In-situ lipid nano preparation and preparation method thereof

Abstract

The invention provides a novel composition comprising a deep eutectic solvent, a lipid material and a nucleic acid drug. The composition is a uniform and stable dispersion system. The administration mode of the composition is not limited to injection administration and can be selected from nasal administration, oral administration, sublingual administration, skin administration and the like. And the medication compliance of a patient using the nucleic acid medicine can be improved by virtue of more selected administration modes. The composition can significantly improve the delivery efficiency of nucleic acid drugs in vivo, especially in cavity mucous membranes and mucus.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and specifically relates to an in-situ lipid nano preparation and a preparation method thereof. Background Art

[0002] Nucleic acid drugs represented by mRNA and siRNA have low transfection efficiency and are easily degraded by ribozymes, so there are huge challenges in drug delivery. Chemical modification of nucleic acid molecules and lipid nanoparticle-based drug delivery systems are currently the main solutions for the clinical application of nucleic acid drugs. However, the above strategies rely on injection administration, and patient compliance is low. In addition, after systemic administration, lipid nanoparticles are easily taken up by the reticuloendothelial system and deposited in the liver, limiting the potential of nucleic acid drugs to treat other lesions. Therefore, improving the local delivery efficiency of nucleic acid drugs, improving patients' medication compliance, and broadening the application prospects of nucleic acid drugs are still difficult problems that need to be solved in the field of nucleic acid delivery. Summary of the invention

[0003] In order to overcome the above problems existing in the prior art, the object of the present invention is to provide a composition comprising a low eutectic solvent, a lipid material and a nucleic acid drug, and a preparation method and application thereof.

[0004] The present invention provides a composition comprising the following components:

[0005] (i) deep eutectic solvent;

[0006] (ii) lipid material;

[0007] (iii) Nucleic acid drugs.

[0008] According to an embodiment of the present invention, in the composition, (ii) the lipid material and (iii) the nucleic acid drug are dispersed in (i) the low eutectic solvent; preferably, the homogenous material is dispersed in (i) the low eutectic solvent.

[0009] According to an embodiment of the present invention, the composition is a uniform and stable dispersion system, such as a solution system, a suspension system, or an emulsion system.

[0010] According to an embodiment of the present invention, lipid nanoparticles are not formed in the composition system.

[0011] According to an embodiment of the present invention, the composition is capable of forming a lipid nanoformulation (eg, lipid nanoparticle) in situ.

[0012] According to an embodiment of the present invention, the composition can form a lipid nanoformulation in situ in a medium. Preferably, the medium is selected from water, a buffer system, a physiological system (e.g., in vivo, cavity mucosa, such as nasal mucosa, vaginal mucosa, oral mucosa, skin mucosa, etc.).

[0013] According to an embodiment of the present invention, the composition can promote (iii) the penetration of nucleic acid drugs into the mucous membranes of the body cavities, such as the nasal mucosa, vaginal mucosa, oral mucosa, skin mucosa, etc.

[0014] According to an embodiment of the present invention, the composition can promote (iii) the delivery efficiency of nucleic acid drugs in vivo (especially in the luminal mucosa and luminal mucus).

[0015] According to an embodiment of the present invention, the administration method of the composition is selected from nasal administration, oral administration, sublingual administration, skin administration, vaginal administration, local injection, etc. The administration method of the composition is not limited to injection.

[0016] According to an embodiment of the present invention, the nucleic acid drug is selected from RNA drugs (such as miRNA drugs, siRNA drugs (such as KCa3.1 siRNA), mRNA drugs, shRNA drugs, lncRNA drugs, ASO drugs, saRNA drugs) or DNA drugs.

[0017] According to an embodiment of the present invention, the lipid material is selected from one or more of ionizable lipids, cationic lipids (e.g., DLin-MC3-DMA, SM-102, ALC-0315, DOTAP), auxiliary lipids (e.g., DSPC, DOPE, DGTS), cholesterol, β-sitosterol, oxidized cholesterol or its derivatives, and PEG-modified lipids (e.g., DMG-PEG 2000, DSPE-PEG 2000, ALC-0159).

[0018] According to an embodiment of the present invention, the deep eutectic solvent is a two-component or three-component eutectic mixture composed of a hydrogen bond acceptor (such as a quaternary ammonium salt, a tertiary amine, and the like) and a hydrogen bond donor (such as a carboxylic acid, a polyol, a sugar, and the like) in a certain stoichiometric ratio. The freezing point of the deep eutectic solvent is significantly lower than the melting point of the pure substances of each component.

[0019] According to an embodiment of the present invention, in the composition, the content of (ii) lipid material is 1% to 50% (mass percentage), preferably 5%-50%, such as 10%, 30%, 50%.

