast agents to enhance magnetic resonance imaging signals, achieving integrated diagnostic and therapeutic functions.

[0085] According to embodiments of the present invention, the nucleic acid drug comprises DNA or RNA.

[0086] According to embodiments of the present invention, the nucleic acid drug includes at least one selected from mRNA, siRNA, tRNA, snRNA, miRNA, piRNA, and circRNA.

[0087] According to embodiments of the present invention, the nucleic acid drug includes those selected from TLR agonists.

[0088] According to embodiments of the present invention, the nucleic acid drug includes at least one selected from Poly IC, CpG, Poly I:U and their derivatives.

[0089] According to an embodiment of the present invention, the nucleic acid drug comprises those selected from Poly IC.

[0090] According to an embodiment of the present invention, the Poly IC includes a selection from LMW Poly IC or HMW Poly IC.

[0091] According to an embodiment of the present invention, the nanogel is prepared from polysaccharides. The nanogel has the potential to promote endosome escape, which can improve the cytoplasmic delivery efficiency of nucleic acid drugs and metal ions, enabling them to more effectively activate the RIG-I / MDA5 signaling pathway and the cGAS-STING signaling pathway in the cytoplasm, resulting in higher levels of type I interferon.

[0092] According to embodiments of the present invention, the polysaccharide comprises at least one selected from hyaluronic acid, sodium alginate, dextran, and their derivatives.

[0093] According to a preferred embodiment of the present invention, the polysaccharide comprises a component selected from hyaluronic acid.

[0094] According to an embodiment of the present invention, the phospholipid layer comprises at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids.

[0095] According to embodiments of the present invention, the phospholipid layer comprises cationic lipids or polyethylene glycol lipids.

[0096] According to embodiments of the present invention, the cationic lipid includes at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, and DC-Chol.

[0097] According to embodiments of the present invention, the polyethylene glycol lipid includes at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG.

[0098] According to an embodiment of the present invention, the phospholipid layer further comprises accessory lipids or steroidal lipids.

[0099] According to an embodiment of the present invention, the auxiliary lipid includes at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, and ALC-0159.

[0100] According to an embodiment of the present invention, the steroidal lipids include cholesterol.

[0101] According to embodiments of the present invention, the phospholipid layer comprises cationic lipids, polyethylene glycol lipids, auxiliary lipids, and steroidal lipids.

[0102] According to an embodiment of the present invention, the molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid and steroidal lipid is (2-6):(0.2-1.5):(0-5):(0-10).

[0103] According to a preferred embodiment of the present invention, the molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid and steroidal lipid is 4:0.5:1:5.

[0104] According to embodiments of the present invention, a second aspect provides a method for preparing the nucleic acid drug delivery system described in the first aspect, comprising:

[0105] (1) Prepare an aqueous solution of nanogel and an aqueous solution of nucleic acid drug by mixing the aqueous solution of nanogel and the aqueous solution of nucleic acid drug to obtain an aqueous phase component;

[0106] (2) Prepare the phospholipid layer to obtain the alcohol phase component;

[0107] (3) The aqueous phase component and the alcohol phase component are mixed to obtain a lipid nanogel encapsulating nucleic acid drugs;

[0108] (4) Add a metal ion solution to the lipid nanogel and heat to incubate to obtain the nucleic acid drug delivery system.

[0109] The inventors discovered that directly mixing metal ion solutions and nucleic acid drugs in an aqueous phase can lead to uncontrollable aggregation, resulting in a formulation with a particle size that does not meet the requirements for intravenous administration. Therefore, a gentler post-incubation method was adopted, in which metal ions are mixed with lipid nanogels and then heated to improve the permeability and fluidity of the phospholipid shell. This allows metal ions to enter the nanogel core along the concentration gradient and form stable chelates with the nucleic acid drugs, thus stably storing the metal ions inside the nucleic acid drug delivery system.

[0110] According to an embodiment of the present invention, step (1) further includes:

[0111] The aqueous solution of the nanogel, the aqueous solution of the nucleic acid drug, and the buffer solution are mixed to obtain the aqueous phase component.

[0112] According to an embodiment of the present invention, the concentration of the nanogel aqueous solution is 0.001-10 mg / ml.

[0113] According to an embodiment of the present invention, the concentration of the nucleic acid drug aqueous solution is 0.001-5 mg / ml.

