Iron-loaded nucleic acid delivery nano platform as well as preparation method and application thereof

Abstract

The invention discloses an iron-loaded nucleic acid delivery nano platform as well as a preparation method and application thereof. The iron-loaded nucleic acid delivery nanometer platform comprises nucleic acid-iron nanometer particles and lipidosome wrapping the surfaces of the nanometer particles. The iron-loaded nucleic acid delivery nano platform can efficiently deliver multiple types of nucleic acid molecules such as siRNA, polyIC and DNA into cells, and the nucleic acid molecules are accurately released in a cytoplasm environment, so that the expected function of the nucleic acid molecules is exerted. The platform has the advantages of high delivery efficiency, good biocompatibility, low cytotoxicity, high stability, wide application range and the like, and is suitable for multiple biomedical fields such as gene regulation and control, immune nucleic acid drug delivery and the like.

Description

Technical Field

[0001] This invention relates to bionanomaterials, specifically to an iron-loaded nucleic acid delivery nanoplatform, its preparation method, and its applications. Background Technology

[0002] Nucleic acid drugs are a class of innovative therapeutic tools based on nucleic acid molecules. By designing specific nucleic acid sequences and employing mechanisms such as editing, activation, replacement, or knockout, they precisely regulate gene expression, thereby intervening in the occurrence and progression of diseases. Nucleic acid drugs include DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA), which, with their unique molecular mechanisms of action and broad potential applications, provide novel solutions for the treatment of intractable diseases. These drugs possess significant advantages such as flexible design, simple synthesis, short development cycles, diverse target selection, strong target specificity, and wide application areas, making them a hot topic in biomedical research. Nucleic acid molecules have demonstrated enormous application potential in fields such as gene editing, disease treatment, and vaccine development.

[0003] However, the unique structure of nucleic acid molecules and the complex physiological environment in vivo pose significant challenges to their delivery. Naked nucleic acid molecules must overcome several obstacles before reaching their target site. First, due to the abundance of nucleases in physiological environments such as blood, naked nucleic acid molecules are easily and rapidly degraded once they enter the systemic circulation. Furthermore, naked nucleic acid molecules may non-specifically bind to proteins in the blood, further reducing their delivery efficiency. Even if nucleic acid drugs successfully reach target cells, the hydrophilic and negatively charged properties of nucleic acid molecules make it difficult for them to cross the phospholipid bilayer of the cell membrane. After being taken up by cells, nucleic acid drugs are usually encapsulated in endosomes, which then enter lysosomes during maturation, where various hydrolases further degrade the nucleic acid molecules. Without a carrier, naked nucleic acid molecules are almost ineffective at acting on target cells. Therefore, developing suitable delivery nanoplatforms is crucial for solving the challenges of nucleic acid drug application. These delivery nanoplatforms need to help improve the stability of nucleic acid molecules in body fluids, reduce degradation, and promote the efficient release and functionalization of nucleic acid molecules at the cellular level.

[0004] Metal nanoparticles, with their high chemical stability, precisely tunable size distribution, excellent surface functional modifiability, and controllable biocompatibility, are considered a promising carrier platform for nucleic acid drug delivery. However, the application of this technology still faces two major challenges: First, the synthesis and functionalization process of traditional metal nanoparticles involves multiple complex reactions, resulting in significant batch-to-batch variability and insufficient process reproducibility. Second, existing metal nanocarriers are generally limited to the delivery capability of single-type nucleic acids, making it difficult to meet the synergistic needs of multimodal nucleic acids in combined gene therapy. These limitations severely restrict the breadth of application of metal nanocarriers in complex disease treatment scenarios.

[0005] Therefore, there is an urgent need to design and develop a delivery nanoplatform that is simple to synthesize, highly stable, and capable of efficiently delivering various types of nucleic acid molecules, in order to expand the applicability of nucleic acid molecules in clinical treatment and meet the treatment needs of a variety of complex diseases. Summary of the Invention

[0006] To address the issues of complex synthesis processes and limited nucleic acid delivery capabilities of traditional metal nanoparticles, this invention provides an iron-supported nucleic acid delivery nanoplatform, its preparation method, and its applications. This platform features a simple synthesis process, high delivery efficiency, and strong versatility, enabling efficient loading, efficient cellular uptake, and precise intracytoplasmic release of nucleic acids.