[0020] According to an embodiment of the present invention, the mass ratio of nucleic acid drugs to (low eutectic solvent + lipid material) is 1:10-5000, preferably 1:100-3000; for example, 1:500, 1:1000, 1:2000.

[0021] According to an embodiment of the present invention, the low eutectic solvent is selected from one or more of an ionic liquid, a non-ionic low eutectic solvent, and a low eutectic mixture; preferably, the low eutectic solvent is an ionic liquid, such as a choline ionic liquid.

[0022] According to an embodiment of the present invention, the preparation method of the low eutectic solvent is: a hydrogen bond donor compound and a hydrogen bond acceptor compound are uniformly mixed in a solid state, and then heated to melt to obtain the low eutectic solvent.

[0023] According to an embodiment of the present invention, the preparation method of the low eutectic solvent is: dissolving a hydrogen bond donor compound and a hydrogen bond acceptor compound in a solvent, mixing and stirring in a certain proportion, and after the reaction is completed, rotary evaporation and vacuum drying are performed to obtain the low eutectic solvent.

[0024] According to an embodiment of the present invention, the hydrogen bond donor compound is selected from any one or more of the following: geranic acid, sorbic acid, malic acid, citric acid, tartaric acid, oxalic acid, lactic acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, acetic acid, malonic acid, succinic acid, caprylic acid, capric acid, oleic acid, linoleic acid, arachidonic acid, retinoic acid, hibiscus acid or chlorogenic acid; preferably, the hydrogen bond donor compound is selected from any one or more of the following: sorbic acid, malic acid, geranic acid, citric acid, oleic acid, linoleic acid, malonic acid; more preferably, the hydrogen bond donor compound is geranic acid.

[0025] According to an embodiment of the present invention, the hydrogen bond acceptor compound is selected from any one or more of the following: choline hydroxide, choline bicarbonate, choline chloride, triethanolamine, diethanolamine, betaine, phospholipid or glycerophosphocholine; preferably, the hydrogen bond acceptor compound is selected from any one or more of the following: choline hydroxide, choline bicarbonate, choline chloride, triethanolamine or diethanolamine; more preferably, the hydrogen bond donor compound is choline bicarbonate.

[0026] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor compound to the hydrogen bond donor compound is 1:0.5-3.

[0027] According to an embodiment of the present invention, the molar ratio of geranic acid to choline bicarbonate is 1:0.5-3, preferably 1:1.

[0028] The present invention also provides a method for preparing the above composition, which comprises the following steps:

[0029] The composition can be obtained by separately preparing a low eutectic solvent system loaded with lipid materials and a low eutectic solvent system loaded with nucleic acid drugs, and then mixing the two.

[0030] According to an embodiment of the present invention, the preparation method comprises the following steps:

[0031] 1) adding lipid material and solvent to a low eutectic solvent, dissolving and mixing, and removing the solvent to obtain a low eutectic solvent system loaded with lipid material;

[0032] 2) adding the nucleic acid drug and the solvent to the deep eutectic solvent, dissolving and mixing, and removing the solvent to obtain a deep eutectic solvent system containing the nucleic acid drug;

[0033] 3) Mixing the low eutectic solvent system respectively loaded with lipid materials and the low eutectic solvent system loaded with nucleic acid drugs to obtain the composition.

[0034] According to an embodiment of the present invention, in step 1), the solvent used is an organic solvent; preferably one or more selected from methanol, ethanol, isopropanol, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and acetone.

[0035] According to an embodiment of the present invention, in step 1), the solvent is removed by vacuum drying or nitrogen blowing drying; preferably, the drying temperature is 25° C. to 60° C.; preferably, the drying time is 20 min to 60 min.

[0036] The lipid material and the low eutectic solvent have high solubility in the organic solvent. The purpose of adding the organic solvent to dissolve is to make the lipid material fully dissolved in the low eutectic solvent.

[0037] According to an embodiment of the present invention, in step 2), the solvent used is enzyme-free water or DEPC water (water treated with diethyl pyrocarbonate).

[0038] According to the embodiment of the present invention, in step 2), the specific operation of removing the solvent is: freezing at -80°C for 2 to 8 hours, and vacuum drying for 30 minutes to 4 hours.

[0039] Nucleic acid drugs and deep eutectic solvents are highly soluble in water. The purpose of adding enzyme-free water or DEPC water to dissolve is to fully dissolve nucleic acid drugs in deep eutectic solvents. The purpose of enzyme-free or DEPC treatment of water is to prevent nucleases in the water from degrading nucleic acid drugs.

[0040] According to an embodiment of the present invention, in step 3), the mixing method is water bath ultrasound; preferably, the power of the water bath ultrasound is 90 Hz; preferably, the water bath temperature is 4° C. to 25° C.; preferably, the water bath time is 5 min to 30 min.

