Method for preparing nucleic acid medicine based on biological ions and application of nucleic acid medicine

Abstract

The invention belongs to the field of biological medicines, relates to delivery and stability of nucleic acid medicines, and particularly relates to a method for preparing a nucleic acid medicine based on biological ions and application of the nucleic acid medicine. The invention provides a zwitterionic biological material which is a cationic polyelectrolyte-nucleic acid micelle formed by inorganic salt and nucleic acid, has biocompatibility, degradability and mild preparation conditions, and can deliver nucleic acid into a living body. Meanwhile, as one of important plant nutrient sources, the plant source has a long coexistence history in the fields of human beings and ecological environments and complements each other. The invention further discusses the structural characteristics of the gene and provides a scheme for multi-scene application of plant genetic transformation, RNA delivery of human medicine mRNA, siRNA and the like, RNA biopesticide on agricultural pest control and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the delivery and stability of nucleic acid drugs. Background Technology

[0002] Plant genetic transformation is a crucial component of agricultural engineering, small molecule synthesis, and bioenergy. Traditional plant DNA delivery tools limit the plant species that can be transformed or exhibit low transformation efficiency and tissue damage due to the use of high external forces. There is an urgent need for novel delivery materials that can deliver genes and proteins into intact cells without external force or assistance. Simultaneously, animal genetic transformation requires the addition of more novel delivery methods to address delivery efficiency and cytotoxicity. Furthermore, the rapid development of mRNA vaccines and RNAi drugs in recent years has further demonstrated the application potential of RNA in organisms. The core issues determining this potential are how to effectively deliver RNA to the lesion site in the organism and how to stably expose RNA molecules to the environment. The emergence of various nucleic acid delivery materials not only makes the production and application of RNA possible but also provides a basis for the feasibility of RNA drug development. Of course, their application faces many challenges related to stability and efficacy. To address these challenges, researchers have developed various delivery materials such as liposomes, virus-like particles, composite nanoparticles, and bio-clays. However, these delivery materials still suffer from problems such as high cost, difficult preparation, and environmental unfriendliness.

[0003] Inorganic ions have a long history of coexistence and mutual support in the human and ecological environments. Examples include macroelements such as nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca), and microelements such as iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), boron (B), molybdenum (Mo), chlorine (Cl), sodium (Na), and nickel (Ni). NO3 - H2PO4 is the main form of nitrogen in plants, used for the synthesis of proteins / chlorophyll. 4- SO4 is the only form of phosphorus in plants, used to form ATP / nucleic acids / phospholipids; 2- It is the main form of sulfonium in plants, used in the synthesis of sulfur-containing amino acids / coenzymes; Cl - They participate in osmotic regulation / photosynthesis and maintain cell turgor pressure. Studies have shown that some inorganic ions also play important roles in genetic information transmission processes such as DNA damage repair and RNA synthesis, suggesting that they may have functions that have not yet been fully explored in the regulation of nucleic acid metabolism and stability.

[0004] Based on this, this study delves into the interaction between inorganic ions and nucleic acids, aiming to explore their potential as a novel, green, and efficient nucleic acid delivery and stabilization system, in order to provide innovative material basis and technical pathways for plant and animal genetic transformation, RNA drug delivery, and agricultural biotechnology. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing nucleic acid drugs based on biological ions and the application of nucleic acid drugs.

[0006] The technical solution of this invention is implemented as follows:

[0007] On one hand, this invention discloses the application of bio-ions in the preparation of nucleic acid drugs, wherein the bio-ions (BIs) are selected from one or more of sodium nitrite (NaNO2), hydrogen chloride (NaCl), sodium sulfate (Na2SO4), sodium selenite (Na2SeO3), sodium phosphate (Na3PO4), ammonium nitrate (NH4NO3), aluminum trichloride (AlCl3), ammonium persulfate ((NH4)2S2O8), ammonium dihydrogen phosphate (NH4H2PO4), ammonium chloride (NH4Cl), potassium chloride (KCl), manganese chloride (MnCl2), manganese sulfate (MnSO4), cobalt chloride (CoCl2), copper sulfate (CuSO4), potassium iodide (KI), sodium silicate (Na2SiO3), and magnesium chloride (MgCl2).