[0007] To achieve the above objectives, the present invention provides an iron-loaded nucleic acid delivery nanoplatform, the core of which is a "core-shell" structure. The core is a nucleic acid-iron nanoparticle formed by the self-assembly of nucleic acid molecules and iron ions, and the outer shell is a liposome layer wrapped around the surface of the nucleic acid-iron nanoparticle. The mass ratio of iron to nucleic acid in the nucleic acid-iron nanoparticles is 1:0.1 to 1:10. A ratio smaller than this range will result in a low nucleic acid loading rate, while a ratio larger than this range will disrupt the system's balance due to excessive nucleic acid input, preventing nanoparticle formation. The mass ratio of nucleic acid-iron nanoparticles to liposomes is 1:2 to 1:10. A ratio smaller than this range will prevent the nucleic acid-iron nanoparticles from lysosome escape, thus preventing nucleic acid molecules from entering the cytoplasm to exert their corresponding effects. A ratio larger than this range will result in an excess of liposomes, causing unnecessary waste. The raw materials for the liposomes, by mass, include the following components: 20-80 parts ionized lipid molecules, 10-40 parts auxiliary lipid molecules, 10-50 parts steroidal lipid molecules, and 1-20 parts PEG-liposome molecules. Preferably, the ratio is 45-60 parts ionized lipid molecules, 10-25 parts auxiliary lipid molecules, 20-30 parts steroidal lipid molecules, and 1-5 parts PEG-liposome molecules. This raw material ratio ensures that the liposomes can be loaded onto the outer layer of the nanoparticles without strong cytotoxicity.

[0008] In nucleic acid-iron nanoparticles, metallic iron coordinates with the phosphate backbone of nucleic acid rather than with internal base sites. This ensures that the hydrogen bonds between base pairs within the nucleic acid molecule are not broken, maximizing the integrity of the nucleic acid molecule and enabling it to perform its biological functions after entering the cell, thus guaranteeing efficient delivery. Furthermore, the phosphate backbone is a common structure for all nucleic acid molecules. The characteristic of metallic iron coordinating with the phosphate backbone in alkaline buffer allows for the encapsulation and delivery of various types of nucleic acid molecules, making this platform highly versatile.

[0009] In liposomes, ionized lipid molecules can alter their charge state under different pH conditions. At neutral pH, they typically exhibit a neutral or weak charge, but in acidic environments (such as endosomes / lysosomes), their charge becomes positive, promoting fusion with the cell membrane or endosome membrane, thus achieving efficient delivery. Auxiliary lipid molecules can enhance the overall stability of liposomes, improve encapsulation efficiency, and enhance their in vivo distribution characteristics. Steroidal lipid molecules can regulate membrane fluidity, stability, and encapsulation efficiency to improve liposome functionality. PEG-lipid molecules can reduce the size of liposome particles, prevent liposome aggregation during storage, and prolong their circulation time in vivo. The various raw materials in liposomes ensure that the iron-nucleic acid nanoparticles encapsulated in liposomes can efficiently deliver various nucleic acid molecules.

[0010] The iron is selected from soluble ferrous ions (Fe2+ or ferric ions), preferably ferrous ions. In alkaline buffer solutions, it can encapsulate nucleic acids and coordinate with the phosphate backbone of nucleic acids, thereby ensuring the integrity of the nucleic acid molecule structure.

[0011] The nucleic acid is selected from at least one of microRNA, siRNA, mRNA, polyIC, and DNA; preferably, the delivered nucleic acid molecule is selected from double-stranded nucleic acid; more preferably, the delivered nucleic acid molecule is siRNA, polyIC, or DNA.

[0012] In some embodiments of the present invention, the ionized lipid molecules in the liposomes are selected from at least one of the following: 1,2-dioleoyl-3-dimethylamino-propane (DODMA), 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-di(oleoyloxy)prop-1-amine (DOBAQ), 1,2-dioleoyl-3-trimethylpropylammonium chloride (DOTAP), methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester (Dlin-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecen-1-yl-1,3-dioxopentane Cyclo-4-ethylamine (Dlin-KC2-DMA) and methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester (Dlin-MC3-DMA); preferably, the ionized lipid molecule is selected from one or more of DODMA, DODAP, DOBAQ, and DOTAP; more preferably, the ionized lipid molecule is DOTAP.