[0041] The purpose of water bath ultrasonic treatment is to evenly disperse the nucleic acid drug and lipid material in the low eutectic solvent.

[0042] The present invention also provides a pharmaceutical composition, which comprises the above composition and a pharmaceutical carrier.

[0043] According to an embodiment of the present invention, the administration method of the pharmaceutical composition is selected from nasal administration, oral administration, sublingual administration, skin administration, vaginal administration, local injection, etc. The administration method of the pharmaceutical composition is not limited to injection.

[0044] According to an embodiment of the present invention, the dosage form of the pharmaceutical composition is selected from gels (eg, in situ gels), ointments, nasal drops, sprays (nasal sprays), emulsions, suspensions, powders, and the like.

[0045] The present invention also provides use of the above composition and the above pharmaceutical composition in preparing products for nucleic acid drug delivery.

[0046] According to an embodiment of the present invention, the delivery method is selected from nasal delivery, oral delivery, sublingual delivery, skin delivery, vaginal delivery, local injection delivery, etc.; for example, non-invasive nasal delivery.

[0047] The present invention also provides the use of the above composition and the above pharmaceutical composition in preparing drugs for treating and / or preventing diseases.

[0048] The present invention also provides an in situ preparation method of a lipid nanoformulation, which comprises: dispersing the above-mentioned composition or pharmaceutical composition in a medium to obtain a lipid nanoformulation (e.g., lipid nanoparticles); preferably, the medium is such as water, a buffer system, or a physiological system (e.g., the nasal cavity, such as nasal mucus, nasal mucosa).

[0049] The present invention also provides a lipid nanoformulation prepared in situ from the above composition or pharmaceutical composition in a medium. Preferably, the medium is water, a buffer system, a physiological system (e.g., in vivo, cavity mucosa, such as nasal mucosa, vaginal mucosa, oral mucosa, etc.).

[0050] Beneficial Effects

[0051] 1) The present invention provides a novel composition comprising a low eutectic solvent, a lipid material, and a nucleic acid drug, wherein the composition is a uniform and stable dispersion system.

[0052] 2) The administration method of the composition of the present invention is not limited to injection administration, and the administration method can be selected from nasal administration, oral administration, sublingual administration, skin administration, vaginal administration, local injection, etc. More selected administration methods can improve the medication compliance of patients using nucleic acid drugs.

[0053] 3) The composition of the present invention can significantly improve the delivery efficiency of nucleic acid drugs in vivo, especially in the luminal mucosa and mucus (promoting penetration and transfection). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0055] Figure 1 The invention discloses a nuclear magnetic spectrum of the choline geranate ionic liquid and raw materials geranic acid and choline bicarbonate, wherein A is choline bicarbonate, B is geranic acid, and C is the choline geranate ionic liquid.

[0056] Figure 2 The system clarification conditions of siRNA-DILs (Example 1) and siRNA-ILs (Comparative Example 2) with different water contents.

[0057] Figure 3 The figure shows the assembly of nanoparticles after siRNA is loaded into the carrier preparation described in Example 1 and hydrated. Wherein, A is the particle size distribution of the self-assembled nanoparticles of the preparation, and B is the zeta potential of the nanoparticles.

[0058] Figure 4 The in vitro release curves of fluorescently labeled FAM-siRNA after being loaded into Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2. Figure 3 In the figure, PBS is comparative example 1, ILs is comparative example 2, 10% DILs is example 1, 30% DILs is example 2, and 50% DILs is example 3.

[0059] Figure 5 TEM image of nanoparticle self-assembly after in vitro release of siRNA-DILs preparation.

[0060] Figure 6 The complex of siRNA and self-assembled CLNs. A is the electrophoresis diagram obtained by agarose gel tissue experiment, and B is the encapsulation efficiency of siRNA in CLNs at different drug loadings determined by RiboGreen method.

[0061] Figure 7 The 20% non-denaturing polyacrylamide gel electrophoresis results of the siRNA-loaded carrier preparations of Comparative Example 1 and Example 1 after being incubated with nuclease for a certain period of time before and after hydration.

[0062] Figure 8 The Materials Studio software was used to construct the hydration process model of the preparation in Example 1, wherein A is the molecular and ionic structure required for model construction, B is the model construction process of 10% DILs encapsulating siRNA diffusing into water, and C is the model used for molecular dynamics simulation.

[0063] Fig. 9 Results and analysis of molecular dynamics simulations performed by Materials Studio. A is the structure at the end of the molecular dynamics simulation (hidden water molecules), B is the structure of the lipid complex encapsulating siRNA, C is the structure of the geranic acid ion micelle formed during the hydration process, D is the radial distribution function analysis between the characteristic groups of each component of the preparation before and after the molecular dynamics simulation, and E is the concentration distribution function analysis of the preparation components before and after the molecular dynamics simulation.