[0008] The nucleic acid drug is a nucleic acid micelle formed by biological ions and nucleic acids. When the above-mentioned biological ion composition is combined with different nucleic acids (NAs) such as DNA, dsRNA, ssDNA, and ssRNA, it has an enzymatic protective effect on this type of nucleic acid, which is beneficial for nucleic acid delivery.

[0009] Secondly, a method for preparing a nucleic acid drug is provided, comprising the following steps: mixing an aqueous nucleic acid solution with a biological ion solution to obtain a BIs@dsRNA complex solution, which is the nucleic acid drug. The nucleic acid is selected from any one of DNA, dsRNA, ssDNA, and ssRNA; the water is pure water without nuclease. The biological ion is selected from one or more of sodium nitrite (NaNO2), hydrogen chloride (NaCl), sodium sulfate (Na2SO4), sodium selenite (Na2SeO3), sodium phosphate (Na3PO4), ammonium nitrate (NH4NO3), aluminum trichloride (AlCl3), ammonium persulfate ((NH4)2S2O8), ammonium dihydrogen phosphate (NH4H2PO4), ammonium chloride (NH4Cl), potassium chloride (KCl), manganese chloride (MnCl2), manganese sulfate (MnSO4), cobalt chloride (CoCl2), copper sulfate (CuSO4), potassium iodide (KI), sodium silicate (Na2SiO3), and magnesium chloride (MgCl2).

[0010] Furthermore, the molar ratio of the nucleic acid to the biological ion is 1:10. -6 -1:1.

[0011] The molar ratio of any one of potassium iodide, aluminum trichloride, and sodium phosphate to the nucleic acid is 1:10. -6 The molar ratio of any one of the following substances—sodium nitrite, sodium chloride, sodium sulfate, sodium selenite, ammonium nitrate, ammonium persulfate, ammonium dihydrogen phosphate, ammonium chloride, potassium chloride, manganese chloride, and copper sulfate—to nucleic acid is 1:10. -6 -1 : 10 -5 The molar ratio of sodium silicate to nucleic acid can be 1:10. -4 The molar ratio of manganese sulfate, magnesium chloride, and nucleic acid can be 1:10. -6 -1 : 10 -4 .

[0012] Thirdly, this application provides a nucleic acid drug prepared using the method described above, wherein the nucleic acid drug is a cationic polyelectrolyte formed by bio-ions and nucleic acids, namely nucleic acid micelles. Furthermore, the bio-ions significantly enhance the stability of nucleic acids at high concentrations of RNase A.

[0013] The bio-ion content of the aforementioned nucleic acid drugs ranges from 0.1% to 39% by mass and the nucleic acid content ranges from 0.1% to 5% by mass and volume.

[0014] When the bio-ion is one or more of sodium selenite, sodium phosphate, magnesium chloride, potassium chloride, sodium chloride, and sodium sulfate, the particle size of the nucleic acid drug is 60-114.4 nm.

[0015] When the bio-ion is one or more of aluminum chloride, ammonium dihydrogen phosphate, ammonium chloride, and sodium silicate, the particle size of the nucleic acid drug is 542-1057 nm.

[0016] For ease of description, the inventors refer to bio-ions as BioIon (BI).

[0017] BI1 is NaNO2, BI2 is NaCl, BI3 is Na2SO4, BI5 is Na2SeO3, BI7 is Na3PO4, BI8 is NH4NO3, BI9 is ​​AlCl3, BI10 is (NH4)2S2O8, BI11 is NH4H2PO4, BI12 is NH4Cl, BI14 is KCl, BI15 is MnCl2, BI16 is MnSO4, BI19 is CoCl2, BI21 is CuSO4, BI24 is KI, BI25 is Na2SiO3, and BI27 is MgCl2.

[0018] The nucleic acid drug is a mixture of one or more of 18 BioIons (BI) with one type of nucleic acid. BioIon (BI) is an innovative bio-ion in this invention, which is a type of zwitterionic biomaterial.

[0019] In some embodiments, the complex contains BIs in the form of a mass-volume percentage as shown in Table 1, such as about 0.1 w / v%, about 0.5 w / v%, 1 w / v%, about 5 w / v%, about 10 w / v%, about 11 w / v%, about 12 w / v%, about 13 w / v%, about 14 w / v%, about 15 w / v%, about 20 w / v%, about 25 w / v%, about 30 w / v%, about 35 w / v%, about 39 w / v%, etc.