[0013] The auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); preferably, the auxiliary lipid molecule is selected from DSPC, DPPC, and DOPE; more preferably, the auxiliary lipid molecule is selected from DOPE.

[0014] The steroidal lipid molecule is selected from at least one of the following: hydroxy cholesterol, cholesterol, dehydro cholesterol, alfalfa sterol, dihydro cholesterol, oleosterol, cholesterol, coprosterol, epicholesterol, ergosterol; preferably, the steroidal lipid molecule is selected from hydroxy cholesterol, cholesterol, dehydro cholesterol, dihydro cholesterol; more preferably, the steroidal lipid molecule is selected from cholesterol.

[0015] The PEG-lipid molecule is selected from at least one of the following: 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distosterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE); preferably, the PEG-lipid molecule is selected from PEG-DMG, PEG-DSPE, PEG-DSG; more preferably, the PEG-lipid molecule is selected from PEG-DSPE.

[0016] A second aspect of the present invention provides a method for preparing the above-mentioned iron-supported nucleic acid delivery nanoplatform, comprising the steps of preparing nucleic acid-iron nanoparticles, liposomes, and liposome-modified nucleic acid-iron nanoparticles, wherein: The preparation method of nucleic acid-iron nanoparticles includes: mixing soluble iron salts and nucleic acids in a certain proportion to form a complex. Then, the complex solution is added to an alkaline buffer. The complex formed by the coordination of iron ions and nucleic acids further coordinates and co-precipitates in the alkaline buffer to form nanoparticles. After allowing the nanoparticles to grow and mature by standing, they are finally collected by centrifugation to obtain well-dispersed nucleic acid-iron nanoparticles. The preparation method of liposomes includes: dissolving ionizable lipid molecules, auxiliary lipid molecules, steroidal lipid molecules and PEG-lipid molecules in an organic solvent in proportion, removing the organic solvent by vacuum rotary evaporation to form a lipid film, and then adding ultrapure water to emulsify to obtain a liposome solution; The method of modifying nucleic acid-iron nanoparticles with liposomes includes: mixing an aqueous solution of nucleic acid-iron nanoparticles with a liposome solution and incubating it under mild heating conditions for a period of time. During this process, the liposomes spontaneously encapsulate the surface of the nucleic acid-iron nanoparticles through hydrophobic interactions, forming the final "core-shell" structured nanoplatform, which can be obtained after centrifugation and purification.

[0017] Iron can stably coordinate with the phosphate backbone of nucleic acids, and its addition to an alkaline buffer system can further coordinate and co-precipitate, thereby driving the self-assembly of nanostructures. Based on this property, an iron-loaded nucleic acid delivery nanoplatform can be constructed in one step under ambient temperature and pressure by simply mixing soluble iron salts with nucleic acids and placing them in an alkaline environment. This process relies on the spontaneous self-coordination of iron and nucleic acids, and can obtain structurally stable and well-dispersed materials without complex multi-step reactions, harsh conditions, or fine processing techniques, significantly simplifying the preparation process.

[0018] Preferably, the soluble iron salt is selected from at least one of ferric chloride, ferrous chloride, ferrous sulfate, ferrous nitrate, ferric sulfate, and ferric nitrate.

[0019] In the preparation process of the above-mentioned nucleic acid-iron nanoparticles, the final concentration of iron ions in the buffer solution is 0.5~20 mM, for example, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 12 mM, 18 mM, 20 mM, etc.; preferably, the concentration of iron ions is 0.5 mM, 1 mM, 2 mM, 4 mM; more preferably, the concentration of iron ions is 5 mM.

[0020] The mass ratio of iron ions to nucleic acids is 1:0.1 to 1:10, for example, 1:0.1, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.0, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.0, 1:4.0, 1:5.0, 1:6.0, 1:7.0, 1:8.0, 1:9.0, 1:10.0, etc.; more preferably, the mass ratio is 1:0.5 to 1:5.0.

[0021] The buffer solution is selected from Tris-HCl, PBS, or Bis-Tris buffer, which are alkaline buffer solutions that can provide additional precipitation ligands. The pH of the buffer solution is 7.5 to 11, for example, pH 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, etc. More preferably, the pH of the buffer solution is 8 to 10.

[0022] The settling time is 5 to 120 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 80 minutes, 100 minutes, or 120 minutes; more preferably, the settling time is 20 to 60 minutes.