[0064] Fig.10 The uptake of the FAM-siRNA prepared in Example 1, Example 4, Example 5, Comparative Example 1 and Comparative Example 3 by HNEpC cells was observed using a laser confocal microscope.

[0065] Fig.11 In order to determine the in vitro uptake efficiency of FAM-siRNA by cells by flow cytometry, the administration preparation was the FAM-siRNA-loaded carrier preparation prepared in Example 1, Example 4, Example 5, Comparative Example 1, and Comparative Example 4.

[0066] Fig.12 This is a rat nasal mucosa section photographed by laser confocal microscopy 4 hours after nasal administration. The delivery preparation is the FAM-siRNA-loaded carrier preparation prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0067] Fig.13 The results are three-dimensional scans of rat nasal mucosa taken by laser confocal microscopy 4 hours after nasal administration. The delivery preparations are carrier preparations prepared in Example 1, Comparative Example 1, and Comparative Example 2 that simultaneously carry Cy3-siRNA and Cy5-siRNA in an equal molar ratio.

[0068] Fig.14 A is the retention effect of Cy5-siRNA in the rat nasal cavity, taken by a small animal living imaging device. Fig.14 B is the drug retention percentage calculated based on the fluorescence intensity.

[0069] Fig.15 The gene silencing effect at the rat nasal mucosa was determined by rt-qPCR. The delivery formulation A is 16 μg of the carrier formulation containing siRNA targeting β-actin prepared in Example 1, Example 4, Example 5, Comparative Example 1, and Comparative Example 3, and the delivery formulation B is 2, 4, 8, and 16 μg of the carrier formulation containing siRNA targeting β-actin prepared in Example 1.

[0070] Fig.16The carrier preparation containing siRNA targeting KCa3.1 prepared in Example 1 is used for the treatment of allergic rhinitis in rats. A is the modeling and treatment plan of allergic rhinitis in rats, B is the statistics of allergic rhinitis symptoms in rats, C, D, and E are the contents of IgE, prostaglandin D2, and histamine in rat nasal lavage fluid and serum, respectively, F, G, and H are the rt-qPCR results of KCa3.1, MUC5AC, and Tryptase in the nasal mucosa, respectively, I is the H&E staining, AB-PAS staining, and toluidine blue staining results of rat nasal mucosa sections, J is the statistics of rat nasal mucosa thickness, and K is the statistics of the number of mast cell infiltration in the nasal mucosa. M is the immunohistochemical staining results of nasal mucosa sections, and the targets are KCa3.1 and ZO-1, respectively. DETAILED DESCRIPTION

[0071] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0072] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0073] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0074] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0075] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0076] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0077] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0078] DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane (methylsulfate).

[0079] DEPC water: Ultrapure water (primary water) treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure, colorless liquid. It is tested to be free of impurities such as RNA, DNA and protein.

[0080] DLin-MC3-DMA: 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester

[0081] SM-102: 1-Octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate

[0082] ALC-0315: ((4-Hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0083] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0084] DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

[0085] DGTS: 1,2-diacylglycerol-O-4'-(N,N,N-trimethyl)homoserine

[0086] DMG-PEG 2000: Dimyristoylglycerol-Polyethylene Glycol 2000

[0087] DSPE-PEG 2000: Distearoylphosphatidylethanolamine-polyethylene glycol 2000

[0088] ALC-0159: 2-[(Polyethylene glycol)-2000]-N,N-tetracosyl acetamide

[0089] KCa3.1: a medium-conductance calcium-activated potassium channel

[0090] The ionic liquid (ILs) used in the following examples is a choline geranate ionic liquid, and its preparation method is as follows: use an electronic balance to weigh 1.6823g (0.01mol) of geranic acid in a 10mL EP tube, add 5mL of anhydrous ethanol to dissolve, and set aside; weigh 2.0716g (0.01mol) of 80% aqueous solution of choline bicarbonate in a 50mL round-bottom flask, stir magnetically at room temperature, rotate at 300rpm, and gradually add dropwise the geranic acid dissolved in the anhydrous ethanol mentioned above. After the addition is completed, when no bubbles are generated in the solution system, continue stirring for 6h, and then use a rotary evaporator to evaporate in a 60°C water bath for 30min. Transfer the ILs in the flask to a 50mL EP tube, place it in a vacuum drying oven and vacuum dry it for 48h to remove moisture and residual solvents to obtain choline geranate ILs. Characterized by hydrogen nuclear magnetic resonance spectrum, the spectrum is as shown Figure 1 As shown in A-1C, A is choline bicarbonate, B is geranic acid, and C is choline geranate ILs. As can be seen from the figure, compared with the raw material, the chemical shifts of the hydrogen atoms on the choline fragment have no significant change, the carboxylic acid hydrogen signal of the lowest field of the geranic acid fragment disappears, and the chemical shifts of the remaining hydrogens move to the high field, indicating that sorbic acid forms ionic bonds and hydrogen bonds with choline to form ILs. The synthesized ILs product is liquid at room temperature, has a good appearance and shape, and is stable in nature.