[0114] According to embodiments of the present invention, the buffer solution includes at least one selected from citrate-sodium citrate buffer, acetic acid-sodium acetate buffer, MES buffer, and glycine-hydrochloric acid buffer.

[0115] According to an embodiment of the present invention, the buffer solution has a pH value of 3-7 and a concentration of 0.001-1M.

[0116] According to an embodiment of the present invention, step (3) further includes:

[0117] The obtained aqueous and alcoholic components are mixed using a microfluidic chip.

[0118] According to an embodiment of the present invention, the flow rate during the mixing process via the microfluidic chip is 5-15 ml / min.

[0119] According to an embodiment of the present invention, the volume ratio of the aqueous phase component to the alcohol phase component is 3:1-9:1.

[0120] According to an embodiment of the present invention, the metal ion solution in step (4) includes at least one selected from soluble manganese ion solution, soluble zinc ion solution, soluble cobalt ion solution, soluble nickel ion solution, soluble iron ion solution, soluble ferrous ion solution, soluble magnesium ion solution, soluble copper ion solution, soluble chromium ion solution, soluble aluminum ion solution, soluble silver ion solution, soluble barium ion solution, and soluble gadolinium ion solution.

[0121] According to an embodiment of the present invention, the soluble manganese ion solution includes at least one selected from manganese sulfate solution, manganese nitrate solution, manganese acetate solution, and manganese citrate solution.

[0122] According to an embodiment of the present invention, the concentration of the soluble manganese ion solution is 0.5-4M.

[0123] According to an embodiment of the present invention, the heating and incubation temperature in step (4) is 37.5-60°C, and the incubation time is 1-6 hours. The incubation temperature and incubation time affect the metal ion loading and formulation stability. Specifically, the incubation temperature should meet the phase transition temperature of phospholipid molecules to satisfy their permeability requirements; the incubation time affects the particle size, zeta potential, metal ion loading, and acid response release of the prepared formulation.

[0124] According to an embodiment of the present invention, the preparation method further includes:

[0125] The obtained nucleic acid drug delivery system was dialyzed, concentrated, filtered, and isotonicized to meet the requirements for subsequent cell processing or animal administration.

[0126] According to embodiments of the present invention, a third aspect of the present invention provides a pharmaceutical composition comprising the nucleic acid drug delivery system described in the first aspect.

[0127] According to embodiments of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0128] According to embodiments of the present invention, a fourth aspect of the present invention provides the use of the nucleic acid drug delivery system of the first aspect or the pharmaceutical composition of the third aspect in the preparation of a medicament, wherein the medicament is used for antitumor immunotherapy or anti-infective therapy.

[0129] According to a specific embodiment of the present invention, the tumor includes, but is not limited to, melanoma, cervical cancer (HPV), colorectal cancer, gastric cancer, breast cancer, and other tumors with specific or universal antigens. The anti-infective treatment includes, but is not limited to, targeted treatment against pathogens such as viruses and bacteria.

[0130] According to embodiments of the present invention, the method of administration of the drug includes at least one selected from intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection.

[0131] According to an embodiment of the present invention, a fifth aspect of the present invention provides a vaccine comprising an antigen and the nucleic acid drug delivery system described in the first aspect.

[0132] According to embodiments of the present invention, a sixth aspect provides the use of the vaccine described in the fifth aspect in the prevention or treatment of tumors or infectious diseases.

[0133] According to a specific embodiment of the present invention, the tumor includes, but is not limited to, melanoma, cervical cancer (HPV), colorectal cancer, gastric cancer, breast cancer, and other tumors with specific or universal antigens. The infectious diseases include, but are not limited to, viral and bacterial infectious diseases, such as diseases caused by cervical cancer (HPV), novel coronavirus, etc.

[0134] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0135] Example 1: Preparation of the LNG-Mn-pIC Nucleic Acid Drug Delivery System

[0136] For detailed procedures, please refer to [link / document / documentation]. Figure 2 Figure A in the diagram.

[0137] (1) Preparation of microfluidic aqueous phase components:

[0138] Mix 2 mg / ml HA aqueous solution (molecular weight between 150 and 250 kDa), 500 μg / ml Poly IC (pIC) aqueous solution, and 100 mM citrate-sodium citrate buffer (pH = 5) in a volume ratio of 3:1:3. Add double-distilled water and bring the volume to 2 ml. Vortex for 30 seconds to mix and let stand on ice.

