A kind of atomized inhalable fluorocarbon nanotransfection reagent for delivering nucleic acid drugs and its preparation method and application

Abstract

The present invention discloses an atomized inhalable fluorocarbon nanotransfection reagent for delivering nucleic acid drugs, and a preparation method and application thereof, and belongs to the field of gene transfection biotechnology. The present invention aims to solve the problem that nucleic acid system delivery is easily degraded by RNase enzymes in plasma and tissues, and the transfection efficiency needs to be improved. The nanotransfection reagent of the present invention includes cationic lipids, neutral phospholipids, cholesterol, auxiliary lipids, stabilizers, and fluorocarbon compounds; and is prepared by thin film dispersion method, ethanol injection method, reverse evaporation method, freeze drying method, freeze-thaw method, organic solvent volatilization method, and ultrasonic dispersion method. It is used for the encapsulation and delivery of nucleic acids. The particle size of the present invention is uniform, nanometer-scale, high in oxygen content, and can be atomized for inhalation administration. At the same time, the atomized nanotransfection reagent has the advantages of small toxic and side effects, good patient compliance, high transfection efficiency, and high encapsulation rate.

Description

Technical Field

[0001] The present invention is to develop a fluorocarbon-based nano drug delivery platform for efficiently delivering nucleic acid drugs, which is used to deliver nucleic acid drugs via atomized inhalation. The present invention comprises cationic lipids, neutral phospholipids, cholesterol, auxiliary lipids, fluorocarbons (PFCs), and a nucleic acid drug delivery system based thereon. The preparation of the fluorocarbon nano transfection nucleic acid drug delivery system is applied to diseases caused by abnormal gene expression. Background Art

[0002] RNA interference (RNAi) technology is a gene blocking technology at the transcription level and is a type of gene therapy. It can specifically interfere with the expression of a particular gene. Studies have shown that a considerable number of diseases are protein problems, and nucleic acid drugs prevent mRNA from being translated into protein through various mechanisms, which can play a good role in treatment at the gene level.

[0003] The advantages of nucleic acid drugs are strong specificity; rich targets, and they can target "untargetable" and "undruggable" diseases; they can act on both the cell nucleus and the cytoplasm; and they are relatively simple to design, requiring only the base sequence of the target gene to be known. Therefore, nucleic acid drugs are expected to become the third largest type of drugs after small molecule drugs and protein drugs. Currently, the FDA has approved two new drugs based on the RNA interference mechanism, namely Patisiran (ONPATTRO TM ) and Givlaari (Givosiran), for the treatment of familial amyloid polyneuropathy and acute hepatic porphyria.

[0004] RNA interference (RNAi) is a naturally occurring phenomenon, which refers to the phenomenon that double-stranded RNA (dsRNA) homologous to the target gene sequence, which is artificially synthesized in vitro or introduced into cells, degrades mRNA, thereby causing gene silencing by mRNA with sequence homology. Small interfering RNA (siRNA) is a double-stranded RNA with a length of 21-25 base pairs. The nuclease Dicer in the cytoplasm processes dsRNA into siRNA with a length of 21-25 nucleotides. siRNA combines with various proteins to form an RNA-induced silencing complex (RISC), which targets and cuts specific mRNA, which is then degraded, thereby blocking the expression of the corresponding gene. It mainly acts on the cytoplasm.

[0005] Although nucleic acid drugs have many advantages, they still have to overcome three major difficulties when entering the human body. First, the large molecular weight and negative charge of nucleic acids prevent them from passing through biological membranes freely. Second, RNA is unstable and easily degraded by RNase enzymes in plasma and tissues, and quickly cleared by the liver and kidneys and recognized by the immune system. Third, after entering the cell, it is "stuck" in the endosome and cannot function (lysosomal escape). The delivery of nucleic acid drugs by aerosol inhalation can be efficiently and targeted to the lungs or specific cells in the lungs, and has inherent advantages for lung diseases. Compared with oral medication and intravenous injection, it can greatly reduce the drug dose required for treatment, reduce liver and kidney toxicity, avoid first-pass effects, and has better compliance, which is suitable for the long-term treatment of chronic diseases. Conventional drugs cannot meet the requirements of aerosol particles, cannot be cleared through the respiratory tract, and then deposited in the lungs, increasing the risk of lung infection.

