Magnetic response nucleic acid drug delivery system and preparation method and application thereof

Abstract

The invention provides a magnetic response nucleic acid drug delivery system and a preparation method and application thereof. Nucleic acid is loaded on the nucleic acid drug delivery system, and then interacts with magnetic nanoparticles of which the surfaces are covalently modified with oligodeoxynucleotide sequences to form the magnetic response nucleic acid drug delivery system. After an external magnetic field is applied to the nucleic acid-loaded magnetic response nucleic acid drug delivery system, cell surface deposition and cell uptake of the nucleic acid drug delivery system can be accelerated under the action of magnetic attraction, so that the aim of improving the nucleic acid drug delivery efficiency and expression level is fulfilled.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a magnetic-responsive nucleic acid drug delivery system, a preparation method thereof, and an application thereof. This delivery system can accelerate the cellular uptake of nucleic acid drugs, thereby improving the delivery efficiency and expression level of nucleic acid drugs. Background Art

[0002] Nucleic acid drugs are a class of negatively charged biological macromolecules that are not easily permeable through biological membranes and are extremely prone to degradation. Therefore, they usually need to rely on delivery systems such as lipid nanoparticles (LNPs) to function. Nucleic acid molecules loaded in the delivery system generally take several hours to be fully taken up by cells, and the unabsorbed delivery systems are gradually degraded. Currently, there is no effective technology for enhancing the uptake rate of nucleic acid molecules loaded in the delivery system. Summary of the Invention

[0003] An object of the present invention is to provide a magnetic-responsive nucleic acid drug delivery system in view of the deficiencies of the prior art, which shortens the nucleic acid uptake process from several hours to dozens of minutes, and ultimately improves the delivery efficiency and expression level of nucleic acids in cells and mice.

[0004] The magnetic-responsive nucleic acid drug delivery system provided by the present invention is composed of a nucleic acid, a nucleic acid drug delivery system, and magnetic nanoparticles covalently modified with oligodeoxynucleotides on the surface;

[0005] Wherein, the nucleic acid is loaded on the nucleic acid drug delivery system to obtain a nucleic acid-loaded nucleic acid drug delivery system,

[0006] The nucleic acid-loaded nucleic acid drug delivery system interacts with the magnetic nanoparticles covalently modified with oligodeoxynucleotides on the surface;

[0007] The nucleic acid drug delivery system can specifically be a lipid nanoparticle;

[0008] The nucleic acid drug delivery system (lipid nanoparticle) can specifically be made of four components or two components;

[0009] The lipid nanoparticle made of four components can specifically be made of components including an ionizable lipid compound, a phospholipid, a structural lipid, and a polyethylene glycol lipid;

[0010] Among them, the ionizable lipid compound is selected from at least one of: quinoline-based ionizable lipid compounds (such as N1,N1,N6-tris((Z)-9-octadecenyl)-N6-(6-((7-(trifluoromethyl)quinolin-4-yl)amino)hexyl)hexane-1,6-diamine), benzene-1,3,5-tricarboxamide-derived ionizable lipid compounds (such as N1,N3,N5-tris(4-dodecylhexadecyl)benzene-1,3,5-tricarboxamide), quaternary ammonium salt lipid compounds (such as (9Z,9'Z,9”Z)-N,N',N”- [benzene-1,3,5-triyltris(methylene)] tris(dimethyl 9-octadecene-1-ammonium) iodide);

[0011] The phospholipids are selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, hydrogenated soy phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, 1-stearoyl-2-oleoyl phosphatidylcholine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, dioleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, sphingomyelin, ceramide, cephalin, cerebroside or diacylglycerol; specifically, it can be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE;

[0012] The structural lipids are selected from any at least one of cholesterol, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl] cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, coprosterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol; specifically, it can be cholesterol;

[0013] The polyethylene glycol lipids are selected from any at least one of PEG-modified phosphatidic acid, PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified dialkylglycerol, and PEG-modified diacylglycerol, and specifically may be 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, distearoyl-rac-glycerol-polyethylene glycol 2000, N-(carbonyloxy-methoxypolyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyloxy-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-diacylglycerol amide, PEG-1,2-dimyristoyloxypropyl-3-amine; specifically may be: 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000;

[0014] Calculated by mole fraction, the ionizable lipid compound is 10-80 parts, the phospholipid is 10-80 parts, the structural lipid is 10-80 parts, and the polyethylene glycol lipid is 0.01-20 parts; specifically, the ionizable lipid compound is 20 parts, the phospholipid is 10-50 parts, the structural lipid is 20-80 parts, and the polyethylene glycol lipid is 0.2-1.45 parts; more specifically, the mole fractions of the ionizable lipid compound: phospholipid: structural lipid: polyethylene glycol lipid are 15.2 parts, 38.2 parts, 45.8 parts, 0.8 parts and 22 parts, 33.1 parts, 44.1 parts, 0.8 parts in sequence.

[0015] The mass ratio of the nucleic acid to the ionizable lipid compound in the nucleic acid drug delivery system may be 1:2 - 1:14, specifically may be 1:4, 1:10.