[0020] In some embodiments, the complex contains NA in the mass-volume percentages shown in Table 1, such as about 0.1 w / v%, about 0.2 w / v%, about 0.3 w / v%, about 0.4 w / v%, about 0.5 w / v%, 1 w / v%, about 2 w / v%, about 3 w / v%, about 5 w / v%, about 5 w / v%, etc.

[0021] In some instances, complex A contains 1 w / v % BI5, 1 w / v % BI7, and 1 w / v dsRNA.

[0022] In some instances, complex B contains 1 w / v % BI9, 1 w / v % BI11, 1 w / v % BI12, 1 w / v % BI25 and 1 w / v dsRNA.

[0023] In some instances, complex C contains 1 w / v % BI27, 1 w / v % BI14, and 1 w / v dsRNA.

[0024] Thirdly, we request protection for the use of the aforementioned nucleic acid drugs in the delivery of nucleic acid drugs.

[0025] The present invention has the following beneficial effects:

[0026] 1. This invention provides a class of zwitterionic biomaterials, which are cationic polyelectrolytes-nucleic acid micelles formed from inorganic salts and nucleic acids. These micelles possess biocompatibility, degradability, and mild preparation conditions, enabling the delivery of nucleic acids into organisms. Furthermore, as an important plant nutrient source, they have a long history of coexistence and mutual support in the human and ecological environments. The invention further explores their structural characteristics and provides solutions for applications in various scenarios, including plant genetic transformation, delivery of human drug mRNA and siRNA, and RNA-based biopesticides for agricultural pests and diseases.

[0027] 2. This invention proposes an innovative biological ion delivery nucleic acid system, providing a new approach for the research and development of novel RNA biopesticides. Simultaneously, this technology also has significant application potential in the delivery of other drugs, such as DNA, and can serve as a reference for a library of materials for the functional modulation or functional addition of new nanomaterials. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This outlines the screening process for bio-ionic materials with nucleic acid stability (BIs).

[0030] Figure 2 This is a schematic diagram of an agarose gel (AGE) assay for screening bio-ionic materials with nucleic acid stability (BIs). There are 18 candidate BIs, listed in ascending order of their serial numbers: 1-sodium nitrite (NaNO2), 2-hydrogen chloride (NaCl), 3-sodium sulfate (Na2SO4), 5-sodium selenite (Na2SeO3), 7-sodium phosphate (Na3PO4), 8-ammonium nitrate (NH4NO3), 9-aluminum trichloride (AlCl3), 10-ammonium persulfate ((NH4)2S2O8), 11-ammonium dihydrogen phosphate (NH4H2PO4), 12-ammonium chloride (NH4Cl), 14-potassium chloride (KCl), 15-manganese chloride (MnCl2), 16-manganese sulfate (MnSO4), 19-cobalt chloride (CoCl2), 21-copper sulfate (CuSO4), 24-potassium iodide (KI), 25-sodium silicate (Na2SiO3), and 27-magnesium chloride (MgCl2).

[0031] Figure 3 A schematic diagram of the mechanism by which BIs stabilize NAs.

[0032] Figure 4 The DLS dynamic light diffraction pattern of the BIs@NAs complex is shown for comparison.

[0033] Figure 5 The PDI and zeta potential diagrams of the BIs@NAs complex are used as a reference.

[0034] Figure 6 To compare the effect of the BIs@NAs complex on RNase A activity.

[0035] Figure 7This serves as a control for the cellular uptake effect of the BIs@NAs complex. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0038] Materials and Methods:

[0039] (1) DNA preparation and purification

[0040] DNA amplification was performed using PCR with 2× Hieff® PCR Master Mix (With Dye) (Yeasen) reagent. Samples with a single amplified band can be directly purified by affinity chromatography, while samples with multiple amplified bands are recovered by gel chromatography. Store at 4°C until use.

[0041] (2) Synthesis and purification of dsRNA

[0042] dsRNA was synthesized using the T7 RNA Transcription Kit Plus (Shanghai Plant Science Biotechnology Co., Ltd., SJ001V2), and the experimental procedures were performed according to the instructions.

[0043] The T7 RNA Polymerase Mix and NTP Mix should be thawed on ice, while the remaining components should be thawed at room temperature.