[0006] Fluorocarbon (PFCs) nanoemulsion is a nanomaterial with unique functions, which is suitable for various biomedical fields such as drug delivery and bioimaging. PFCs nanoemulsion has the characteristics of low toxicity and stable chemical properties. PFCs nanoemulsion also has high oxygen solubility and is developed as a blood substitute. However, due to the strong chemical inertness of fluorocarbon compounds, it is difficult to load nucleic acid drugs, which leads to technical difficulties such as failure to protect nucleic acid molecules, inability of nucleic acid molecules to enter biological membranes, and difficulty in achieving lysosomal escape. Summary of the invention

[0007] The purpose of the present invention is to prepare an aerosolized inhalable fluorocarbon nanotransfection reagent, which can encapsulate nucleic acid (siRNA) and deliver it into cells after combining with nucleic acid, avoiding degradation by nucleases before transfection, and playing a role in alleviating pulmonary fibrosis.

[0008] The inhalable fluorocarbon nanotransfection reagent of the present invention comprises cationic lipids, neutral phospholipids, cholesterol, auxiliary lipids, stabilizers and fluorocarbons (PFCs).

[0009] It is further defined that the molar ratio of the cationic lipid, neutral phospholipid, cholesterol and auxiliary lipid is 60%-20%: 40%-10%: 40%-10%: 30%-0%.

[0010] It is further defined that the stabilizer accounts for 1%-10% by volume.

[0011] It is further defined that the volume proportion of the fluorocarbon compounds (PFCs) is 1% to 30%.

[0012] It is further defined that the cationic lipid is at least one of didecyl dimethyl ammonium bromide (DDAB), DLin-MC3-DMA, dioleoyl trimethyl ammonium chloride (DOTMA), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and dimethylaminoethyl formyl-cholesterol (DC-cholesterol).

[0013] It is further defined that the neutral phospholipid is at least one of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine DSPE.

[0014] It is further defined that the auxiliary lipid is at least one of PEGylated lipid and dioleoylphosphatidylethanolamine (DOPE).

[0015] It is further defined that the stabilizer is at least one of glycerol, sucrose and trehalose.

[0016] It is further defined that the fluorocarbon compound (PFCs) is at least one of perfluoro-15-crown-5-ether, perfluorooctane bromide, perfluorobutyric acid, heptacosyltributylamine, and iodinated fluorocarbon compounds.

[0017] The application of the nanotransfection reagent is for the encapsulation and delivery of nucleic acids, and the siRNA sequence is: CCGTGAACTTAACTTTCAA.

[0018] It is further defined that the particle size of the delivered siRNA / PFCs is 100nm-160nm; preferably, the particle size of the delivered siRNA / PFCs is 110nm-130nm.

[0019] It is further defined that the Zeta potential of the delivered siRNA / PFCs is 40mV-80mV.

[0020] The atomized inhalable fluorocarbon nanotransfection reagent of the present invention adopts at least one of the preparation methods well known in the technical field, such as thin film dispersion method, ethanol injection method, reverse evaporation method, freeze drying method, freeze-thaw method, organic solvent volatilization method, and ultrasonic dispersion method to prepare the fluorocarbon nanotransfection reagent.

[0021] It is further defined that the PFCs described in the present invention are prepared by ultrasonic dispersion, specifically by the following method:

[0022] Step 1: Accurately weigh the cationic lipid, neutral phospholipid, cholesterol, and auxiliary lipid, dissolve them in a mixed organic solvent, remove the organic solvent by rotary evaporation at 40°C-45°C under reduced pressure for 2h-4h, and dry them in a constant temperature vacuum incubator at 37°C-45°C overnight.