[0016] The lipid nanoparticles made of two components may specifically be made of a two-component including a lipid compound and a phospholipid;

[0017] Among them, the lipid compound may specifically be a quaternized lipid compound, and more specifically may be (9Z,9'Z,9”Z)-N,N',N”- [benzene-1,3,5-triyltris(methylene)] tris(dimethyl 9-octadecene-1-ammonium) iodide;

[0018] The phospholipid can be selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, hydrogenated soy phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, 1-stearoyl-2-oleoyl phosphatidylcholine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, dioleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, 1,2-distearoyl-sn-glycero-3-phosphoglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, sphingomyelin, ceramide, cephalin, cerebroside or diacylglycerol; specifically, it can be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE;

[0019] By mole fraction, 10-90 parts of lipid compound and 10-90 parts of phospholipid; more specifically, the lipid compound and phospholipid are 50 parts and 50 parts in turn by mole fraction;

[0020] The nitrogen-phosphorus ratio of the lipid compound to the nucleic acid can be 1:1 - 9:1, specifically 1.5:1, 4.5:1.

[0021] The nucleic acid is selected from: DNA, RNA (including any at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, lncRNA9).

[0022] The magnetic nanoparticles with oligodeoxynucleotides covalently modified on the surface can be specifically magnetic beads with oligothymidine deoxynucleotides covalently modified on the surface; more specifically, magnetic beads with a 25-mer dT sequence covalently modified on the surface;

[0023] The mass ratio of the nucleic acid to the magnetic beads is 1:0.3 - 1:250, specifically 1:25 - 1:56, 1:25 - 1:50, and more specifically 1:25.

[0024] The present invention also provides a preparation method of the above magnetic-responsive nucleic acid drug delivery system.

[0025] The preparation method of the magnetic-responsive nucleic acid drug delivery system provided by the present invention includes the following steps:

[0026] 1) Preparation of a nucleic acid-loaded nucleic acid drug delivery system (specifically, a nucleic acid-loaded lipid nanoparticle)

[0027] a) Dissolve an ionizable lipid compound, a phospholipid, a structural lipid, and a polyethylene glycol lipid in ethanol according to a molar ratio to obtain an ethanol phase containing the four lipids; mix the ethanol phase containing the four lipids with a nucleic acid solution, blow and beat well to obtain a mixture, and finally add PBS buffer, mix well, and let stand to obtain a nucleic acid-loaded nucleic acid drug delivery system;

[0028] Or b) Dissolve a lipid compound and a phospholipid in ethanol respectively, mix to obtain an organic phase; mix the organic phase with water to obtain a mixed phase; then mix the mixed phase with a nucleic acid solution and let stand to obtain a nucleic acid-loaded nucleic acid drug delivery system;

[0029] 2) Preparation of a magnetically responsive nucleic acid drug delivery system

[0030] Ultrasonically disperse magnetically nanoparticles covalently modified with oligodeoxynucleotides on the surface, and resuspend with PBS buffer. Mix the suspension of magnetically nanoparticles covalently modified with oligodeoxynucleotides on the surface obtained with the nucleic acid-loaded nucleic acid drug delivery system prepared in step 1), mix well, and let stand to obtain a magnetically responsive nucleic acid drug delivery system.

[0031] In step 1) a) of the above method, the volume ratio of the ethanol phase to the nucleic acid solution is 2:1 - 1:5, specifically 1:1;

[0032] The nucleic acid solution can be specifically prepared by diluting nucleic acid in a citrate buffer (50 mM, pH = 3);

[0033] The addition amount of the PBS buffer is 1 - 10 times (by volume) relative to the mixture, specifically 2 times;

[0034] The standing time can be 0 - 60 min, specifically 10 min;

[0035] In step 1) b) of the above method, the volume ratio of the organic phase to water can be 1:1 - 1:10, specifically 1:9;

[0036] The volume ratio of the mixed phase to the nucleic acid solution is 1:1 - 1:10, specifically 1:9;

[0037] The nucleic acid solution can be specifically prepared by diluting nucleic acid in a PBS buffer (pH = 7.4);

[0038] In step 2) of the above method, the parameters of the ultrasonic treatment can be: 50% ultrasonic power percentage, ultrasonic time of 10 minutes, and the working time - interval time is 2 seconds - 3 seconds;

[0039] The mixing is carried out by vigorously pipetting with a pipette gun or vortexing with a vortex mixer for 10 - 90 s (specifically, it can be 30 s);

[0040] The standing time can be 0 - 30 min, specifically it can be 5 min.

[0041] The present invention also provides a nucleic acid drug delivery promoting uptake system.

[0042] The nucleic acid drug delivery promoting uptake system provided by the present invention includes the above-mentioned magnetic-responsive nucleic acid drug delivery system and a magnet.

[0043] Specifically, the magnetic field intensity of the magnet is N28 - N52, specifically it can be N35 - N42, and the magnetic attraction time can be 5 - 60 min.

[0044] In the present invention, nucleic acids are loaded onto the nucleic acid drug delivery system, and then interact with magnetic nanoparticles covalently modified with poly-thymidine deoxynucleotide sequences on the surface to form a magnetic-responsive nucleic acid drug delivery system. Under the action of an external magnetic field, the magnetic-responsive nucleic acid drug delivery system is added to cells, and the effects of different preparation processes and magnetic treatment parameters on nucleic acid delivery efficiency are investigated. The optimal magnetic-responsive nucleic acid drug delivery system is optimized, and finally the efficacy of the optimal magnetic-responsive nucleic acid drug delivery system is confirmed at the animal level. After adopting the technical solution of the present invention, the time required for the nucleic acid drug delivery system to be taken up by cells is greatly reduced, and the nucleic acid delivery efficiency and expression level are significantly improved, which can be increased by 26.7 times (delivering siRNA) and 33.4 times (delivering mRNA) in cells and mice, respectively.