[0091] Example 1

[0092] Preparation of carrier combination formulations (lipid-ionic liquid combined formulations) containing siRNA:

[0093] 1) Add 1.6 mg DOTAP and 50 μL anhydrous ethanol to 6.4 mg ILs, vortex for 30 seconds to mix, and dry under nitrogen for 1 hour at room temperature to remove ethanol to obtain ILs loaded with DOTAP (DOTAP-ILs, i.e., blank DILs); 2) Add 8 μg siRNA and 50 μL DEPC water to 8 mg ILs, gently blow and mix, freeze at -80°C for 4 hours, and then vacuum dry for 2 hours to remove DEPC water to obtain ILs loaded with siRNA (siRNA-ILs); 3) Mix the DOTAP-ILs obtained in the above steps with siRNA-ILs, and ultrasonicate in a water bath for 5 minutes at room temperature to evenly disperse DOTAP and siRNA in the ILs to obtain the final siRNA-loaded carrier combination preparation, named siRNA-DILs.

[0094] From the above preparation process, it can be seen that in the final siRNA-carrying carrier combination preparation, the content of the lipid material DOTAP in the siRNA-DILs preparation is 10% (mass percentage), and the mass ratio of siRNA to siRNA-DILs is 1:2000.

[0095] Example 2

[0096] Same as Example 1, except that in step 1), 4.8 mg of DOTAP is loaded into 3.2 mg of ILs. In the final preparation, the content of DOTAP in the siRNA-DILs preparation is 30%.

[0097] Example 3

[0098] Same as Example 1, except that the amount of DOTAP used in step 1) is 8.0 mg, and no ILs is added. In the final preparation, the content of DOTAP in the siRNA-DILs preparation is 50%.

[0099] Example 4

[0100] Same as Example 1, except that step 2) is: adding 32 μg siRNA to 8 mg ILs. In the final preparation, the mass ratio of siRNA to (ILs+DOTAP) is 1:500.

[0101] Example 5

[0102] Same as Example 1, except that step 2) is: adding 16 μg siRNA to 8 mg ILs. In the final preparation, the mass ratio of siRNA to (ILs+DOTAP) is 1:1000.

[0103] Comparative Example 1

[0104] siRNA was dissolved in phosphate buffered saline (PBS) to set siRNA:PBS=1:2000 (mass ratio).

[0105] Comparative Example 2

[0106] 8 μg siRNA was added to 16 mg ILs, and 50 μL DEPC water was added, and the mixture was gently pipetted and mixed, and the mixture was frozen at -80°C for 4 h, and vacuum dried for 2 h to remove the DEPC water, thereby obtaining ILs loaded with siRNA. In the final preparation, siRNA:ILs=1:2000 (mass ratio).

[0107] Comparative Example 3

[0108] 8 μg siRNA was added to 4 mg ILs, and 50 μL DEPC water was added, and the mixture was gently pipetted and mixed, and the mixture was frozen at -80°C for 4 hours, and vacuum dried for 2 hours to remove the DEPC water, thereby obtaining ILs loaded with siRNA. In the final preparation, siRNA:ILs=1:500 (mass ratio).

[0109] Comparative Example 4

[0110] The commercially available transfection reagent Lipofectamine 2000 (Lipo2000) and siRNA were added to the Opti-MEM culture medium and mixed by pipetting, and the ratio of siRNA:Lipo2000 was set to 1:2 (m / v).

[0111] Effect verification example

[0112] (1) Determination of particle size and surface potential of siRNA-DILs after in vitro hydration

[0113] Depend on Figure 2 As shown, the prepared siRNA-DILs (Example 1) and siRNA-ILs (Comparative Example 2) are both clear and transparent. As the water content increases, both gradually become turbid and then become clear. When the water content reaches 99.9%, there is no visible precipitation in the system. The reason for the turbidity of siRNA-ILs is that at a specific water content, the geranyl anions in the ILs form micelles, which are relatively unstable. Therefore, as the water content increases, the micelle structure is destroyed, and ultimately no nanostructure is detected. siRNA-DILs gradually hydrate as the water content increases, and self-assemble into uniformly dispersed nanoparticles without obvious aggregation.