[0023] Step 2: Then add pure water for hydration, add stabilizer and fluorocarbons (PFCs), and perform ultrasonic treatment for 10-20 minutes in an ice bath.

[0024] Step 3: Then use a liposome extruder to extrude a 200 nm membrane, extrude 5-20 times, encapsulate with a stable nitrogen, argon or other inert gas flow, and store at 4°C

[0025] It is further defined that the organic solvent described in step 1 is a mixture of chloroform and methanol in a volume ratio of 3:1.

[0026] It is further defined that the hydration time in step 1 is 50 min.

[0027] It is further defined that the ultrasonic condition in step 1 is 110 W, working for 3 min, 4 times.

[0028] The preparation method of the siRNA fluorocarbon nanotransfection reagent (siRNA / PFCs) complex of the present invention is carried out according to the following steps:

[0029] The above-mentioned fluorocarbon nanotransfection reagent is specifically operated according to the following steps: 0.01 μL-16 μL of fluorocarbon nanotransfection reagent is mixed with 0.01 μL-1 μL of siRNA solution, slightly vortexed, and incubated at room temperature for 15 min-30 min to obtain siRNA fluorocarbon nanotransfection reagent complex (siRNA / PFCs).

[0030] The principle of gene transfection of the fluorocarbon nanotransfection reagent of the present invention is:

[0031] The surface of the fluorocarbon nanotransfection reagent carries a positive charge, while the siRNA drug carries a negative charge. The two combine with each other through electrostatic interaction to form a siRNA fluorocarbon nanotransfection reagent complex; the surface of the complex carries a positive charge and is also easy to combine with the negatively charged cell surface, which is beneficial for the delivery of nucleic acid drugs into cells using endocytosis.

[0032] Cationic lipids can efficiently encapsulate siRNA drugs, provide positive charges, and assist in in vivo transfection.

[0033] Neutral phospholipids are membrane skeleton materials and the main components of lipid membranes.

[0034] Cholesterol can increase the stable nanoparticle structure of liposomes, regulate membrane fluidity, improve particle stability, and thus reduce permeability.

[0035] Auxiliary phospholipids can stabilize the double-layer membrane and reduce the toxicity of positive components. It can also make the endosomal membrane unstable, promote the release of nucleic acids in cells, and assist cell penetration. Maintain the microscopic morphology of liposomes and make the lysosomal membrane unstable.

[0036] Stabilizer, improves the low-temperature stability of nanotransfection reagents and the stability of siRNA drugs.

[0037] Perfluorocarbons (PFCs) are suitable for drug delivery and have the advantages of low toxicity, stable chemical properties, high oxygen solubility, and are being developed as blood substitutes.

[0038] The siRNA / PFCs prepared by the present invention for delivery of aerosol inhalation have the following advantages: uniform and small particle size, suitable for aerosol administration, and the aerosolized nanotransfection reagent has the advantages of small toxic and side effects, good patient compliance, high transfection efficiency, etc.

[0039] In order to further understand the features and technical contents of the present invention, please refer to the following detailed description and drawings of the present invention. However, the attached drawings are only provided for reference and illustration and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The agarose gel electrophoresis experiment was used to investigate the binding ability of the siRNA / PFCs complex;

[0041] Figure 2 It is the particle size distribution diagram of PFCs nanotransfection reagent;

[0042] Figure 3 It is the Zeta potential diagram of PFCs nanotransfection reagent;

[0043] Figure 4 This is a transmission electron microscope morphology photo of the PFCs nanotransfection reagent;

[0044] Figure 5 This is the result of MTT toxicity experiment of NIH-3T3 cell line co-incubated with transfected probe;

[0045] Figure 6 This is a comparison chart of siRNA / PFCs transfection efficiency results;

[0046] Figure 7 This is a comparison chart of the effects of gene silencing on pulmonary fibrosis markers;

[0047] Figure 8It is the PFCs nanotransfection reagent. The nanotransfection reagent has a stable placement and protective effect on nucleic acids.