[0045] The present invention utilizes magnetic attraction force to apply an external magnetic field to the magnetic-responsive nucleic acid drug delivery system loaded with nucleic acids to accelerate the deposition of the nucleic acid drug delivery system on the cell surface and cell uptake, and improve the nucleic acid drug delivery efficiency and expression level. Compared with the traditional non-magnetic-responsive nucleic acid drug delivery system, the magnetic-responsive nucleic acid drug delivery system can be highly enriched and rapidly taken up at the site where the external magnetic field is applied (at the cell and in vivo levels), and the whole process often only takes dozens of minutes, so as to achieve better delivery efficiency in a shorter time. The present invention improves the cellular uptake and intracellular delivery of nucleic acids through the magnetic-responsive nucleic acid drug delivery system, and improves the nucleic acid delivery efficiency and expression level in cells and mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA prepared in Example 1 of the present invention under magnetic attraction. (The scale bar is 50 microns, and for the rest of the pictures, if not otherwise specified, the scale bars are all the same as Figure 1 consistent).

[0047] Figure 2 To study the delivery efficiency of the lipid nanoparticles loaded with eGFP mRNA in Comparative Example 1 under magnetic attraction.

[0048] Figure 3 To study the effect of the mass ratio of eGFP mRNA (maintaining 100 ng per group unchanged) to magnetic beads in the eGFP mRNA lipid nanoparticles on the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system under magnetic attraction in Example 2 of the present invention.

[0049] Figure 4 To study the effect of the mixing method of magnetic beads and eGFP mRNA lipid nanoparticles on the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system under magnetic attraction in Example 3 of the present invention.

[0050] Figure 5 To study the effect of the standing time at room temperature on the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system after magnetic beads and eGFP mRNA lipid nanoparticles are uniformly mixed under magnetic attraction in Example 4 of the present invention.

[0051] Figure 6 To study the effect of the magnetic attraction time on the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system after the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA is added to cells in Example 5 of the present invention.

[0052] Figure 7 To compare the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system prepared in Example 6 of the present invention, which is loaded with firefly luciferase mRNA (FLuc mRNA), with that of the FLuc mRNA lipid nanoparticles without magnetic beads under magnetic attraction.

[0053] Figure 8 To compare the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA with that of the eGFP mRNA lipid nanoparticles without magnetic beads (control group) for different cell types under magnetic attraction in Example 7 of the present invention.

[0054] Figure 9 To compare the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA with that of the eGFP mRNA lipid nanoparticles without magnetic beads (control group) for 3D cell spheroids under magnetic attraction in Example 8 of the present invention.

[0055] Figure 10 To compare the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system loaded with secreted Gauss luciferase mRNA (GLucmRNA) with that of the GLuc mRNA lipid nanoparticles without magnetic beads under magnetic attraction in Example 9 of the present invention.

[0056] Figure 11Comparison of the delivery efficiency between the magnetic-responsive nucleic acid drug delivery system loaded with secreted human vascular endothelial growth factor receptor mRNA (sFlt-1mRNA) and the sFlt-1mRNA lipid nanoparticles without magnetic beads under magnetic attraction in Example 10 of the present invention.

[0057] Figure 12 Comparison of the delivery efficiency between the TT3 magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA and the eGFP mRNA TT3 lipid nanoparticles (control group) without magnetic beads under magnetic attraction in Example 11 of the present invention.

[0058] Figure 13 Comparison of the delivery efficiency between the qtB-UC18 magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA and the eGFP mRNA qtB-UC18 lipid nanoparticles (control group) without magnetic beads under magnetic attraction in Example 12 of the present invention.

[0059] Figure 14 Comparison of the silencing efficiency between the magnetic-responsive nucleic acid drug delivery system loaded with siRNA (FLuc siRNA) that silences the expression of firefly luciferase and the FLuc siRNA lipid nanoparticles without magnetic beads under magnetic attraction in Example 13 of the present invention.

[0060] Figure 15 Comparison of the cell uptake time between the magnetic-responsive nucleic acid drug delivery system loaded with Cy3 mRNA and the Cy3 mRNA lipid nanoparticles (control group) without magnetic beads (scale bar is 20 microns) in Example 14 of the present invention.

[0061] Figure 16 Effect of magnetic attraction of magnets with different intensities on the delivery efficiency of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA in Example 15 of the present invention.

[0062] Figure 17 a is the bioluminescence imaging diagram after intraperitoneal administration of the lipid nanoparticles loaded with firefly luciferase mRNA (FLuc mRNA) in Example 16 of the present invention; b is the bioluminescence imaging diagram after intraperitoneal administration of the magnetic-responsive nucleic acid drug delivery system loaded with firefly luciferase mRNA (FLuc mRNA) without magnetic attraction of the N42 magnet; c is the bioluminescence imaging diagram after intraperitoneal administration of the magnetic-responsive nucleic acid drug delivery system loaded with firefly luciferase mRNA (FLuc mRNA) under magnetic attraction of the N42 magnet.