[0023] The organic solvent is selected from chloromethane, dichloromethane, trichloromethane, and anhydrous ethanol; preferably, the organic solvent is selected from trichloromethane.

[0024] The heating temperature is 30℃ to 80℃, for example, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃; more preferably, the heating temperature is set to 35℃ to 60℃.

[0025] The heating time is set to 5~120 min, for example 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 11 min, 120 min; more preferably, the heating time is set to 30~60 min.

[0026] In the above technical solutions, the static state is to allow nanoparticles to form; if the time is too short, centrifugation will not result in precipitation. The same applies to heating temperature and time; if the temperature is not suitable, liposomes cannot coat the surface of the material.

[0027] The centrifugation speed is 4000~13000 rpm, for example 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm or 13000 rpm; more preferably, the centrifugation speed is 8000~12000 rpm; more preferably, the centrifugation speed is 10000 rpm.

[0028] The third aspect of this invention provides the application of the above-described iron-supported nucleic acid delivery nanoplatform or the iron-supported nucleic acid delivery nanoplatform prepared by the above-described method in any of the following: (1) Prepare FesiRNPlip, which is used to deliver different target genes to different cell lines to achieve gene silencing function; the cells include but are not limited to U78MG, MDA-MB-231, CT-26, MCF7-GFP, SW480-GFP, and A375; the genes include but are not limited to CD146, Bcl2, GFP, PLK1, EGFR, and EPHA2.

[0029] (2) Prepare FepolyIClip for delivering the immune-activating nucleic acid molecule polyIC to immune cells to achieve cellular immune activation. Cells include, but are not limited to, RAW264.7, DC2.4, and THP-1; immune-activating nucleic acid molecules include, but are not limited to, polyIC, polyAU, and ISD.

[0030] (3) Preparation of FeDN[lip] for delivering exogenous DNA to immune cells to achieve cellular immune activation. Cells include, but are not limited to, RAW264.7, DC2.4, and THP-1; such DNA includes, but is not limited to, interferon-stimulated DNA (ISD) of various lengths.

[0031] Through the above technical solution, the present invention achieves the following beneficial effects: This invention develops an iron-supported nucleic acid delivery nanoplatform using a metal ion coordination strategy. The platform is simple to synthesize and can efficiently deliver various types of nucleic acid molecules into cells, where they are precisely released into the cytoplasm to exert their specific biological functions. Furthermore, this iron-supported nucleic acid delivery nanoplatform effectively protects nucleic acid molecules from external degradation, significantly improving their stability. This platform provides an innovative technical solution for nucleic acid drug delivery and possesses significant potential for clinical application. Attached Figure Description

[0032] Figure 1 The images show the TEM (A), TEM-EDS (B), and FT-IR (C) characterizations of FesiRNP. Figure 2 The results are for the stability test of FesiRNP. Figure 3 The TEM (A, B) and Zeta potential (C) characterization diagrams of FesiRNPlip are shown. Figure 4 The results of detecting FesiRNPlip's delivery of siRNA into cells to perform gene silencing function are shown in Figure A, where A is the fluorescence image result and B and C are the Western blot results. Figure 5 The results show the stability of the liposome / siRNA complex (A) and FesiRNPlip (B). Figure 6 The results of the detection of the activation of cellular immune effects by polyIClip delivery of FeNANPlip are shown, where A is the WB result and B is the CLSM result. Figure 7 Western blot (WB) results of DNA delivery to FeNANPlip to activate cellular immune responses. Detailed Implementation

[0033] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0034] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The invention is further described below with reference to specific embodiments, but is not intended to limit the scope of the invention.

[0035] In this invention, for ease of understanding of the terminology used, the siRNA designed for a specific target gene is labeled as "abbreviation of target gene name + siRNA", for example, the siRNA designed for the GFP gene is labeled as GFP siRNA.

[0036] In this invention, the nucleic acid delivery nanoplatform ultimately formed by nucleic acid-iron nanoparticles is labeled FeNANP, wherein the siRNA-loaded nanoparticles are named FesiRNP, the polyIC-loaded nanoparticles are named FepolyIC, and the DNA-loaded nanoparticles are named FeDNP; for more detailed labeling, the siRNA-loaded with a target gene can be directly labeled with the name of the target gene, such as the nanoparticles formed by GFP siRNA can be named FeGFP.