[0139] (2) Preparation of microfluidic alcohol phase components:

[0140] Mix 140ul of 10mg / ml ethanol solution containing DSPC, cholesterol, DSPE-PEG2000, and Dlin-MC3-DMA (molar ratio of 1:5:0.5:4) with 560ul of ethanol, mix thoroughly by pipetting, and let stand at room temperature.

[0141] (3) Microfluidic preparation of LNG-pIC:

[0142] 2 ml of aqueous phase component and 700 μl of alcohol phase component were mixed through a microfluidic chip at a flow rate of 10 ml / min and an alcohol-water ratio of 1:3 to prepare LNG-pIC.

[0143] (4) Incubation of manganese ions after heating:

[0144] 500 μL of 3M MnCl2 aqueous solution was slowly added to the LNG-pIC obtained in step (3), and the mixture was rapidly mixed while adding. The mixture was incubated in a water bath at 47.5 °C for 2 h, and then cooled on ice for 10 min to obtain LNG-Mn-pIC.

[0145] To meet the requirements for intravenous administration, the preparation method of the nucleic acid drug delivery system may further include:

[0146] (5) Dialysis:

[0147] This operation was performed in a cold room. The incubated LNG-Mn-pIC formulation was dialyzed in single-distilled water for 24 hours, with the dialysate replaced every 8 hours. After dialyzing, the particle size and zeta potential of the formulation were measured using a Malvern particle size analyzer. The results showed that the LNG-pIC particle size before incubation was approximately 60 nm, and the zeta potential was approximately -3 mV; after incubation, the particle size increased to approximately 80 nm, and the zeta potential decreased to approximately -10 mV. Figure 2 (Figures B and C in the diagram) This may be due to the mineralization of manganese ions and pIC in the core, as well as the adsorption of a small amount of HA on the surface of the formulation, leading to an increase in particle size and a decrease in potential.

[0148] (6) Concentration and Quantification:

[0149] The dialysis-completed formulation was then collected into a 10kD ultrafiltration tube for concentration. After concentration to one-third of the original aqueous phase volume, the formulation was collected into an EP tube. The manganese in the formulation was initially quantified by potassium periodate oxidation method, and the accurate manganese content in the formulation was detected by inductively coupled plasma mass spectrometry (ICP-MS). The encapsulation efficiency of pIC was then detected by agarose gel electrophoresis.

[0150] (7) Filtration and isotonic conditioning:

[0151] After quantification, the formulation is adjusted to isotonic and neutral pH using 10×PBS at pH 7.4, and then sterilized by filtration through a 0.22µm filter membrane before subsequent cell treatment or animal administration.

[0152] Example 2 Effect Verification

[0153] Compare the differences in physicochemical properties and biological activity between the HA-containing formulation (LNG-Mn-pIC) and the HA-free formulation (LNP-Mn-pIC).

[0154] (1) Particle size

[0155] LNP-Mn-pIC has a slightly smaller particle size than LNG-Mn-pIC, approximately 70 nm. Figure 2 (See Figure D), while the potential is close to neutral, approximately -2.5mV. Figure 2 (China E diagram).

[0156] (2) Manganese ion loading

[0157] The manganese content in the formulation was quantified by potassium permanganate oxidation. The results showed that there was no significant difference in manganese ion loading between the two methods after incubation, both being around 1.5 mM. Figure 2 (Figure F in the middle)

[0158] (3) pIC encapsulation rate

[0159] Quantification of pICs in the formulation was performed using agarose gel electrophoresis. Since free pICs cannot be removed by dialysis during preparation, the pIC encapsulation efficiency can be calculated by detecting the pIC content inside and outside the formulation. Specifically, a standard curve was prepared using a free pIC solution. 1% Triton-X100 solution was used as the lysis buffer to release the pICs from the formulation. The bands in the experimental group without lysis buffer showed free pICs not encapsulated in the core, while the bands in the experimental group with lysis buffer showed all pICs. The resulting electrophoresis images were used to calculate grayscale values ​​using ImageJ software to plot the standard curve, and then the pIC content of each experimental group was calculated. The results are as follows: Figure 2As shown in Figure I, the pIC encapsulation efficiency of both LNP-Mn-pIC and LNG-Mn-pIC is close to 100%, and the pIC is not degraded in the formulation after heating and incubation with manganese ions, but is stably present in the nanogel core crosslinked with HA and manganese ions.