[0048] Fig. 9 This is a Micro-CT image of the lungs of mice with pulmonary fibrosis after treatment with siDnm3os / PFCs aerosol nanotransfection reagent, a pulmonary fibrosis treatment drug. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0050] Example 1: The preparation method of the fluorocarbon nanotransfection reagent in this example is carried out according to the following steps:

[0051] Step 1: Accurately weigh 12 mg of cationic lipid, 5 mg of neutral phospholipid, 3 mg of cholesterol, and 2 mg of auxiliary lipid, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 45°C under reduced pressure for 2.5 h, and dry in a constant temperature vacuum incubator at 45°C overnight.

[0052] Step 2: Then add pure water for hydration for 50 minutes, add 0.04 mL of stabilizer and 0.2 mL of fluorocarbons (PFCs), and perform ultrasonic treatment at 110 W for 3 minutes under ice bath conditions, with an interval of 5 minutes for 4 times.

[0053] Step 3: Then, a liposome extruder was used to extrude a 200 nm membrane, which was extruded 20 times, and the membrane was packaged with a nitrogen gas flow and stored at 4°C.

[0054] Wherein, the cationic lipid is (2,3-dioleoyl-propyl)-trimethylamine (DOTAP).

[0055] The neutral phospholipid is dipalmitoylphosphatidylcholine (DPPC).

[0056] The dioleoylphosphatidylethanolamine (DOPE).

[0057] The stabilizer is glycerol.

[0058] The fluorocarbon compound (PFCs) is perfluorooctyl bromide (PFOB).

[0059] Example 2: The preparation method of the siRNA fluorocarbon nanoemulsion (siRNA / PFCs) complex in this example is carried out according to the following steps:

[0060] 12 μl of fluorocarbon nanoemulsion was mixed with 1 μl of siRNA solution, vortexed slightly, and incubated at room temperature for 15 min-30 min to obtain siRNA fluorocarbon nanotransfection complex (siRNA / PFCs).

[0061] The following test was used to verify the effect of the siRNA fluorocarbon nanotransfection reagent invention

[0062] Experiment 1: An agarose gel electrophoresis experiment was used to examine the binding ability of the siRNA fluorocarbon nanotransfection reagent of the present invention.

[0063] Weigh 1.6g agarose and dissolve it in 40mL 1xTBE solution. Heat it in a microwave oven to completely dissolve the agarose particles. After cooling, add 4uL GelRed to the agarose gel. Pour the 50°C agarose gel into the gel tank until it is completely cooled. Mix the fluorocarbon nanotransfection reagent and siRNA in mass ratios of 0:1, 4:1, 8:1, 12:1, 16:1, etc., mix with 2uL 5xDNAloading Buffer and add to the loading well. Set the electrophoresis voltage to 100v and perform electrophoresis for 10 minutes. The experimental results were analyzed by a UV gel imaging system. The results show that the gel retardation bands of siRNA fluorocarbon nanotransfection reagents formed at different mass ratios are different. Figure 1 It can be seen that lane 1 is a nucleic acid with a mass ratio of 0:1, and the band is the brightest, indicating that it contains the most nucleic acid and is not coated. As the mass of the nanotransfection reagent increases, the band gradually becomes darker. When the mass ratio of the nanotransfection reagent to the nucleic acid is 12:1 or above, the gel electrophoresis blocking experiment shows a gel blocking phenomenon, indicating that when the mass ratio is 12:1 or above, the nanotransfection reagent and the nucleic acid are completely fused. We chose to use a mass ratio of 12:1 for the following experiments.