[0063] Figure 18This is a quantitative bioluminescence image of the whole body and organs after intraperitoneal administration of a magnetic-responsive nucleic acid drug delivery system loaded with firefly luciferase mRNA (FLuc mRNA) and lipid nanoparticles in Example 16 of the present invention. The dashed line represents the average bioluminescence of intraperitoneal administration of the magnetic-responsive nucleic acid drug delivery system loaded with firefly luciferase mRNA (FLuc mRNA) (without N42 magnet magnetic attraction). Detailed implementation manners

[0064] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0065] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0066] Example 1: Preparation of a magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA and in vitro delivery activity test

[0067] (1) Preparation of lipid nanoparticles loaded with eGFP mRNA: Dissolve the ionizable lipid compound N1,N1,N6-tris((Z)-9-octadecenyl)-N6-(6-((7-(trifluoromethyl)quinolin-4-yl)amino)hexyl)hexane-1,6-diamine (abbreviated as CF3-3N6-UC18), phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE), structural lipid (cholesterol) and polyethylene glycol lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000) in ethanol at a molar ratio of 20:50:60:1.1 to obtain an ethanol phase containing four lipids. Mix the above ethanol phase and enhanced green fluorescent protein (eGFP) mRNA (Shanghai Weihuan Biotechnology Co., Ltd., product number R1016) diluted in citrate buffer (50 mM, pH = 3) in equal volume (the mass ratio of the ionizable lipid compound to mRNA is 4:1), blow and beat well to obtain a mixed solution, and finally add 2 volumes (relative to the mixed solution) of PBS buffer, gently mix and let stand for 10 min to obtain lipid nanoparticles loaded with eGFP mRNA.

[0068] (2) Preparation of a magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA: Mix 10 μL of magnetic bead suspension (5 mg / mL, Biotium Inc., catalog number R0075) covalently modified with a 25-mer dT sequence (oligothymidine) on the surface and 200 μL of PBS buffer in a 1.5 mL centrifuge tube. Under ice bath conditions, use an ultrasonic crusher (Ningbo Weicheng Ultrasonic Equipment Technology Co., Ltd., model USA35-1000) for ultrasonic treatment. The ultrasonic parameters are: 50% ultrasonic power percentage, ultrasonic time of 10 minutes, and a working time - interval time of 2 seconds - 3 seconds. Place the ultrasonicated magnetic beads on a magnetic separation rack (Biotium Inc., catalog number FMS024) for 1 minute. After aspirating the supernatant, add 200 μL of PBS to resuspend the magnetic beads and pipette evenly. Repeat this step twice. After the last aspiration of the supernatant, add 10 μL of PBS buffer to resuspend. Add the lipid nanoparticles containing 2000 ng of eGFP mRNA prepared in step (1) to 10 μL of magnetic beads (the mass ratio of mRNA to magnetic beads is 1:25), and vigorously pipette and mix well. Let it stand for 5 minutes to obtain a magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA.

[0069] (3) In vitro delivery activity test: Seed 293T cells at a density of 20,000 cells per well in a 96-well plate. After overnight culture, add the magnetic-responsive nucleic acid drug delivery system loaded with 100 ng of eGFP mRNA prepared in step (2) to each well. Place the plate on a magnetic plate of the same size as the plate (Biotium Inc., catalog number FMS085), and place both in a CO 2 incubator. After magnetic attraction for 20 minutes, remove the magnetic plate. Use a pipette to aspirate the original culture medium, add fresh culture medium, and continue culturing for 24 hours. Then, use an inverted fluorescence microscope (Zeiss, Axio Vert A1) to observe the expression of eGFP.

[0070] The results are as Figure 1 shown. The magnetic-responsive nucleic acid drug delivery system can be rapidly taken up by cells within only 20 minutes of magnetic attraction, achieving effective delivery and expression of eGFP mRNA. The above results indicate that the lipid nanoparticles with magnetic beads, namely the magnetic-responsive nucleic acid drug delivery system, have magnetic responsiveness.

[0071] Comparative Example 1: In vitro delivery activity test of lipid nanoparticles loaded with eGFP mRNA

[0072] The difference from Example 1 is only that the lipid nanoparticles loaded with eGFP mRNA prepared in step (1) of Example 1 instead of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA prepared in step (2) of Example 1 are added to the 96-well plate, and other in vitro delivery activity test conditions are the same.

[0073] The results are as Figure 2 shown. When there is a magnetic attraction, the lipid nanoparticles without magnetic beads cannot be rapidly taken up by cells, resulting in basically undetectable expression of eGFP mRNA. The above results indicate that the lipid nanoparticles without magnetic beads do not have magnetic responsiveness.

[0074] Example 2. Optimize the mass ratio of mRNA and magnetic beads in eGFP mRNA lipid nanoparticles

[0075] The difference between this example and Example 1 is only that the lipid nanoparticles containing 2000 ng of eGFP mRNA are added to 0.156 μL, 0.625 μL, 2.5 μL, 20 μL, 50 μL or 100 μL of magnetic beads (the mass ratio of mRNA to magnetic beads is 1:0.39, 1:1.56, 1:6.25, 1:50, 1:125 or 1:250) for mixing. Example 1 (1:25) is used as a control.