[0160] (4) Manganese ion release behavior

[0161] One ml of LNG-Mn-pIC and LNP-Mn-pIC formulations were placed in dialysis columns with a pore size of 1000 kD and immersed in 50 ml of PBS buffer (pH 7.4, to simulate the in vivo plasma environment) and 50 ml of PBS buffer (pH 4.5, to simulate the endosome environment), respectively. Release experiments were conducted at 37℃ and 100 rpm. The remaining manganese content in the dialysis column was measured at different time points, and manganese ion release curves for both formulations over 24 hours were plotted. The results showed that LNG-Mn-pIC exhibited a significantly more acid-responsive release capacity than LNP-Mn-pIC. Figure 2 (See Figure G in the middle), which will facilitate the rapid release of manganese ions by LNG-Mn-pIC in the acidic environment of endosomes to activate downstream signaling pathways, thereby generating a more effective immune response.

[0162] (5) Biological activity

[0163] In vitro ELISA experiments confirmed that LNG-Mn-pIC indeed had a greater advantage in biological activity than LNP-Mn-pIC. BMDC cells were isolated from mouse bone marrow and induced to differentiate in vitro with granulocyte-monocyte colony-stimulating factor (GM-CSF) until day 6, at a concentration of 1.2 × 10⁶ cells per well. 5 Dendritic cells (DCs) were seeded at a density of [number] cells per well in 96-well plates and stimulated with the same concentrations of LNG-Mn-pIC and LNP-Mn-pIC. After 24 hours, the supernatant was collected, and INF-β secreted by DCs was detected using the LumiKine Xpress mIFN-β2.0luex-mifnbv2 (InvivoGen) kit. The results showed that LNG-Mn-pIC significantly activated higher levels of INF-β compared to LNP-Mn-pIC. Figure 2 (See Figure H in the middle), which also proves that HA can help manganese ions achieve more effective biological activity with pIC.

[0164] Example 3: Optimization of LNG-Mn-pIC Preparation Process

[0165] (1) Effects of phospholipid type and manganese ion incubation temperature on manganese ion loading

[0166] Phospholipid bilayers possess fluidity, and heating helps improve the permeability of the lipid shell, thereby facilitating the exchange of ions between the inner and outer layers. The principle utilized in this invention is to promote the diffusion of manganese ions from the outer aqueous phase to the inner aqueous phase of the lipid nanogel by increasing the temperature. When the concentrations of the inner and outer ions reach equilibrium, rapid cooling "locks" the ions in the inner aqueous phase within the lipid bilayer, preventing leakage. Since different phospholipid molecules have different phase transition temperatures, different temperatures have varying effects on the permeability of the lipid bilayer. To obtain higher manganese ion loading, the effects of different incubation temperatures and times on manganese ion loading, as well as the particle size and potential of the formulation, were investigated. The differences between DSPC and DOPE, two phospholipids with different phase transition temperatures, in this process were also compared, providing important information for the final determination of the LNG-Mn-pIC composition and process.

[0167] The incubation temperature range was set to 25℃~60℃. Figure 3 Figures A, B, and C show the differences in manganese ion loading, particle size, and zeta potential of LNG-Mn-pIC prepared at different incubation temperatures when DSPC was used as an auxiliary phospholipid. The results indicate that when the incubation time is constant (2 h), the manganese ion loading first increases and then decreases with increasing incubation temperature, reaching a peak at 47.5 °C, approximately 1.5 mM (…). Figure 3 (Figure A in the middle); Figure 3 Figure B shows that the particle size of LNG-Mn-pIC gradually increases with increasing temperature. When the incubation temperature exceeds 50℃, the particle size more than doubles, and when the incubation temperature exceeds 60℃, the particle size increases to four times its original size. Figure 3 The results in Figure C show that as the incubation temperature increases, the surface charge of the formulation gradually becomes more negative in a regular manner. This may be because the higher the incubation temperature, the stronger the interaction between phospholipid molecules and HA, and the more HA remains on the surface of the formulation. Figure 3 Figures D, E, and F show the differences in manganese ion loading, particle size, and zeta potential of LNG-Mn-pIC prepared at different incubation temperatures when DOPE is used as an auxiliary phospholipid. The results show that the manganese ion loading of the formulation obtained when DOPE is used as an auxiliary phospholipid increases with increasing temperature, the particle size gradually increases, and the potential change is similar to that of DSPC. However, the manganese ion loading of the formulation obtained when DOPE is used as an auxiliary phospholipid is significantly lower than that of the DSPC formulation overall.