[0037] Example 1: Synthesis and Application of FesiRNPlip (1) Synthesis and characterization of FesiRNP Weigh out Tris-base and dissolve it in water. Adjust the pH to 8-10 with concentrated hydrochloric acid to prepare a 10 mM Tris-HCl alkaline buffer solution. Take 10 μL of a solution containing 56 μg Fe. 2+FeCl2 aqueous solution was added to 40 μg of siRNA, mixed well, and then 10 mM Tris-HCl buffer was added to 1 mL. After standing at room temperature for 5 min, the mixture was centrifuged at 12000 rpm for 10 min to obtain nucleic acid-iron nanoparticles. The particles encapsulated with siRNA were named FesiRNP. The obtained FesiRNPs were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR).

[0038] result: Figure 1 Transmission electron microscopy (TEM) image (A) shows that FesiRNP is a nanoparticle structure intermediate between spherical and lamellar shapes. TEM-EDS energy dispersive spectroscopy analysis (B) shows characteristic excitation energy peaks of Fe and P elements in FesiRNP, confirming that FesiRNP successfully encapsulates siRNA. FT-IR spectrum (C) shows that the antisymmetric stretching vibration peak of the phosphate group in siRNA is at 1110 cm⁻¹. -1 At that location, the peak in FesiRNP shifted to approximately 1077 cm⁻¹. -1 This indicates that during the formation of FesiRNP, the phosphate groups in the siRNA phosphate backbone participate in the coordination reaction with iron ions.

[0039] (2) Stability testing of FesiRNP in different environments Stability assay of FesiRNP in serum: FesiRNP nanoparticles were added to fetal bovine serum (FBS) and incubated at 37°C for 0, 6, 12, 24, 36, and 48 h, respectively. After centrifugation at 13000 rpm for 5 min, the FBS in the supernatant was removed. The nanoparticles were washed twice with anhydrous ethanol and then twice with enzyme-free water. PBS solution was added and the FesiRNP precipitate was co-incubated for 6 h, followed by centrifugation at 13000 rpm for 5 min. The supernatant was collected, and a portion of the supernatant was subjected to agarose gel electrophoresis to detect siRNA release. Simultaneously, an equal volume of naked siRNA solution was added to FBS and treated in the same way. The treated naked siRNA was then detected by agarose gel electrophoresis to assess the degradation of naked siRNA in FBS.

[0040] Stability assay of FesiRNP in RNase A: FesiRNP nanoparticles were added to RNase A and incubated at 37℃ for 0, 1, 6, 12, and 24 h, respectively. After incubation, the nanoparticles were centrifuged at 13000 rpm for 5 min to remove RNase A from the supernatant. The FesiRNP nanoparticles were washed twice with anhydrous ethanol, then twice with enzyme-free water. PBS was then added and co-incubated with the FesiRNP nanoparticles for 6 h to release siRNA. The nanoparticles were centrifuged at 13000 rpm for 5 min. An equal volume of naked siRNA solution was added to RNase A and incubated at 37℃ for 0, 15 min, 30 min, 1 h, and 2 h, respectively. After incubation, the reaction solution was subjected to agarose gel electrophoresis to detect the degradation of naked siRNA in RNase A.

[0041] result: Figure 2 Agarose gel electrophoresis results showed that naked siRNA gradually degraded in FBS over time, and was completely degraded after 48 h (A); while FesiRNP could still release siRNA after co-incubation with FBS for 48 h (B), indicating that FesiRNP can protect siRNA from serum degradation. Naked siRNA was completely degraded within 15 min of co-incubation with RNase A (C), while FesiRNP could still release siRNA after co-incubation with RNase A for 24 h (D), indicating that FesiRNP can protect siRNA from nuclease degradation.

[0042] (3) Synthesis and characterization of FesiRNPlip Liposome synthesis: Weigh 59 parts DOTAP, 10 parts DOPE, 30 parts cholesterol, and 1 part PEG-DSPE. Dissolve each component in 1 mL of chloroform and transfer the solution to a 100 mL round-bottom flask. Add another 30 mL of chloroform and evaporate the organic solvent using a vacuum rotary evaporator to form a thin lipid film. Add sterile water to the lipid film and hydrate overnight to form the liposome solution.