[0076] The results are as Figure 3 shown. When the mass ratio of mRNA to magnetic beads is 1:1.56 - 1:50, the eGFP positive cell rate and fluorescence intensity are slightly lower than 1:25, and other ratios are significantly lower than 1:25.

[0077] Example 3. Optimize the mixing method of eGFP mRNA lipid nanoparticles and magnetic beads

[0078] The difference between this example and Example 1 is only that the lipid nanoparticles loaded with eGFP mRNA and magnetic beads are mixed by gently pipetting or vortexing with a vortex mixer (Scilogex, model: SCILOGEX-Vortex Mixer-MX-S) for 30 seconds. To reduce the difference between batches, Example 1 (vigorously pipetting and mixing) is repeated and used as a control.

[0079] The results are as Figure 4 shown. Under the mixing methods of vigorously pipetting with a pipette and vortexing with a vortex mixer for 30 seconds, the expression level of enhanced green fluorescent protein is relatively high.

[0080] Example 4. Optimize the standing time after mixing eGFP mRNA lipid nanoparticles and magnetic beads

[0081] The difference between this example and Example 1 is only that after the lipid nanoparticles loaded with eGFP mRNA and the magnetic beads are mixed, they are allowed to stand at room temperature for 30 minutes, 20 minutes, 10 minutes respectively, or administered immediately after mixing. To reduce the differences between batches, Example 1 (standing for 5 minutes) is repeated and used as a control.

[0082] The results are as Figure 5 shown that the expression levels of enhanced green fluorescent protein under each standing time condition are basically the same.

[0083] Example 5. Optimization of the magnetic attraction time of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA

[0084] The difference between this example and Example 1 is only that after the magnetic-responsive nucleic acid drug delivery system is added to the 293T cell wells (the dosing dose is 100 ng mRNA / well), magnetic attraction is performed for 60 minutes or 5 minutes. To reduce the differences between batches, Example 1 (magnetic attraction time is 20 minutes) is repeated and used as a control group.

[0085] The results are as Figure 6 shown that when the magnetic attraction time is 20 and 60 minutes, the expression levels of enhanced green fluorescent protein are relatively high.

[0086] Example 6. Preparation of the magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA and in vitro delivery activity test

[0087] The difference between this example and Example 1 is only that the mRNA used and the method for detecting the expression of the target protein are different. The mRNA is Firefly luciferase mRNA (abbreviated as FLuc mRNA) (Shanghai Weihuan Biotechnology Co., Ltd., product number R1013), and the target protein detection kit is One-Lumi Firefly luciferase reporter gene detection kit (Shanghai Beyotime Biotechnology Co., Ltd., product number RG055M). An enzyme-linked immunosorbent assay instrument (BioTek, model: SYNERGY LX) is used to detect the expression level of the Firefly luciferase protein. The lipid nanoparticles loaded with FLuc mRNA without magnetic beads are used as a control group.

[0088] The results are as Figure 7 shown that the bioluminescence intensity of the magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA is approximately 6.6 times that of the FLuc mRNA lipid nanoparticles without magnetic beads.

[0089] Example 7. In vitro delivery activity test of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA on different types of cells

[0090] The difference between this example and Example 1 is only that the cells used are NIH / 3T3 cells (20,000 cells / well), MC38 cells (20,000 cells / well), and THP-1 cells (40,000 cells / well). The lipid nanoparticles loaded with eGFP mRNA without magnetic beads were used as the control group.

[0091] The results are as Figure 8 shown. For different types of adherent cells and suspension cells, the magnetic-responsive nucleic acid drug delivery system can improve the expression of eGFP through magnetic attraction under the condition of magnetic attraction for 20 minutes.

[0092] Example 8. In vitro delivery activity test of the magnetic-responsive nucleic acid drug delivery system loaded with eGFP mRNA on 3D cell spheroids

[0093] The difference between this example and Example 1 is only that the 293T cells used are replaced with 293T 3D cell spheroids. In a laminar flow hood or biosafety cabinet, add 40 μL of coating solution to each well of a U-bottom 96-well plate according to the steps in the instruction manual of the 3D cell culture coating kit (Shanghai Beyotime Biotechnology Co., Ltd., product number C0366S), cover the lid of the U-bottom 96-well plate, and place it in a carbon dioxide incubator for 30 minutes. Take out the culture plate, carefully aspirate the coating solution, avoiding touching the bottom coating area. Keep the culture plate still in the laminar flow hood for another 30 minutes to dry, and then perform cell seeding. Seed 100 μL of cells containing 1,000 293T cells into each well and continue culturing for 24 hours to form 293T 3D cell spheroids. The lipid nanoparticles loaded with eGFP mRNA without magnetic beads were used as the control group.

[0094] The results are as Figure 9 shown. The magnetic-responsive nucleic acid drug delivery system can deliver eGFP mRNA to more and deeper cells and the expression level of fluorescent protein is higher under the condition of magnetic attraction for 20 minutes.

[0095] Example 9. Preparation and in vitro delivery activity test of the magnetic-responsive nucleic acid drug delivery system loaded with secreted Gauss luciferase mRNA

[0096] The difference between this example and Example 1 is only that the mRNA used and the method for detecting the expression of the target protein are different. The mRNA is secreted Gauss luciferase mRNA (abbreviated as Gluc mRNA) (Shanghai Weihuan Biotechnology Co., Ltd., product number R1999). The Gauss luciferase reporter gene detection kit (Beyotime, RG072M) was used for detection, and then a microplate reader (BioTek, model: SYNERGY LX) was used to detect the bioluminescence value. The lipid nanoparticles loaded with Gluc mRNA without magnetic beads were used as the control group.