[0168] (2) Effects of phospholipid type and manganese ion incubation time on manganese ion loading

[0169] After selecting the optimal incubation temperature for each manganese ion loading, the trend of manganese ion loading with incubation temperature at that temperature was further investigated. Figure 3Figures G, H, and I show the effects of different incubation times at 47.5℃ on the manganese ion loading, particle size, and zeta potential of LNG-Mn-pIC prepared with DSPC as an auxiliary phospholipid. The results show that with the increase of incubation time, the manganese ion loading first increases and then decreases, reaching a peak of about 1.5 mM at 2 h. At the same time, the higher the incubation temperature, the larger the particle size, and the zeta potential stabilizes at -8 mV to -10 mV after the incubation time exceeds 2 h.

[0170] akin, Figure 3 Figures J, K, and L show the effects of different incubation times at 60℃ on the manganese ion loading, particle size, and zeta potential of LNG-Mn-pIC prepared with DOPE as an auxiliary phospholipid. The results show that the manganese ion loading of LNG-Mn-pIC prepared with DOPE as an auxiliary phospholipid also shows a trend of first increasing and then decreasing with increasing incubation time, reaching a peak of about 1 mM at 1 h; the particle size gradually increases with increasing incubation time, and the zeta potential stabilizes at about -17 mV after the incubation time exceeds 3 h.

[0171] (3) Effects of phospholipid type and manganese ion incubation time on formulation stability

[0172] To compare the stability and manganese ion release of LNG-Mn-pIC prepared using DSPC as a cofactor phospholipid and LNG-Mn-pIC prepared using DOPE as a cofactor phospholipid, an in vitro manganese ion release experiment was conducted to determine the stability of the two formulations in the blood and their degradation and release behavior after being phagocytosed by cells and entering endosomes. Figure 3 (M and N diagrams). The release experiment method was similar to that used in Example 2 to study the manganese ion release behavior. The formulation was placed in a 1000kD dialysis column, and the pH of the release solution (PBS buffer containing 10% FBS) was controlled to simulate the entry of the formulation into the bloodstream (pH=7.4) and its phagocytosis into endosomes by cells (pH=4.5). The results showed that LNG-Mn-pIC prepared with DSPC as a cofactor phospholipid had higher stability in plasma than LNG-Mn-pIC prepared with DOPE as a cofactor phospholipid. The stability was better than that of LNG-Mn-pIC prepared with DSPC as a cofactor phospholipid in 24h. When LNG-Mn-pIC was prepared using DSPC as an auxiliary phospholipid, only about 20% was released, while LNG-Mn-pIC prepared using DOPE as an auxiliary phospholipid released nearly 80%. In addition, there was a significant difference in the acid response release between the two. At 24h, LNG-Mn-pIC prepared using DSPC as an auxiliary phospholipid could release nearly 80%, while LNG-Mn-pIC prepared using DOPE as an auxiliary phospholipid was more stable under acidic conditions than under neutral conditions, releasing about 50%.

[0173] In summary, phospholipids with different transition temperatures exhibit significantly different properties during the incubation process of loading manganese ions after heating. Furthermore, different incubation times and temperatures also affect the stability of the formulation. Therefore, the components and preparation process of LNG-Mn-pIC can be rationally selected and adjusted based on actual needs.

[0174] Example 4: Investigation of the bioactivity of LNG-Mn-pIC in activating BMDC cells and killing tumor cells in vitro.

[0175] (1) In order to investigate whether manganese ions and pIC can synergistically stimulate DC cells to produce type I interferon, the activation effects of the two components on DC cells in different forms were investigated.

[0176] First, a two-component concentration-dependent experiment was conducted on free manganese ions and free pIC. Figure 4 (Figures C and F) BMDC cells were isolated from mouse bone marrow and induced to differentiate in vitro with GM-CSF until day 6, at a density of 1.2 × 10⁶ cells per well. 5 Cells were densely seeded into 96-well plates and stimulated with different concentrations of free manganese ions (100 μM–400 μM) and pIC (1 μg / ml–16 μg / ml). After 24 h, the supernatant was collected, and the INF-β content in the culture medium was detected using the LumiKine Xpress mIFN-β2.0luex-mifnbv2 (InvivoGen) kit. The results showed that when the concentration of manganese ions was 200 μM or lower, the synergistic effect between manganese ions and pIC was not significant. When the concentration of manganese ions reached 300 μM, the two showed a pIC concentration-dependent synergistic activation effect. When the pIC concentration exceeded 4 μg / ml, the activation effect reached saturation. This confirmed that free manganese ions and pIC can synergistically activate BMDCs within a certain concentration range.