[0043] Synthesis of FesiRNPlip from liposome-modified nucleic acid-iron nanoparticles: Following a nucleic acid to primary liposome mass ratio of 1:5, nanoparticles containing 40 μg of nucleic acid were mixed with 200 μg of primary liposomes. The mixture was incubated at 30℃ for 120 min, centrifuged at 10000 rpm for 5 min, the supernatant was discarded, sterile water was added, and the mixture was thoroughly mixed by pipetting to obtain liposome-modified nucleic acid-iron nanoparticles, labeled FesiRNPlip.

[0044] Characterization of liposome-modified nucleic acid-iron nanoparticles: FesiRNP and FesiRNPlip were negatively stained and their morphology was observed by TEM; the surface potential of FesiRNP and FesiRNPlip was analyzed by Zeta potential analyzer.

[0045] result: Figure 3 TEM images showed that, compared to the dark black surface of FesiRNP(A) after negative staining, FesiRNPlip(B) exhibited brightness after negative staining due to liposome modification, indicating that the liposomes were successfully modified onto the surface of the nucleic acid-iron nanoparticles. Zeta potential results showed that siRNA carries a negative charge in aqueous solution due to the dissociation of H+ ions on its phosphate backbone, while FesiRNP as a whole exhibits a positive charge, with a Zeta potential of +16.4 mV. The Zeta potential of the liposome-modified FesiRNPlip increased to +74.8 mV, indicating that the liposomes successfully modified the surface of FesiRNP, forming FesiRNPlip.

[0046] (4) Verification of gene silencing function of FesiRNPlip delivered siRNA FeGFPlip was synthesized using GFP siRNA as the nucleic acid raw material and used to treat SW480-GFP cells that stably express GFP protein. Changes in GFP fluorescence intensity in both cell types were observed using CLSM fluorescence microscopy. FeEGFRlip was synthesized using EGFR siRNA as the nucleic acid raw material and used to treat U89MG cells. Changes in EGFR protein expression levels in the cells were detected using Western blotting (WB). FeCD146lip was synthesized using CD146 siRNA as the nucleic acid raw material and used to treat A375 cells. Changes in CD146 protein expression levels in the cells were detected using WB. FeNClip was synthesized using NC siRNA as the nucleic acid raw material as a negative control.

[0047] Negative control siRNA (NC siRNA) and siRNA sequences targeting the gene (5'→3'): NC siRNA: UUCUCCGAACGUGUCACGUdTdT; Cy5-modified NC siRNA: Cy5-UUCUCCGAACGUGUCACGUdTdT; CD146 siRNA:GAGCGAACUUGUAGUUGAAdTdT; GFP siRNA:GGCUACGUCCAGGAGCGCAdTdT; EGFR siRNA:GCAAGUGUAAGAAGUGCGAdTdT.

[0048] result: Figure 4 A fluorescence image results showed that, compared with the control group FeNClip, the fluorescence intensity of SW480-GFP cells in the FeGFPlip treatment group was significantly reduced, indicating that FeGFPlip successfully delivered GFP siRNA to both cell types and achieved gene silencing. Figure 4 B-WB results showed that, compared with the control group FeNClip, the expression level of EGFR protein in U89MG cells treated with FeEGFRlip was significantly downregulated, indicating that FeEGFRlip can deliver EGFR siRNA into cells to perform gene silencing. Figure 4 C-WB results showed that, compared with the control group FeNClip, the expression level of CD146 protein in the FeCD146lip treatment group was significantly downregulated, indicating that FeCD146lip can deliver CD146 siRNA into cells to perform gene silencing. These results systematically demonstrate that the iron-loaded nucleic acid delivery nanoplatform of this invention can deliver siRNA sequences of different types to different cell lines to perform gene silencing, showcasing the wide applicability of this iron-loaded nucleic acid delivery nanoplatform.

[0049] (5) Stability testing of FesiRNPlip FeCD146lip was synthesized using CD146 siRNA as the nucleic acid raw material. At the same time, a liposome / siRNA complex system was prepared. A375 cells were treated with FeCD146lip and the liposome / siRNA complex system at room temperature for 0, 1 and 2 days. The stability of the FesiRNA / siRNA complex system was evaluated by detecting the changes in CD146 protein expression level by Western blotting.