[0097] The results are as follows Figure 10 shown. When the magnetic-responsive nucleic acid drug delivery system was magnetically attracted for 20 minutes, the bioluminescence intensity of the magnetic-responsive nucleic acid drug delivery system loaded with GLuc mRNA was approximately 2.4 times that of the GLuc mRNA-loaded lipid nanoparticles without magnetic beads.

[0098] Example 10: Preparation and in vitro delivery activity test of a magnetic-responsive nucleic acid drug delivery system loaded with soluble Fms-like tyrosine kinase-1 mRNA

[0099] The difference between this example and Example 1 is only that the mRNA used and the method for detecting the expression of the target protein are different. The mRNA is soluble Fms-like tyrosine kinase-1 mRNA (abbreviated as sFlt-1 mRNA) (Shanghai Weihuan Biotechnology Co., Ltd., product number R1999). The protein expression level in the supernatant was detected using a human vascular endothelial growth factor receptor enzyme-linked immunosorbent assay kit (Elabscience, E-EL-H6175), and then the absorbance at 450 nm was detected using a microplate reader (BioTek, model: SYNERGY LX). Lipid nanoparticles loaded with sFlt-1 mRNA without magnetic beads were used as the control group.

[0100] The results are as follows Figure 11 shown. When the magnetic-responsive nucleic acid drug delivery system was magnetically attracted for 20 minutes, the sFlt-1 protein expression level of the magnetic-responsive nucleic acid drug delivery system loaded with sFlt-1 mRNA was approximately 10.4 times that of the sFlt-1 mRNA-loaded lipid nanoparticles without magnetic beads.

[0101] Example 11: Preparation and in vitro delivery activity test of a TT3 magnetic-responsive nucleic acid drug delivery system

[0102] The difference between this example and Example 1 is only that the mRNA-loaded lipid nanoparticles used are TT3 lipid nanoparticles. Lipid nanoparticles loaded with eGFP mRNA without magnetic beads were used as the control group.

[0103] The specific preparation method of TT3 lipid nanoparticles loaded with eGFP mRNA is as follows: The ionizable lipid compound N1,N3,N5-tris(4-dodecylhexadecyl)benzene-1,3,5-tricarboxamide (abbreviated as TT3), phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE), structural lipid (cholesterol), and polyethylene glycol lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000) were dissolved in ethanol at a molar ratio of 20:30:40:0.75 to obtain an ethanol phase containing four lipids. The above ethanol phase was mixed with eGFP mRNA diluted in citrate buffer (50 mM, pH = 3) in equal volume (the mass ratio of TT3 to mRNA was 10:1), and the mixture was thoroughly pipetted to obtain a mixed solution. Finally, 2 volumes (relative to the mixed solution) of PBS buffer was added, and after gently mixing, it was left standing for 10 min to obtain a lipid nanoparticle solution loaded with eGFP mRNA.

[0104] The synthesis route of TT3 can be as follows: 1,3,5-benzenetricarbonyl chloride and N-Boc-1,3-propanediamine were added to a solution of dichloromethane and pyridine at a molar equivalent of 1:4, and stirred at room temperature overnight. Dichloromethane was added and washed twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered and rotary evaporated. The obtained oily compound was purified by column chromatography to obtain intermediate 1. Intermediate 1 was dissolved in dichloromethane, and trifluoroacetic acid equimolar to intermediate 1 was slowly added dropwise, and the reaction was carried out at room temperature for 2 hours. Dichloromethane was added and washed twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered and rotary evaporated to obtain intermediate 2. Intermediate 2, triethylamine, dodecyl aldehyde, and sodium triacetoxyborohydride were dissolved in anhydrous tetrahydrofuran at a ratio of 1:4:9:10, and the reaction was carried out at room temperature for 48 hours. After the reaction was detected to be complete by thin layer chromatography, the solvent was evaporated to dryness, and the final product TT3 was obtained after purification by column chromatography.

[0105] The results are as Figure 12 shown. The TT3 magnetic-responsive nucleic acid drug delivery system can also improve the expression of enhanced green fluorescent protein under the condition of magnetic attraction for 20 minutes.

[0106] Example 12. Preparation and in vitro delivery activity test of qtB-UC18 magnetic-responsive nucleic acid drug delivery system

[0107] The difference between this example and Example 1 is only that the mRNA lipid nanoparticles used are qtB-UC18 lipid nanoparticles. The qtB-UC18 lipid nanoparticles loaded with eGFP mRNA without magnetic beads were used as the control group.

[0108] The specific preparation method of qtB-UC18 lipid nanoparticles loaded with eGFP mRNA is as follows: Mix the main lipid molecule (9Z,9'Z,9”Z)-N,N',N”-[benzene-1,3,5-triyltris(methylene)]tris(dimethyl9-octadec-9-en-1-ammonium) iodide (qtB-UC18, dissolved in ethanol) with a concentration of 3 mg / mL and 1.6 mg / mL DOPE (dissolved in ethanol) in a volume ratio of 1:1 to obtain a homogeneous organic phase. Mix the organic phase and water in a volume ratio of 1:9 to obtain a mixed phase; quickly mix the mixed phase with eGFP mRNA (eGFP mRNA diluted in PBS buffer with a pH of 7.4) in a volume ratio of 3:7 (the nitrogen-phosphorus ratio of qtB-UC18 and mRNA is 1.5:1), and then let it stand for 10 min to obtain a lipid nanoparticle solution loaded with eGFP mRNA.