[0177] Subsequently, LNG-Mn and LNG-pIC were prepared using microfluidic technology, and the two were administered in combination to stimulate DC cells. Free manganese ions were used as a reference to investigate whether free manganese ions and LNG-Mn could synergistically activate DC cells with LNG-pIC. Figure 4(Figure A). The results showed that LNG-pIC alone could activate DC cells in a dose-dependent manner, but the activation level was low. Co-administration with free manganese ions (200 μM) significantly amplified the activation effect, showing a significant "from zero to one" enhancement of activity even at a pIC concentration of only 0.1 μg / ml. However, co-administration of LNG-Mn and LNG-pIC did not enhance the activation effect of pIC, indicating that the separate delivery of the two formulations could not achieve the purpose of synergistic activation of DC cells. This may be because LNG-Mn itself is too stable and has poor release properties, and cannot be rapidly decomposed into free manganese ions to exert its effect after entering the cell.

[0178] Furthermore, in vitro experiments confirmed that LNG-Mn-pIC significantly activated DC cells compared to LNG-Mn and LNG-pIC, and this activation was manganese ion and pIC concentration-dependent. Figure 4 (Figure B). To more systematically reveal the effects of manganese ion and pIC concentrations on LNG-Mn-pIC activation of DC cells, LNG-Mn-pIC formulations containing different ratios of manganese ions and pIC were prepared, and DC cell activation was detected in vitro. Figure 4 Figures D and G show that both manganese ions and pIC in the LNG-Mn-pIC formulation can produce a significant concentration-dependent activation effect on DC cells. However, no DC cell activation was detected even at a concentration of 400 μM for LNG-Mn alone. Furthermore, even if LNG-pIC alone can activate DC cells in a dose-dependent manner, its activation level is significantly lower than that of the formulation combined with manganese ions. Therefore, the synergistic effect of the two in this process is further confirmed.

[0179] (2) Since the formulation is engulfed by a large number of tumor cells when it accumulates in tumor tissue, the effect of the formulation on tumor cells was investigated.

[0180] Using mouse colon cancer cell line MC38 as the model cell line, cells were seeded at 2 × 10⁶ cells per well in a 96-well plate. 4 Tumor cells were stimulated with LNG-Mn-pIC, and the toxicity of this preparation to tumor cells was detected by a CCK8 assay kit (GLPBIO) after 24 hours. Figure 4 (Figures E and H). The results showed that the LNG-Mn-pIC formulation exhibited manganese ion-dependent cytotoxicity; the higher the manganese ion concentration, the stronger the cytotoxicity. However, pIC (below 16 μg / ml) had no significant effect on the viability of tumor cells. Therefore, the LNG-Mn-pIC formulation provided by this invention can effectively activate dendritic cells (DCs) and also effectively kill tumor cells.

[0181] To demonstrate that LNG-Mn-pIC formulations function through two pathways—namely, manganese ions primarily activate the cGAS-STING signaling pathway to produce type I interferon in dendritic cells (DCs), and pIC primarily activates DCs to produce type I interferon through the cytoplasmic MDA5 / RIG-I-MAVS signaling pathway—mouse BMDCs with STING and MAVS knockout were extracted and cultured in vitro until day six. The DCs were then stimulated with LNG-Mn-pIC in the same manner. The results showed that MAVS knockout DCs completely eliminated their ability to respond to LNG-Mn-pIC, while STING knockout DCs showed a significantly reduced ability to produce type I interferon. Figure 4 (See Figure I in the original text), thus proving that both pathways played an important role in the process.

[0182] Example 5: Investigation of in vivo tissue distribution and antitumor activity of LNG-Mn-pIC

[0183] (1) After the bioactivity of LNG-Mn-pIC was initially demonstrated in vitro, the antitumor effect of LNG-Mn-pIC was investigated in vivo using the MC38 mouse subcutaneous colon cancer tumor model as an example.