[0064] Experiment 2: Determination of particle size and surface potential Zeta of fluorocarbon nanotransfection reagents

[0065] After the nanotransfection reagent of Example 1 was diluted with 3 mL of ultrapure water, its particle size distribution and surface potential were measured using a Malvern particle size analyzer. The results are as follows: Figure 2 , 3 shown.

[0066] Experiment 3: siRNA / PFCs transmission electron microscopy (TEM) detection

[0067] The prepared PFCs nanotransfection reagent was subjected to transmission electron microscopy detection using transmission electron microscopy negative staining technology, in order to observe whether the morphology of the PFCs nanotransfection reagent prepared in Example 1 is stable.

[0068] TEM of the PFCs nanotransfection reagent prepared in Example 1 is as follows Figure 4 As shown, the prepared PFCs have a regular spherical appearance and are evenly dispersed.

[0069] Experiment 4: Cytotoxicity of siRNA / PFCs

[0070] The cytotoxicity test of the fluorocarbon nanotransfection reagent was performed using the MTT method. The present invention uses NIH-3T3 as a cell model to detect the cytotoxicity of the siRNA fluorocarbon nanotransfection reagent under different mass ratios of the siRNA fluorocarbon nanotransfection reagent. NIH-3T3 cells were cultured at 4x10 4 The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured for 24 hours. A blank control was set up. Complete culture medium was added to the cationic liposome / nucleic acid complex solution at a mass ratio of 4:1, 8:1, 12:1, and 16:1, and cultured for 24 hours. The operation was performed in triplicate. 10 μl of MTT was added to the culture medium in each well and incubated at 37°C for 4 hours. The culture medium was removed, 150 μl of DMSO was added, and the cells were incubated at 37°C for 10 minutes to dissolve the MTT crystals and shaken for 10 minutes. The cytotoxicity was detected by MTT using an enzyme marker at a wavelength of 490 nm. The results are as follows: Figure 5 shown.

[0071] Cell survival rate = absorbance value of drug group - absorbance value of zero-adjustment group / absorbance value of control group (blank) - absorbance value of zero-adjustment group x 100%

[0072] Experiment 5: Transfection efficiency experiment

[0073] To verify the transfection effect of the fluorocarbon nanotransfection reagent of the present invention, we selected lnc Dnm3os as the target gene, and used the fluorocarbon nanotransfection reagent prepared by the method described in Example 1 to transfect a siRNA targeting lnc DNM3OS and a control siRNA into lung fibroblast NIH-3T3 cells. The results are as follows: Figure 6 shown.

[0074] Compared with the control siRNA group, the unprotected naked siDnm3os group had no significant statistical difference. Compared with the siDnm3os not loaded with PFCs, the siDnm3os / PFCs group had the highest silencing efficiency, indicating that fluorocarbon nanotransfection reagents can improve gene transfection efficiency.

[0075] 24 hours after siRNA transfection of NIH-3T3 cells, the relative expression of lnc Dnm3os in NIH-3T3 cells was detected by real-time quantitative PCR.

[0076] 1. RNA Extraction and cDNA Reverse Transcription

[0077] Remove the culture medium, wash with PBS buffer, add 1ml Trizol, blow thoroughly with a gun, let it stand at room temperature for 5 minutes, add 200μl chloroform per 1ml Trizol, cover the EP tube tightly and shake vigorously for 15 seconds, centrifuge at 12000rpm at 4℃ for 10min, take the upper aqueous phase to a new RNase-free EP tube, add an equal volume of isopropanol, and gently invert to mix. Let it stand at room temperature for 10min to precipitate RNA, and centrifuge at 12000rpm for 10min. Carefully discard the supernatant, mix the precipitated RNA with 1ml 75% ethanol, centrifuge at 12000rpm at 4℃ for 5min, and wash with isopropanol. Wash 2-3 times. Discard the supernatant and dry the RNA at room temperature. Add 10μl DEPC water to dissolve. Take 1μl of RNA and use Nanodrop to measure the absorbance at 260nm to determine the concentration and purity of the RNA. Take 500 ng of total RNA and reverse transcribe the RNA into cDNA using an RNA reverse transcription kit.