[0109] The synthesis route of qtB-UC18 can be as follows: Add tB-UC18, methyl iodide, and potassium carbonate to an ethanol solution in molar equivalents of 1:21:4.5, stir at 40 °C for 72 h, add water, extract 3 times with dichloromethane, add anhydrous sodium sulfate, filter, and rotary evaporate to obtain qtB-UC18.

[0110] The results are as Figure 13 shown that the qtB-UC18 magnetic-responsive nucleic acid drug delivery system can also improve the expression of enhanced green fluorescent protein under magnetic attraction for 20 minutes.

[0111] Example 13: Preparation and in vitro activity test of a magnetic-responsive nucleic acid drug delivery system loaded with siRNA (FLuc siRNA) that silences the expression of firefly luciferase

[0112] (1) Dissolve the ionizable lipid compound CF3-3N6-UC18, phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated as DOPE), structural lipid (cholesterol), and polyethylene glycol lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000) in ethanol in a molar ratio of 20:50:60:1.1 to obtain an ethanol phase containing four lipids.

[0113] (2) Mix the above ethanol phase and siRNA (FLuc siRNA) that silences firefly luciferase expression and is diluted in citrate buffer (50 mM, pH = 3) (GenScript Biotech Corporation, base sequence from 5' to 3': GCUAUGGGCUGAAUACAAAdTdT; UUUGUAUUCAGCCCAUAGCdTdT) in equal volumes (at this time, the mass ratio of CF3-3N6-UC18 to siRNA is 75:1). Vigorously pipette to obtain a mixed solution. Finally, add 2 volumes (relative to the mixed solution) of PBS buffer, gently mix, and let stand for 10 min to obtain a lipid nanoparticle solution loaded with FLuc siRNA. Use eGFP siRNA as an irrelevant siRNA (GenScript Biotech Corporation, base sequence from 5' to 3': GGCUACGUCCAGGAGCGCACCdTdT; GGUGCGCUCCUGGACGUAGCCdTdT).

[0114] (3) Mix according to the ratio of 30 μL of a formulation with a siRNA concentration of 33.3 nM and 5 μL of magnetic beads. After mixing, vigorously pipette and let stand at room temperature for 5 minutes. In 293T cells (293T-luc2-tdT cells, Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences, 1101HUM-PUMC000622) that are stably expressing luc2 at 20,000 cells per well and have been plated overnight, add the above siRNA biological agent so that the final concentration of siRNA per well is 1 nM. After magnetic adsorption for 20 minutes, remove the magnetic plate. Then, use a pipette to aspirate the original cell culture supernatant, add fresh medium, and continue culturing for 48 hours. Then, use a microplate reader to detect the expression level of firefly luciferase protein. Use the lipid nanoparticle loaded with FLuc siRNA without magnetic beads as the control group. The calculation method for gene silencing efficiency is 100% - (bioluminescence intensity of the well administered with FLuc siRNA / bioluminescence intensity of the well administered with irrelevant siRNA) × 100%.

[0115] The results are as Figure 14 shown. In the case of magnetic adsorption for 20 minutes, the relative silencing activity of the magnetic-responsive nucleic acid drug delivery system loaded with FLuc siRNA is approximately 26.7 times that of the lipid nanoparticle loaded with FLuc siRNA without magnetic beads.

[0116] Example 14. Cellular Uptake of Magnetic-Responsive Nucleic Acid Drug Delivery System

[0117] The difference between this example and Example 1 is only that the mRNA in the preparation process of Example 1 is replaced with Cy3-labeled mRNA (Shanghai Weihuan Biotechnology Co., Ltd., product number R1008) to prepare a magnetic-responsive nucleic acid drug delivery system loaded with Cy3 mRNA. An inverted fluorescence microscope was used to observe the uptake of mRNA lipid nanoparticles by cells. Lipid nanoparticles loaded with Cy3 mRNA without magnetic beads were used as the control group.

[0118] The results are as Figure 15 shown. In the case of magnetic attraction for 20 minutes, the magnetic-responsive nucleic acid drug delivery system accelerated the uptake rate of mRNA.

[0119] Example 15. Optimization of magnet strength

[0120] The difference between this example and Example 1 is only that a magnet with a magnetic strength of N28, N35, N42 or N52 (Ningbo Cibao Technology Co., Ltd., 10×10×5 mm) was used for magnetic attraction.

[0121] The results are as Figure 16 shown. When the magnet strength was N35 and N42, the expression level of enhanced green fluorescent protein was relatively high.