[0184] To enhance the accumulation of the formulation at the tumor site and reduce the "first-pass effect" caused by hepatic metabolism, intravenous administration was used. To demonstrate that LNG-Mn-pIC can accumulate at the tumor site through the high permeability and retention effect of solid tumors, the hydrophobic near-infrared fluorescent dye IR780 was encapsulated in the phospholipid bilayer of LNG-Mn-pIC as a tracer to show the accumulation of the formulation in various tissues of mice. First, 5 × 10⁻⁶... 5 MC38 tumor cells were subcutaneously inoculated into the upper right leg of C57BL / 6 mice. When the tumor size reached 300 mm, 3 ~400mm 3 LNG-Mn-pIC-IR780 (containing 25 μg manganese ions, 10 μg pIC, and 5 μg IR780) was intravenously injected into mice. After 48 hours, the mice were euthanized, and heart, liver, spleen, lung, kidney, and tumor tissues were removed. IR780 in each tissue was imaged using an IVIS live imaging system. The results showed that the accumulation of LNG-Mn-pIC in tumor tissue was significantly higher than in other tissues. Figure 5 (See Figure A in the middle), thus indicating that the preparation can accumulate well at the tumor site.

[0185] (2) A dose escalation experiment of LNG-Mn-pIC was conducted in MC38 tumor-bearing mice to preliminarily explore the therapeutic effect and safety of the preparation in vivo.

[0186] Treatment plan such as Figure 5 As shown in Figure B, first, 5×10 5 MC38 tumor cells were subcutaneously inoculated into the upper right leg of C57BL / 6 mice, and the tumors were allowed to grow to 80 mm in size. 3 At approximately 10:00 AM, mice were randomly divided into two groups: an untreated group (NT) and three treated groups (manganese ion and pIC doses of 25 μg and 5 μg, 25 μg and 10 μg, and 25 μg and 20 μg, respectively), with n=3 in each group. Tumor growth and mouse weight changes were monitored every 2-3 days. Figure 5 (Figures C and D) The results showed that LNG-Mn-pIC at all three doses effectively inhibited tumor growth. The experimental groups with manganese ion and pIC doses of 25 μg and 5 μg, respectively, showed a significant rebound after 10 days of administration. However, the inhibitory effects of manganese ion and pIC doses of 25 μg and 10 μg, and 25 μg and 20 μg, respectively, persisted for nearly 20 days after treatment, with no significant difference in inhibitory efficacy. Simultaneously, mice at all three doses experienced reversible weight loss after administration, but this phenomenon was not dose-dependent and gradually returned to normal levels after treatment. Based on these results, subsequent treatments used fixed doses of manganese ion and pIC of 25 μg and 10 μg, respectively.

[0187] To demonstrate that the antitumor therapeutic effect of LNG-Mn-pIC is component-dependent, C57BL / 6 mice were inoculated with MC38 tumors using the same method. Figure 5 (Figure E) When the tumor size grows to 80mm 3 At approximately 8:00 AM, mice were randomly divided into four groups: untreated group (NT), LNG-Mn group (manganese ion dose of 25 μg), LNG-pIC group (pIC dose of 10 μg), and LNG-Mn-pIC group (manganese ion dose and pIC dose of 25 μg and 10 μg, respectively), with n=5 in each group. Drug administration was performed on days 8 and 14, and tumor growth and mouse weight changes were monitored every 2-3 days. The results showed that LNG-Mn-pIC had the most significant tumor growth inhibitory effect compared to the other three groups. Figure 5 (See Figure F). The LNG-Mn group showed almost no inhibitory effect compared to the NT group, while the LNG-pIC group, although showing more significant initial inhibition, experienced a rapid rebound, indicating a significantly weaker inhibitory effect than the LNG-Mn-pIC group. Regarding changes in body weight (…), Figure 5 (Figure G) Both the LNG-pIC and LNG-Mn-pIC groups experienced transient and reversible weight loss after administration, while the LNG-Mn group showed no effect. Therefore, it can be concluded that the side effects of the LNG-Mn-pIC formulation may be caused by the pIC itself. From the survival curves ( Figure 5(Figure H-L) Although the LNG-Mn-pIC group could not cure the tumor, the survival time of the animals in this group was significantly longer than that of the control group. Therefore, the above in vivo experiments confirmed that the LNG-Mn-pIC preparation has significant anti-tumor activity, and this activity depends on the combined action of manganese ions and pIC.