[0078] 2. RT-qPCR detection

[0079] The reaction system was 10 μl, 5 μl SYBR, 1 μl cDNA, 1 μl primer, and 3 μl DEPC water, and the reaction conditions were 95°C, 2 min, 94°C, 30 s, 60°C, 1 min, and 40 cycles.

[0080] 3. Data analysis: The ΔΔCT method was used to analyze the expression of target genes.

[0081] Experiment 6: Effects of silencing Dnm3os on pulmonary fibrosis markers

[0082] Functional effects of silencing Dnm3os on TGF-β signaling in lung fibroblasts. NIH-3T3 cells were transfected with control siRNA, naked siDnm3os, unloaded PFCs siDnm3os, and siDnm3os / PFCs, and the mRNA expression of pulmonary fibrosis markers Col1a1 and Col3a1 was detected. 24 h after transfection, total RNA was isolated from cells, and quantitative RT-PCR was used to detect Col1a1 and Col3a1 mRNA, and normalized to the corresponding level of β-actin mRNA. The results are shown in Figure 7 shown.

[0083] Compared with the control group siRNA, naked siDnm3os, and siDnm3os not loaded with PFCs, the expression of Col1a1 and Col3a1 mRNA in the siDnm3os / PFCs group was the lowest. The results showed that silencing Dnm3os through transfection with fluorocarbon nanotransfection reagents could alleviate pulmonary fibrosis to a certain extent.

[0084] Experiment 7: Storage stability of fluorocarbon nanotransfection reagents and their protective effect on nucleic acid (siRNA)

[0085] The fluorocarbon nanotransfection reagent of Example 1 was stored in a refrigerator at 4°C, taken out at regular intervals within two weeks, diluted with ultrapure water and measured for particle size and Zeta potential. The results are shown in Table 1, indicating that the particle size and Zeta potential of the nanotransfection reagent of Example 1 did not change significantly within 2 weeks, and had good stability under storage conditions of 4°C.

[0086] Table 1: Stability of Fluorocarbon Nanotransfection Reagent in Example 1

[0087] Detection time (days) Average particle size (nm) PDI Potential (mV) 1 120.6 0.131 57.3 3 119.8 0.115 58.2 5 127.0 0.297 34.7 7 136.3 0.296 42.2 14 133.8 0.254 56.1

[0088] The fluorocarbon nanotransfection reagent of Example 1 was placed at 4° C. for 24 hours at a mass ratio of siRNA to fluorocarbon nanotransfection reagent of 12:1, and the protective effect of the fluorocarbon nanotransfection reagent on siRNA was investigated by agarose gel electrophoresis.

[0089] Agarose gel electrophoresis results Figure 8 As shown, compared with the standard nucleic acid solution, the siRNA / PFCs agent of the present invention still has no free nucleic acid after being placed for 24 hours, and the brightness of the electrophoresis band after demulsification with Triton X-100 is equivalent to that of the standard nucleic acid solution band, indicating that the fluorocarbon nanotransfection reagent of the present invention can protect nucleic acids and has high stability.

[0090] Experiment 8: Micro-CT images of the lungs of mice with pulmonary fibrosis after treatment with aerosolized inhaled drug siDnm3os / PFCs

[0091] In this example, a mouse pulmonary fibrosis model was established to investigate the efficacy of the pulmonary fibrosis therapeutic drug siDnm3os / PFCs prepared in Example 1 on pulmonary fibrosis.

[0092] Grouping and drug delivery:

[0093] The mice were randomly divided into three groups: healthy group, BLM group, siNC / PFCs group and pulmonary fibrosis treatment group siDnm3os / PFCs group.

[0094] After 7 days of bleomycin induction, the mice were randomly divided into groups according to the above grouping, and treatment and administration began on the 8th day for a total of 3 times.