[0044] Prepare the reaction mixture as follows: 2 µL of T7 RNA Transcription Kit Plus, 2 µL of 10×T7 transcription buffer, 8 µL of NTP mixture, 1 µg of DNA template, and enzyme-free deionized water to a final volume of 20 µL.

[0045] Gently mix the reaction mixture thoroughly and incubate at 37°C for 2-4 hours to allow the reaction to proceed fully. For samples shorter than 400 nt, the reaction time can be appropriately extended, but should not exceed 16 hours, pending purification of the dsRNA sample.

[0046] Purification was performed using the MagBeads RNA Purification Kit (Shanghai Plant Science Biotechnology Co., Ltd, PU002), by vortexing or inverting the binding magnetic beads several times to ensure thorough mixing.

[0047] ① Add an equal volume of binding magnetic beads to the dsRNA sample to be purified, pipette until fully mixed, and incubate at room temperature for 8 min;

[0048] ② Place the sample on the magnetic rack and magnetically attract it for 5 minutes. After the magnetic beads have completely gathered, remove the supernatant (the centrifuge tube is still placed on the magnetic rack).

[0049] ③ Rinse the magnetic beads with Wash Buffer, incubate at room temperature for 30 seconds, and carefully aspirate the supernatant;

[0050] ④ Repeat the previous step, rinsing twice in total;

[0051] ⑤ Keep the sample on the magnetic rack and open the lid to dry the magnetic beads at room temperature for 3-5 minutes to allow any residual ethanol or other liquids to evaporate completely.

[0052] ⑥ Remove the sample from the magnetic rack, add 50µL of enzyme-free deionized water, gently mix with a pipette, and incubate at room temperature for 3 min;

[0053] ⑦ Place the sample on a magnetic rack and wait for the magnetic beads to completely aggregate (about 2 minutes). Carefully aspirate the supernatant into a new enzyme-free centrifuge tube. This solution is the purified RNA sample. The purified dsRNA can be directly used in the next step of the experiment or stored at -20°C.

[0054] (3) Synthesis and purification of ssRNA

[0055] Take a small number of 3rd-5th instar cotton bollworm larvae, freeze them in liquid nitrogen, grind them, and then use the TRIeasy™ Total RNA Extraction Reagent (Yeasen) to extract total RNA from the cotton bollworm larvae according to the manufacturer's instructions to obtain ssRNA.

[0056] (4) The original solutions of each component of the BIs@NAs series solutions used in this invention: see Table 1. Dissolve the analytical grade product in enzyme-free deionized water and stir until completely dissolved.

[0057] Table 1. BIs@NAs Component Stock Solution Formulation

[0058]

[0059] (5) Lyophilized NAs to be compounded: NAs with a concentration of 10 mg / ml were lyophilized in a freeze dryer.

[0060] (6) During the experiment, the nucleic acid (NA) stock solution was diluted to the specified concentration and then filtered and sterilized using a 0.22 μm filter membrane. The BI stock solution was diluted to the specified concentration. Then, one or more BIs were mixed with the nucleic acid at a volume ratio of 1:1 to obtain the desired BIs@NAs composite solution.

[0061] Example

[0062] The preparation method of the nucleic acid drug (BIs@dsRNA) in this embodiment includes the following steps:

[0063] (1) Dissolve 1 mg of dsRNA lyophilized powder in 10 mL of nuclease-free water. Add the dsRNA solution to a 30 kDa ultrafiltration centrifuge tube, centrifuge at high speed for 2-5 minutes, and discard the waste liquid. Add an appropriate amount of fresh nuclease-free water to the upper chamber, mix well, and then centrifuge to concentrate to the target volume. Repeat this step 2-3 times to remove other inorganic salts remaining during dsRNA synthesis and purification.

[0064] (2) Adjust the concentration of dsRNA solution to 1 mg / ml with deionized enzyme-free water, and then mix it with BIs;

[0065] (3) The BI stock solution (see Table 1) was serially diluted 5-6 times to obtain stock solution 2 M (or 0.2 M), diluents 0.2 M, 0.02 M, 2 mM, 0.2 mM, 0.02 mM, and 2 μM BIs. The molar ratio of BIs to 1 mg / ml (2 μM) dsRNA was 1:1. -6 1:10 -5 1:10 -4 1:10 -3 1:10 -2 1:10 -1 A 1:1 solution of BIs@dsRNA complex.