[0050] result: Figure 5 Western blot results showed that, compared with the control group, both the liposome / siRNA complex (A) and FesiRNPlip (B) delivery methods exhibited a gene silencing efficiency of approximately 65% ​​at day 0. However, after 1 day at room temperature, the gene silencing efficiency of the liposome / siRNA complex significantly decreased to 25%, and further decreased to 22% after 2 days. In contrast, FeCD146lip maintained a silencing efficiency of 56% after 2 days at room temperature. These data indicate that FeCD146lip has significantly better stability than the liposome / siRNA complex system.

[0051] Example 2: Synthesis and Application of FepolyIClip (1) Synthesis of FepolyIClip Take 10 μL containing 56 μg Fe 2+ FeCl2 aqueous solution was added to 40 μg of polyIC and mixed thoroughly. Then, 10 mM Tris-HCl buffer from "Example 1" was added to 1 mL. After standing at room temperature for 5 min, the mixture was centrifuged at 12000 rpm for 10 min to obtain nucleic acid-iron nanoparticles. The particles encapsulated with polyIC were named FepolyIC. The liposomes prepared in "Example 1" were taken, and at a nucleic acid to primary liposome mass ratio of 1:5, FepolyIC nanoparticles containing 40 μg of polyIC were mixed thoroughly with 200 μg of primary liposomes. The mixture was incubated at 80℃ for 5 min, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, sterile water was added, and the mixture was mixed thoroughly by pipetting to obtain liposome-modified nucleic acid-iron nanoparticles, labeled FepolyIClip.

[0052] (2) Verification of FeNANPlip delivery of polyIC to activate cellular immune function After co-incubating FepolyIClip with RAW264.7 cells for 0, 1, 2, 4, 6, 8, 10, 12, and 24 h, cells were collected. Western blotting was used to detect the dynamic changes in phosphorylation of key proteins in the innate immune signaling pathway (p-IRF3, p-IKKα / β, p-IκBα) and the expression levels of pattern recognition receptors (RIG-I, MDA-5). CLSM was used to observe the nuclear translocation of the NF-κBp65 subunit after 6 h of FepolyIClip treatment.

[0053] result: Figure 6 Western blot (WB) results (A) showed that, compared to 0 h, significant IRF3 phosphorylation signals were detected in the FepolyIClip treatment group at 2 h, 4 h, and 6 h. Significant phosphorylation signals were also detected in the IKKα / β complex at 2 h and 4 h. The phosphorylation process of the downstream signaling molecule IκBα showed a time-delayed characteristic, starting at 6 h and continuing until 24 h. The expression levels of the RIG-I and MDA-5 receptor proteins also increased with increasing treatment time. CLSM results (B) showed that the NF-κB p65 subunit underwent significant nuclear translocation after IκBα phosphorylation. These results demonstrate that the iron-loaded nucleic acid delivery nanoplatform of this invention can deliver polyIC into cells and successfully release it into the cytoplasm, activating related immune signaling pathways.

[0054] Example 3: Synthesis and Application of FeDNPlip (1) Synthesis of FeDNAlip: Take 10 μL containing 56 μg Fe 2+FeCl2 aqueous solution was added to 40 μg of interferon-stimulated DNA (ISD), mixed thoroughly, and then 10 mM Tris-HCl buffer from "Example 1" was added to 1 mL. After standing at room temperature for 5 min, the mixture was centrifuged at 12000 rpm for 10 min to obtain nucleic acid-iron nanoparticles. The particles encapsulated with DNA were named FeDNA. The liposomes prepared in "Example 1" were taken, and the DNA-iron nanoparticles containing 40 µg of DNA were mixed with 200 µg of primary liposomes at a mass ratio of 1:5 (nucleic acid to primary liposomes in the nucleic acid nanoparticles). After mixing thoroughly, the mixture was incubated at 60°C for 60 min, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, sterile water was added, and the mixture was mixed thoroughly by pipetting to obtain liposome-modified nucleic acid-iron nanoparticles, labeled FeDNPlip.

[0055] (2) Verification of DNA delivery by FeDNAPlip to activate cellular immune function FeDNAlip was co-incubated with RAW264.7 cells for 0, 1, 2, 4, 6, 8, 10, 12, and 24 h, and the expression levels of key proteins (STING, TBK1, and IRF3) in the cGAS-STING pathway were detected by Western blotting.