[0095] Drug administration for each group:

[0096] Control group: mice were allowed to breathe freely in the nebulizer inhalation device and inhale normal saline in a conscious state;

[0097] BLM group: Bleomycin-induced mice (same as above) were allowed to breathe freely with aerosolized normal saline;

[0098] siNC / PFCs group: mice induced by bleomycin were allowed to breathe freely and aerosolized with an equal volume of siNC / PFCs as saline;

[0099] siDnm3os / PFCs group: Bleomycin-induced mice were allowed to breathe freely with aerosolized siDnm3os / PFCs in an equal volume to that of normal saline.

[0100] Micro-CT imaging after treatment:

[0101] Micro-CT imaging was performed on the mice in the healthy group, BLM group, siNC / PFCs group, and siDnm3os / PFCs group 7, 14, and 21 days after treatment.

[0102] Micro-CT imaging results are as follows Fig. 9 The results showed that compared with the lungs of healthy mice, the degree of pulmonary fibrosis in the BLM group and the siNC / PFCs group was significantly aggravated, while the degree of pulmonary fibrosis in the siDnm3os / PFCs group was milder than that in the BLM group and the siNC / PFCs group, indicating that the siDnm3os / PFCs group, when inhaled into the lungs of mice through aerosolization, has the effect of slowing down pulmonary fibrosis in mice.

[0103] Sequence Listing, Stand-alone Text

[0104] The sequence of the siRNA used in the specific embodiment is: CCGTGAACTTAACTTTCAA.

Claims

1. A method for preparing a siRNA fluorocarbon nanotransfection reagent complex, characterized in that: The preparation method is carried out according to the following steps: 0.01 μL-16 μL of an atomizable inhalable fluorocarbon nanotransfection reagent for delivering nucleic acid drugs is mixed with 0.01 μL-1 μL of siRNA solution, vortexed slightly, and incubated at room temperature for 15 min-30 min to obtain a siRNA fluorocarbon nanotransfection reagent complex; The mass ratio of siRNA to aerosolizable inhalable fluorocarbon nanotransfection reagent for delivering nucleic acid drugs is 1:12; The nanotransfection reagent comprises cationic lipid, neutral phospholipid, cholesterol and auxiliary lipid, wherein, in molar ratio, cationic lipid: 20% to 60%, neutral phospholipid: 10% to 40%, cholesterol: 10% to 40%, auxiliary lipid: 0% to 30%; the stabilizer accounts for 1% to 10% of the volume of the nanotransfection reagent, and the fluorocarbon compound accounts for 1% to 30% of the volume of the nanotransfection reagent; The cationic lipid is didecyl dimethyl ammonium bromide, DLin-MC3-DMA, dioleoyl trimethyl ammonium chloride, (2,3-dioleoyl-propyl)-trimethylamine or dimethylaminoethyl formyl-cholesterol; The neutral phospholipid is dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine or distearoylphosphatidylethanolamine; The helper lipid is a PEGylated lipid or dioleoylphosphatidylethanolamine; The stabilizer is glycerol, Tween 80, sucrose or trehalose; The fluorocarbon compound is perfluoro-15-crown-5-ether, perfluorooctane bromide, perfluorobutyric acid, heptacosyltributylamine, or iodinated perfluorocarbon; The siRNA sequence is: CCGTGAACTTAACTTTCAA.

2. The preparation method according to claim 1 is characterized in that the atomizable inhalable fluorocarbon nanotransfection reagent for delivering nucleic acid drugs is prepared by a thin film dispersion method, an ethanol injection method, a reverse evaporation method, a freeze-drying method, a freeze-thaw method, an organic solvent volatilization method, or an ultrasonic dispersion method.

3. The preparation method according to claim 1, characterized in that: The particle size of the siRNA fluorocarbon nanotransfection reagent complex is 100nm-160nm, and the Zeta potential is 40mV-80mV.

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