[0122] Example 16. In vivo delivery activity test of a magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA

[0123] An N42 magnet was fixed to the abdomen of a mouse with medical tape. The magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA prepared in Example 6 was dialyzed in a dialysis cup (Thermo Fisher Scientific, product number 88403) with PBS for 30 minutes, and then injected intraperitoneally into the C57BL / 6J mouse below the magnet fixation site at a dose of 2.5 μg mRNA per mouse. After 30 minutes, the magnet was removed, and the luciferase substrate was injected intraperitoneally at a dose of 150 mg / kg. After 10 minutes, anesthetized mice were imaged using a small animal imager (PerkinElmer, model: IVIS Lumina LT). After in vivo imaging, the main tissues of the mice were taken out for ex vivo imaging. The imaging pictures were quantitatively analyzed using PerkinElmer in vivo imaging software to obtain the bioluminescence of the whole body and each organ. Intraperitoneal injection of lipid nanoparticles loaded with the same dose of FLuc mRNA and intraperitoneal injection of the magnetic-responsive nucleic acid drug delivery system loaded with the same dose of FLuc mRNA (without magnetic attraction by the N42 magnet) were used as the control groups.

[0124] The results are as Figure 17 and 18As shown, under the magnetic attraction, the overall bioluminescence intensity of the mice intraperitoneally injected with the magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA was approximately 33.4 times that of the lipid nanoparticles loaded with FLuc mRNA; under the magnetic attraction, the bioluminescence intensities of the heart, liver, spleen, lung, kidney, pancreas and remains of the mice intraperitoneally injected with the magnetic-responsive nucleic acid drug delivery system loaded with FLuc mRNA were 2.0, 20.5, 44.3, 5.4, 2.7, 33.8 and 17.7 times that of the lipid nanoparticles loaded with FLuc mRNA, respectively.

[0125] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. A magnetically responsive nucleic acid drug delivery system, comprising a nucleic acid, a nucleic acid drug delivery system, and magnetic nanoparticles with oligodeoxynucleotides covalently modified on the surface; in, The nucleic acid is loaded on the nucleic acid drug delivery system to obtain a nucleic acid-loaded nucleic acid drug delivery system, and the nucleic acid-loaded nucleic acid drug delivery system interacts with the magnetic nanoparticles whose surfaces are covalently modified with oligodeoxynucleotides.

2. The magnetic responsive nucleic acid drug delivery system according to claim 1, characterized in that: The nucleic acid drug delivery system is a lipid nanoparticle; The magnetic nanoparticles with oligodeoxynucleotides covalently modified on the surface are magnetic beads with oligothymidine deoxynucleotides covalently modified on the surface.

3. The magnetic-responsive nucleic acid drug delivery system according to claim 2, characterized in that: The lipid nanoparticles are lipid nanoparticles made of four components or two components.

4. The magnetic-responsive nucleic acid drug delivery system according to claim 3, characterized in that: The lipid nanoparticles made of four components are made of four components including ionizable lipid compounds, phospholipids, structural lipids, and polyethylene glycol lipids; in terms of molar fractions, the ionizable lipid compounds are 10 to 80 parts, the phospholipids are 10 to 80 parts, the structural lipids are 10 to 80 parts, and the polyethylene glycol lipids are 0.01 to 20 parts; The mass ratio of the nucleic acid to the ionizable lipid compound in the nucleic acid drug delivery system is 1:2-1:

14.

5. The magnetic-responsive nucleic acid drug delivery system according to claim 3, characterized in that: The lipid nanoparticles made of two components are made of two components including lipid compounds and phospholipids; in terms of molar fractions, the lipid compounds are 10 to 90 parts and the phospholipids are 10 to 90 parts; The nitrogen-phosphorus ratio of the lipid compound to the nucleic acid is 1:1-9:

1.

6. The magnetic-responsive nucleic acid drug delivery system according to claim 2, characterized in that: The nucleic acid is selected from DNA and RNA; In the magnetic-responsive nucleic acid drug delivery system, the mass ratio of the nucleic acid to the magnetic beads is 1:0.3-1:

250.

7. A method for preparing a magnetically responsive nucleic acid drug delivery system according to any one of claims 1 to 6, comprising the following steps: 1) preparing a nucleic acid-loaded nucleic acid drug delivery system a) dissolving an ionizable lipid compound, a phospholipid, a structural lipid and a polyethylene glycol lipid in ethanol according to a certain molar ratio to obtain an ethanol phase containing four lipids; mixing the ethanol phase containing the four lipids with a nucleic acid solution, fully blowing to obtain a mixed solution, and finally adding a PBS buffer, mixing, and standing to obtain a nucleic acid-loaded nucleic acid drug delivery system; or b) dissolving the lipid compound and the phospholipid in ethanol respectively, mixing to obtain an organic phase; mixing the organic phase with water to obtain a mixed phase; and then mixing the mixed phase with a nucleic acid solution, allowing to stand, to obtain a nucleic acid-loaded nucleic acid drug delivery system; 2) Preparation of magnetic responsive nucleic acid drug delivery system The magnetic nanoparticles with oligodeoxynucleotides covalently modified on the surface are ultrasonically dispersed, resuspended with PBS buffer, mixed with the nucleic acid-loaded nucleic acid drug delivery system prepared in step 1), mixed evenly, and allowed to stand to obtain a magnetically responsive nucleic acid drug delivery system.

8. The method according to claim 7, characterized in that In step 2), the mixing is performed by vigorous blowing with a pipette or vortexing with a vortexer for 10-90 seconds; The standing time is 0-30 min.

9. A nucleic acid drug delivery and uptake-enhancing system, comprising the magnetic-responsive nucleic acid drug delivery system according to any one of claims 1 to 6 and a magnet.

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