[0114] 400 μg of the siRNA-DILs preparation prepared in Example 1 was taken, 1 mL of DEPC water was added, and the mixture was gently blown to completely hydrate it. The particle size and potential of the lipid complex after hydration were measured using a nanoparticle size potentiometer.

[0115] The results are as follows Figure 3 As shown, Figure 3 A is the particle size distribution of the self-assembled nanoparticles of the preparation, Figure 3 B is the zeta potential of the nanoparticles. Figure 3 It can be seen that the particle size of the self-assembled nanoparticles is uniform, the particle size distribution shows a single peak of 138.7nm, and the zeta potential is 23.3mV.

[0116] (2) Evaluation of in vitro release of siRNA after loading into DILs

[0117] Fluorescently labeled FAM-siRNA was used instead of conventional siRNA to investigate the drug release in PBS of the carrier combination preparations loaded with FAM-siRNA prepared in Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2. A Franz in vitro diffusion cell was selected as a model, and a 50 nm pore size polycarbonate semipermeable membrane was used instead of the nasal mucosa to screen out possible aggregates in the preparation before diffusion into water, ensuring that the siRNA and the preparation group were diffused into water and then self-assembled into lipid nanoparticles.

[0118] The results are as follows Figure 4As shown, compared with the PBS solution of naked siRNA (PBS-Comparative Example 1), ILs (Comparative Example 2) can promote the diffusion of siRNA into water, and siRNA almost completely diffuses from ILs into the receiving pool within 60 minutes. After siRNA was encapsulated in DILs with 10%, 30%, and 50% DOTAP content (Examples 1, 2, and 3), the in vitro release amounts were 35%, 18%, and 13%, respectively, and the diffusion reached equilibrium within 30 minutes.

[0119] Figure 4 In the figure, PBS-Comparative Example 1, ILs are Comparative Example 2, 10% DILs are Example 1, 30% DILs are Example 2, and 50% DILs are Example 3.

[0120] (3) TEM characterization of lipid nanoparticles obtained by in vitro release of siRNA-DILs

[0121] The lipid nanoparticles formed in the receiving pool after 4 hours of in vitro release in Example 1 were characterized by TEM.

[0122] The results are as follows Figure 5 As shown in the figure, after siRNA-DILs diffuse into water through the semipermeable membrane, lipid nanoparticles with a particle size of about 100 nm can be formed, with uniform particle size and regular shape. This result shows that the DILs preparation components can diffuse into the receiving pool together with siRNA and self-assemble to form nanoparticles after hydration.

[0123] (4) Effect of siRNA loading on the encapsulation efficiency of self-assembled siRNA-CLNs

[0124] The siRNA loading capacity is defined as the mass ratio of siRNA to DILs (siRNA:DILs, w / w), and the encapsulation efficiency characterizes the complexing and encapsulation capacity of self-assembled CLNs for siRNA. Figure 6 As shown in the gel retardation experiment, when the drug loading is ≤1:500, siRNA is almost completely complexed with lipid nanoparticles, and its negative charge is neutralized, so it is retained in the sample tank in the agarose gel electrophoresis experiment. In addition, the RiboGreen method further determined that when the drug loading is 1:500, the encapsulation efficiency is 90% ( Figure 6 B).

[0125] (5) Evaluation of DILs preparations against ribozyme degradation

[0126] The PBS solution of siRNA prepared in Comparative Example 1, the combined carrier preparation loaded with siRNA prepared in Example 1, and the hydrated combined carrier preparation loaded with siRNA in Example 1 were incubated with RNase A for a certain period of time, and the degradation of siRNA was determined by 20% non-denaturing polyacrylamide gel electrophoresis.

[0127] The results are as follows Figure 7 As shown. Figure 7 It can be seen that naked siRNA was completely degraded by RNase A within 15 minutes, while the DILs preparation and the lipid nanoparticles formed after hydration could protect siRNA from RNase A hydrolysis within 4 hours.

[0128] (6) Molecular dynamics simulation of the self-assembly process of siRNA-DILs nanoparticles after hydration

[0129] Materials Studio software was used for model construction and molecular dynamics simulation. Figure 8 A is the molecular structure of each component of siRNA-DILs, Figure 8 B is the process of building the preparation hydration model. Figure 8 C is the model used for molecular dynamics simulation. In brief, the siRNA-DILs prepared in Example 1 were placed in the center of the box, and water molecules were filled around it. The force field was selected as COMPASSIII, the ensemble was selected as NVT, and the molecular dynamics simulation time was set to 3 ns.