[0066] Implementation effect

[0067] 1. Enzymatic hydrolysis and screening yield stable BIs.

[0068] We prepared inorganic salt ion libraries of BIs and combined them with one type of nucleic acid dsRNA. BIs were then screened using RNase A enzymatic digestion. Starting with a high-concentration inorganic salt ion library (I0), different BIs were mixed with control dsRNA to obtain BIs@dsRNA complexes. These complexes were then incubated with RNase A at room temperature for enzymatic digestion experiments, and nucleic acid band degradation was verified using agarose gel electrophoresis (15% AGE).

[0069] The results are as follows Figure 1 As shown, after 30 minutes of enzymatic digestion, almost no full-length naked dsRNA bands were detected, but some BIs@dsRNAs significantly retained full-length dsRNA bands. Subsequently, BIs from I0 that retained full-length bands were selected and their concentration reduced by 10-fold to form a new BIs library (I1). The above operation was repeated with the newly obtained BIs library (I1) in a continuous cycle until all BIs@dsRNA major bands completely disappeared. Figure 1 ).

[0070] The agarose gel electrophoresis (AGE) results showed that the molar ratio of BIs to dsRNA was 1:10. -6 -1:10 -4 The schematic diagram is as follows Figure 2 The results showed that most BIs@dsRNAs were at a high molar ratio of 1:10. -6 Compared to the control naked dsRNA, there was a significant retention of the main band; a few BIs, such as BI25, showed retention at a higher molar ratio of 1:10. -5 This will promote the degradation of the dsRNA mainband, while at a low molar ratio of 1:10... -4 There was a clear retention of the main band. We speculate that different BIs generally enhance the RNase A digestion stability of dsRNA. Meanwhile, as the molar ratio decreased, the retention of the main band of different BIs@dsRNA after digestion varied; for example, BI25 / BI27 showed better retention at lower BIs@dsRNA molar ratios (1:10). -4 At low concentrations, the main band of dsRNA showed significant retention compared to naked dsRNA, while other biochemical inhibitors (BIs) did not exhibit obvious dsRNA stabilization. Therefore, at low concentrations, different BIs showed varying stabilizing effects on dsRNA. We also found that the introduction of different BIs resulted in different electrophoretic band patterns of dsRNA, such as well as differences in gel well blockage, main band migration distance, and the location and intensity of degradation bands. Therefore, we speculate that the structure-activity relationship between different BIs and dsRNA may differ. Figure 2 ).

[0071] According to the research by Mo et al., Cd2+ Zn 2+ These compounds possess the ability to bind to small DNA molecules, and this binding is concentration-dependent. Combined with the enzymatic digestion experiments described above, our results also show that the protective effect of these three BIs on dsRNA is concentration-dependent. Therefore, we hypothesize that BIs enhance the structural rigidity of dsRNA through electrostatic interactions. By analogy, we can infer that BIs can generate electrostatic interactions with multiple types of nucleic acid backbones, including DNA, ssDNA, and ssRNA, thereby improving the stability of various nucleic acid types. When the molar ratio of BIs to NAs is greater than or equal to a critical value, the counterions acting as electrostatic shielding around the nucleic acid are saturated. This critical molar ratio is defined as the "critical load" of BIs. Within a range not less than the critical load, BIs exhibit a strong ability to improve nucleic acid stability. Figure 3 We obtained a critical load of 1:10 for improving nucleic acid stability at BI7 / 24. -6 The molar ratio of BI1 / 2 / 3 / 4 / 5 / 6 / 10 / 11 / 12 / 14 / 15 for improving nucleic acid stability is 1:10. -5 The molar ratio for improving nucleic acid stability in BI16 / 17 / 18 / 25 / 27 is 1:10. -4 The "critical molar ratio" of BI8 / 9 is difficult to determine and needs to be verified through further experiments.

[0072] In summary, we obtained BIs with nucleic acid stability, as well as a "critical load" that can improve nucleic acid stability.

[0073] Application Example 1

[0074] Next, based on the BIs we selected, we arbitrarily choose the type and quantity, and according to their "critical loading" molar ratio, we combine them with one of the nucleic acid types, NAs:

[0075] Complex A contains 1 w / v % BI5, 1 w / v % BI7 and 1 w / v dsRNA.