[0056] Figure 7 Western blot results showed that, compared to 0 h, after 1 hour of co-incubation of FeISDlip with RAW264.7 cells, STING protein began to phosphorylate, reaching its maximum phosphorylation level at 2 hours. Simultaneously, TBK1 and IRF3 proteins were also gradually activated and phosphorylated, with their phosphorylation levels peaking within 2 hours. These results demonstrate that the iron-loaded nucleic acid delivery nanoplatform of this invention can deliver DNA into cells and successfully release it into the cytoplasm, activating relevant immune signaling pathways.

[0057] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0059] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An iron-supported nucleic acid delivery nanoplatform, characterized in that, The invention comprises nucleic acid-iron nanoparticles and liposomes coated on the surface of the nanoparticles. The mass ratio of iron to nucleic acid in the nucleic acid-iron nanoparticles is 1:0.1 to 1:10, and the mass ratio of nucleic acid to liposomes is 1:2 to 1:

10. By mass parts, the raw materials of the liposomes include the following components: 20 to 80 parts of ionized lipid molecules, 10 to 40 parts of auxiliary lipid molecules, 10 to 50 parts of steroidal lipid molecules, and 1 to 20 parts of PEG-liposome molecules.

2. The iron-supported nucleic acid delivery nanoplatform according to claim 1, characterized in that, Iron is selected from divalent iron ions or trivalent iron ions.

3. The iron-supported nucleic acid delivery nanoplatform according to claim 1, characterized in that, Nucleic acid is selected from at least one of microRNA, siRNA, mRNA, polyIC, and DNA.

4. The iron-supported nucleic acid delivery nanoplatform according to claim 1, characterized in that, The ionized lipid molecule is selected from at least one of the following: 1,2-dioleoyl-3-dimethylamino-propane, 1,2-dioleoyloxy-3-(dimethylamino)propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-di(oleoyloxy)prop-1-amine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecen-1-yl-1,3-dioxolane-4-ethylamine, and methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester; The auxiliary lipid molecule is selected from at least one of the following: 1,2-distearate-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), oleoylphosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylethanolamine; The steroidal lipid molecule is selected from at least one of the following: hydroxy cholesterol, cholesterol, dehydrocholesterol, alfalfa sterol, dihydrocholesterol, oleosterol, cholesterol, coccosterol, epicholesterol, ergosterol; The PEG-lipid molecule is selected from at least one of the following: 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoyl, PEG-dipalmitoyl phosphatidylethanolamine.

5. The method for preparing the iron-supported nucleic acid delivery nanoplatform according to any one of claims 1 to 4, characterized in that, The process includes steps for preparing nucleic acid-iron nanoparticles, liposomes, and liposome-modified nucleic acid-iron nanoparticles, wherein: The preparation method of nucleic acid-iron nanoparticles includes: mixing soluble iron salt and nucleic acid in a certain proportion to form a complex, then adding the complex to a buffer solution, allowing it to stand, centrifuging, and obtaining nucleic acid-iron nanoparticles; The preparation method of liposomes includes: dissolving ionizable lipid molecules, auxiliary lipid molecules, steroidal lipid molecules and PEG-lipid molecules in an organic solvent in a certain proportion, then removing the solvent, adding ultrapure water and emulsifying to obtain a liposome solution; The method for modifying nucleic acid-iron nanoparticles with liposomes includes: mixing an aqueous solution of nucleic acid-iron nanoparticles with a liposome solution, heating, and centrifuging to obtain liposome-modified nucleic acid-iron nanoparticles.

6. The preparation method according to claim 5, characterized in that, The soluble iron salt is selected from at least one of ferric chloride, ferrous chloride, ferrous sulfate, ferrous nitrate, ferric sulfate, and ferric nitrate.

7. The preparation method according to claim 5, characterized in that, The buffer solution is selected from Tris-HCl, PBS or Bis-Tris buffer, with a pH of 8-10.

8. The preparation method according to claim 5, characterized in that, The settling time is 5 to 120 minutes.

9. The preparation method according to claim 5, characterized in that, The heating temperature is 30℃ ~ 80℃, and the time is 5~60 min.

10. The application of the iron-supported nucleic acid delivery nanoplatform according to any one of claims 1 to 4 or the iron-supported nucleic acid delivery nanoplatform prepared by the preparation method according to any one of claims 5 to 9 in any of the following: (1) Prepare nucleic acids for delivering siRNA to achieve gene silencing function; (2) Preparation of nucleic acids for delivery of polyIC to achieve immune activation function; (3) Prepare nucleic acids for delivering exogenous DNA to achieve immune activation.

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