[0130] Processing of molecular dynamics simulation results ( Fig. 9 A), from which the lipid nanoparticle structure encapsulating siRNA was cut out ( Fig. 9 B). DOTAP cations are all concentrated at the ends of siRNA, with the hydrophilic quaternary ammonium groups facing the inside and outside, while the hydrophobic fatty long chains are concentrated together. In comparison, the diffusion behavior of choline and geranyl ions in water is more complicated. A portion of geranyl and choline ions diffuse into the water from the DILs preparation, while another portion of choline ions are embedded in the siRNA structure. It is worth noting that the geranyl anions that diffuse into the water spontaneously aggregate to form micelle structures ( Fig. 9 C), containing 4 to 10 geranyl anions, with the hydrophilic carboxyl end facing outward. At the same time, there are also a few geranyl micelles around siRNA and in the gaps of DOTAP aggregates, which may stabilize the lipid nanoparticles. Fig. 9 D is the radial distribution function analysis between the characteristic groups of different components. After hydration, the phosphate group of siRNA is most likely to interact with the hydroxyl group of choline, and the maximum probability of interaction distance is In addition, in the original preparation, the most significant interaction between geranic acid and choline occurs between the hydroxyl group of choline and the carboxyl group of geranic acid, with an interaction distance of After the preparation diffused into water, the probability of interaction between the two groups was reduced to half, indicating that choline and geranic acid ions separated from each other during the diffusion process. Fig. 9 E is the spatial concentration analysis of the components of the DILs preparation, which again shows that some geranic acid and choline ions diffuse into the water, while DOTAP tends to aggregate.

[0131] (7) Evaluation of DILs in promoting cellular uptake of siRNA Human nasal mucosal epithelial cell line HNEpC, human lung adenocarcinoma epithelial cell line Calu-3, and mouse mast cell line P815 were selected as models for cellular uptake evaluation. Fluorescently labeled FAM-siRNA was used instead of conventional siRNA to prepare the siRNA-loaded preparations prepared in Example 1, Example 4, Example 5, Comparative Example 1, Comparative Example 3, and Comparative Example 4. The above preparations were diluted with the culture medium Opt-MEM to a siRNA concentration of 100 nM and incubated with HNEpC and Calu-3 cells for 4 h. The above preparations were diluted with the culture medium Opt-MEM to a siRNA concentration of 20 nM and incubated with P815 cells for 4 h. Then, the uptake of FAM-siRNA by cells was observed under laser confocal microscopy, and the siRNA transfection was measured by flow cytometry and the transfection efficiency was calculated.

[0132] The results of laser confocal microscopy were as follows: Fig.10 As shown in Figure 3, ILs failed to promote the uptake of siRNA by HNEpC cells (Comparative Example 3), but after adding DOTAP, the uptake of siRNA by HNEpC cells increased significantly, and increased accordingly with the increase of drug loading. Fig.11 As shown, when the siRNA loading in DILs was 1:500 (Example 4), the uptake efficiency of HNEpC, Calu-3 and P815 for siRNA reached 96.92%, 69.86% and 77.17%, respectively. When the loading was reduced to 1:2000 (Example 1), the uptake efficiency of the three cells decreased to 46.85%, 13.64% and 0.61%, respectively. It is worth noting that for P815 cells, when the loading was as high as 1:1000 (Example 5), the effect of DILs in promoting cell uptake of siRNA was better than that of the commercially available transfection reagent Lipofectamine 2000 (Comparative Example 4).

[0133] (8) Evaluation of the effect of DILs on the penetration of siRNA-loaded nasal mucosa

[0134] Fluorescently labeled FAM-siRNA was used instead of conventional siRNA, and a siRNA-loaded preparation was prepared according to Example 1, Comparative Example 1, and Comparative Example 2 for standby use. Before nasal administration, male SD rats were anesthetized and intubated through the oral cavity to prevent the liquid from flowing into the airway and causing suffocation. After intubation, a carrier preparation containing 8 μg of FAM-siRNA was delivered to the rat nasal cavity. The rat nasal mucosa was taken 4 hours after administration, and the penetration of siRNA in the nasal mucosa was observed under a laser confocal microscope after frozen sectioning.

[0135] The results are as follows Fig.12As shown, 4 hours after nasal delivery of the preparation, both ILs and DILs can promote siRNA penetration into the deep mucosa. Careful observation revealed that after delivery with ILs (Comparative Example 2), siRNA was diffusely distributed between mucosal epithelial cells, while siRNA delivered with DILs (Example 1) was concentrated around the cell nucleus, which indirectly indicates that DILs have the dual ability of promoting penetration and transfection.