[0076] Complex B contains 1 w / v % BI9, 1 w / v % BI11, 1 w / v % BI12, 1 w / v % BI25 and 1 w / v dsRNA.

[0077] Complex C contains 1 w / v % BI27, 1 w / v % BI14 and 1 w / v dsRNA.

[0078] The control group contained dsRNA 1 w / v %.

[0079] 1. Measurement of particle size and zeta potential of the prepared BIs@dsRNA

[0080] The particle size and zeta potential of the BIs@NAs complex (containing NAs at a concentration of 0.5 g / L) were tested at 25 °C using a Zetasizer Nano ZS (Malvin Instruments, USA) with 3-5 replicates.

[0081] The obtained complexes A, B, and C were used as a control group, and the control group, complex A, complex B, and complex C were subjected to DLS dynamic light diffraction experiments.

[0082] The results are as follows Figure 4 As shown, the experimental results indicate that the particle size of complex A is 87.72 ± 26.69 nm, which is 27.14 nm smaller than that of the control group (114.86 ± 22.48 nm); the particle size of complex B is 799.55 ± 257.51 nm, which is 600% larger than that of the control group; and the particle size of complex C is 84.46 ± 24.34 nm, which is 30.40 nm smaller than that of the control group. This demonstrates that different BIs bind to NAs at different particle sizes, allowing for the selection of BIs@NAs with different particle sizes based on different application scenarios. Furthermore, compared to the control group, the PDI of complexes A, B, and C are all reduced, indicating that these three BIs@NAs complexes exhibit better dispersibility than NAs. Figure 5 (Left image).

[0083] At the same time, zeta potential such as Figure 5 The right figure shows that, compared to the control group (-0.96 ± 0.32 mV), the charges of complexes A, B, and C are all increased, at -0.46 ± 0.22 mV, -0.35 ± 0.59 mV, and 0.32 ± 0.54 mV, respectively. This reduces the electrostatic repulsion between the dsRNA complex and the negatively charged biomembrane, facilitating endosome escape. Therefore, BIs@NAs can be selected with different particle sizes and charges depending on the specific application scenario. This zwitterionic biomaterial can achieve better transfection efficiency and endosome escape strategies, and can be used in plant and animal genetic transformation, human drug mRNA delivery, and RNA biopesticides for agricultural pests and diseases.

[0084] 2. RNase A activity assay was performed on the prepared BIs@NAs complex.

[0085] We performed RNase A enzyme activity tests on the control group, complex A, complex B, and complex C, and the results are as follows: Figure 6 As shown, the enzyme activity of complex A was significantly higher than that of the control, while the enzyme activity of complex B and complex C was significantly lower than that of the control. This indicates that different combinations of enzyme bioassays (BIs) and NAs produce different activity effects on enzymes. Depending on the specific application requirements, different BIs can be selected and combined with NAs to form complexes, ultimately stabilizing nucleic acid structures, promoting nucleic acid internalization, and enhancing the efficacy of nucleic acid mechanisms in vivo.

[0086] 3. Cell culture, dsRNA incubation, and microscopic observation.

[0087] The silkworm ovary cell line (BmN) was cultured at 27°C in TC-100 insect medium containing 10% FBS. Following the manufacturer's recommendations, dsRNA was labeled with Cy3 using the Silencer siRNA labeling kit (Life Technologies), and then different complexes were prepared according to the formulation. These complexes were stored at room temperature, protected from light, and aseptically until used for cell incubation.

[0088] To determine the intracellular uptake of BIs@NAs, 24-well BmN cells were cultured to 80% density, the old culture medium was discarded, and the cells were washed twice with 1×PBS, followed by 200 μL of freshly prepared culture medium. The control group consisted of 4 μL of Cy3-NAs containing 20 ng / μL dsRNA plus 25 μL of freshly prepared culture medium. The experimental group consisted of 4 μL of Cy3-labeled complexes A, B, and C (each containing 20 ng / μL dsRNA) plus 25 μL of freshly prepared culture medium. Incubation time was 24 h. Fluorescence intensity was then captured using a Leica stereomicroscope, and images were processed using ImageJ. Results are shown below. Figure 7 The results showed that the intracellular uptake of nucleic acids by the three complexes in the BIs@NAs delivery system was much higher than that of the control group naked NAs, indicating that BIs@NAs has a good cell delivery effect.