[0188] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0189] 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.

Claims

1. A nucleic acid drug delivery system, characterized by, The nucleic acid drug delivery system comprises lipid nanogels and metal ions; The lipid nanogel comprises a nanogel and a phospholipid layer, wherein the phospholipid layer coats the outer surface of the nanogel; The nucleic acid drug and metal ions are placed in the nanogel; The metal ion is a manganese ion; The nucleic acid drug is PolyIC; The nanogel is prepared from hyaluronic acid.

2. The nucleic acid drug delivery system according to claim 1, wherein The phospholipid layer includes at least one selected from neutral phospholipids, negatively charged phospholipids, positively charged phospholipids, or ionizable phospholipids.

3. The nucleic acid drug delivery system according to claim 1, characterized in that, The phospholipid layer comprises cationic lipids or polyethylene glycol lipids.

4. The nucleic acid drug delivery system according to claim 3, characterized in that, The cationic lipids include at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, and DC-Chol; Optionally, the polyethylene glycol lipid includes at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG.

5. The nucleic acid drug delivery system according to claim 1, characterized in that, The phospholipid layer further includes accessory lipids or steroidal lipids.

6. The nucleic acid drug delivery system according to claim 5, characterized in that, The auxiliary lipids include at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, and ALC-0159; Optionally, the steroidal lipids include cholesterol.

7. The nucleic acid drug delivery system according to claim 1, characterized in that, The phospholipid layer includes cationic lipids, polyethylene glycol lipids, auxiliary lipids, and steroidal lipids.

8. The nucleic acid drug delivery system according to claim 7, characterized in that, The molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid and steroid lipid is (2-6):(0.2-1.5):(0-5):(0-10).

9. The nucleic acid drug delivery system according to claim 7, characterized in that, The molar ratio of the cationic lipid, polyethylene glycol lipid, auxiliary lipid, and steroidal lipid is 4:0.5:1:

5.

10. A method for preparing a nucleic acid drug delivery system according to any one of claims 1-9, characterized in that, include: (1) Prepare an aqueous solution of nanogel and an aqueous solution of nucleic acid drug, and mix the aqueous solution of nanogel and the aqueous solution of nucleic acid drug to obtain an aqueous phase component; (2) Prepare the phospholipid layer to obtain the alcohol phase component; (3) The aqueous phase component and the alcohol phase component are mixed to obtain a lipid nanogel encapsulating nucleic acid drugs; (4) Add a metal ion solution to the lipid nanogel and heat to incubate to obtain the nucleic acid drug delivery system; The metal ion solution is a soluble manganese ion solution.

11. The preparation method according to claim 10, characterized in that, Step (1) further includes: The aqueous solution of the nanogel, the aqueous solution of the nucleic acid drug, and the buffer solution were mixed to obtain an aqueous phase component; Optionally, the concentration of the aqueous nanogel solution is 0.001-10 mg / ml; Optionally, the concentration of the nucleic acid drug aqueous solution is 0.001-5 mg / ml; Optionally, the buffer solution comprises at least one selected from citrate-sodium citrate buffer, acetate-sodium acetate buffer, MES buffer, and glycine-hydrochloric acid buffer. Optionally, the buffer solution has a pH of 3-7 and a concentration of 0.001-1M.

12. The preparation method according to claim 10, characterized in that, Step (3) further includes: The obtained aqueous phase component and alcohol phase component are mixed using a microfluidic chip; Optionally, the flow rate during the mixing process via the microfluidic chip is 5-15 ml / min; Optionally, the volume ratio of the aqueous phase component to the alcohol phase component is 3:1 to 9:

1.

13. The preparation method according to claim 10, characterized in that, The soluble manganese ion solution includes at least one selected from manganese sulfate solution, manganese nitrate solution, manganese acetate solution, and manganese citrate solution; Optionally, the concentration of the soluble manganese ion solution is 0.5-4M.

14. The preparation method according to claim 10, characterized in that, The temperature for heating and incubation in step (4) is 37.5-60℃, and the incubation time is 1-6h.

15. The preparation method according to any one of claims 10-14, characterized in that, The preparation method further includes: The obtained nucleic acid drug delivery system was subjected to dialysis, concentration, filtration, and isotonic adjustment.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: the nucleic acid drug delivery system according to any one of claims 1-9; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

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