[0089] In summary, it is hypothesized that the addition of BIs reduces the electronegativity of nucleic acids, thereby decreasing electrostatic repulsion with the negatively charged cell membrane and promoting nucleic acid uptake by the cell membrane. This invention demonstrates that the BIs@NAs delivery system enhances nucleic acid stability and promotes cell delivery, suggesting that its mechanism involves promoting nucleic acid internalization and possessing the potential for endosome escape. This patent provides an important opportunity to enrich and improve nucleic acid delivery systems. Furthermore, BIs can also serve as a reference for a library of new nanomaterials for functional modulation or functional enhancement.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of biological ions in the preparation of nucleic acid drugs, characterized by: The bio-ion is selected from one or more of sodium nitrite, sodium chloride, sodium sulfate, sodium selenite, sodium phosphate, ammonium nitrate, aluminum trichloride, ammonium persulfate, ammonium dihydrogen phosphate, ammonium chloride, potassium chloride, manganese chloride, manganese sulfate, copper sulfate, cobalt chloride, potassium iodide, sodium silicate, and magnesium chloride.

2. The application of the bio-ion in the preparation of nucleic acid drugs according to claim 1, characterized in that: The nucleic acid drug is a nucleic acid micelle formed by biological ions and nucleic acids.

3. The application of the bio-ion in the preparation of nucleic acid drugs according to claim 2, characterized in that: The nucleic acid is selected from any one of DNA, dsRNA, ssDNA, and ssRNA.

4. A method for preparing a nucleic acid drug, characterized in that, The steps are as follows: Mix the nucleic acid aqueous solution with the biological ion solution to obtain the BIs@dsRNA complex solution, which is the nucleic acid drug.

5. The method for preparing the nucleic acid drug according to claim 4, characterized in that: The nucleic acid is selected from any one of DNA, dsRNA, ssDNA, and ssRNA; the water is pure water without nuclease.

6. The method for preparing the nucleic acid drug according to claim 4, characterized in that: The bio-ion is selected from one or more of sodium nitrite, sodium chloride, sodium sulfate, sodium selenite, sodium phosphate, ammonium nitrate, aluminum trichloride, ammonium persulfate, ammonium dihydrogen phosphate, ammonium chloride, potassium chloride, manganese chloride, manganese sulfate, copper sulfate, cobalt chloride, potassium iodide, sodium silicate, and magnesium chloride; the molar ratio of the bio-ion to nucleic acid is in the range of 1:

10. -6 -1:10 -3 .

7. The method for preparing the nucleic acid drug according to claim 6, characterized in that: The molar ratio of any one of potassium iodide, aluminum trichloride, and sodium phosphate to the nucleic acid is 1:

10. -6 The molar ratio of any one of the following—sodium nitrite, sodium chloride, sodium sulfate, sodium selenite, ammonium nitrate, ammonium persulfate, ammonium dihydrogen phosphate, ammonium chloride, potassium chloride, manganese chloride, and copper sulfate—to nucleic acid is 1:

10. -6 -1 : 10 -5 The molar ratio of sodium silicate to nucleic acid can be 1:

10. -4 The molar ratio of manganese sulfate, magnesium chloride, and nucleic acid is 1:

10. -6 -1 : 10 -4 .

8. A nucleic acid drug prepared using the method according to any one of claims 4-7, characterized in that: The nucleic acid drug is a cationic polyelectrolyte formed by biological ions and nucleic acids, namely nucleic acid micelles.

9. The nucleic acid drug according to claim 8, characterized in that: The bio-ion content of the nucleic acid drug is 0.1%-48% by mass and volume; the nucleic acid content is 0.1%-10% by mass and volume. When the bio-ion is one or more of sodium selenite, sodium phosphate, magnesium chloride, potassium chloride, sodium chloride, and sodium sulfate, the particle size of the nucleic acid drug is 60-114.4 nm. When the bio-ion is one or more of aluminum chloride, ammonium hydrogen phosphate, ammonium chloride, and sodium silicate, the particle size of the nucleic acid drug is 542-1057 nm.

10. The use of the nucleic acid drug of claim 8 in the delivery of nucleic acid drugs.

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