[0136] (9) In vivo verification of siRNA-DILs self-assembly into nanoparticles in the nasal mucosa

[0137] The siRNA was fluorescently labeled with Cy3 and Cy5, respectively, and the molar ratio of Cy3-siRNA to Cy5-siRNA was set to 1:1. The preparation was loaded according to Example 1 and Comparative Example 1-2 for standby use. Male SD rats were anesthetized, and the above-mentioned carrier preparation containing a total of 8 μg Cy3 / Cy5-siRNA was delivered to the nasal cavity through oral airway intubation. After 4 hours of administration, the nasal mucosa at the nasal septum of the rat was completely removed, placed on a slide, and sealed with a sealing agent containing DAPI, and immediately observed and photographed with the Z-stack module of CLSM.

[0138] The results are as follows Fig.13 As shown, the DILs preparation can effectively deliver Cy3 / Cy5-siRNA to the deep part of the nasal mucosa and produce a significant fluorescence resonance energy transfer (FRET) effect. This phenomenon shows that siRNA-DILs self-assembles in situ to produce lipid nanoparticles during the penetration of the nasal mucosa, so that the Cy3-siRNA and Cy5-siRNA originally dispersed in the DILs are aggregated in the in situ nanoparticles, thereby producing a FRET effect. In contrast, the Cy3 / Cy5-siRNA-PBS solution (Comparative Example 1, PBS group) failed to effectively penetrate into the nasal mucosal tissue. Cy3 / Cy5-siRNA-ILs (Comparative Example 2, ILs group) can effectively penetrate the nasal mucosa under the permeation-promoting effect of ILs, but no obvious FRET effect is produced, indicating that the siRNA delivered only by ILs does not undergo in situ self-assembly at the nasal mucosa.

[0139] (10) Evaluation of siRNA nasal retention ability

[0140] Fluorescently labeled Cy5-siRNA was used instead of conventional siRNA, and a carrier preparation loaded with siRNA was prepared according to Example 1, Comparative Example 1, and Comparative Example 2 for later use. Male SD rats were anesthetized and intubated via the oral cavity, and the carrier preparation loaded with 8 μg of Cy5-siRNA was delivered to the rat nasal cavity. The retention of siRNA in the rat nasal cavity was observed by a small animal in vivo imaging device at 0, 1, 2, 4, and 12 hours after administration ( Fig.14 A).

[0141] The statistical results are as follows Fig.14 As shown in Figure B, only 24.4% of the drug in the PBS solution of naked siRNA (Comparative Example 1) was retained in the nasal cavity after 1h of administration, and only 5.4% remained after 12h, indicating that the transfection and penetration ability of siRNA itself was poor, and thus it was easily removed in the nasal cavity. With ILs encapsulated siRNA (Comparative Example 2), 91.5% of siRNA was still retained in the nasal cavity after 4h of nasal administration, but only 20.3% remained after 12h, which proved that ILs could effectively promote siRNA penetration and thus be retained in the nasal mucosa. The reason why the retention amount in the ILs group decreased significantly after 12h of administration was that siRNA was not effectively transfected into the cell after penetrating into the nasal mucosa, and was diffusely distributed among the epithelial cells of the nasal mucosa, and thus was gradually removed. Compared with the above two groups, 40.0% of siRNA was still retained in the nasal mucosa after 12h of siRNA-DILs administration, and the retention amount was significantly higher than that of the naked siRNA group and the ILs group, which once again confirmed that DILs could promote siRNA penetration while improving its transfection efficiency.

[0142] (11) Evaluation of gene silencing effects of siRNA-DILs in nasal mucosa

[0143] The siRNA targeting rat β-actin was loaded into the preparation according to Example 1, Example 4, Example 5, Comparative Example 1, and Comparative Example 3 for standby use. SD rats were anesthetized, and the above-mentioned vector preparation containing 16 μg was delivered to the nasal cavity through oral airway intubation, with 6 rats in each group. 48 hours after administration, the rat nasal mucosa was taken, total RNA was extracted, and rt-qPCR detection was performed after reverse transcription.

[0144] The results are as follows Fig.15 As shown in A, after 48 hours of administration of siRNA-DILs preparations with drug loading of 1:500, 1:1000, and 1:2000, the mRNA level of β-actin in the nasal mucosa of rats was reduced by about 50% compared with that of blank rats, and there was no significant difference in the gene silencing effect of the above three preparation groups, indicating that the preparations with the above three drug loading amounts can all cause significant gene silencing in the nasal cavity of rats. At the same time, nasal delivery of siRNA-ILs (Comparative Example 3) failed to cause significant gene silencing effect, and the β-actin level was not significantly different from that of the blank control group. This result once again proves the importance of lipid materials in promoting siRNA transfection.

[0145] The drug loading was fixed at 1:2000 to further explore the effect of the dosage on the gene silencing effect. A carrier combination preparation containing siRNA targeting rat β-actin was prepared according to Example 1 and set aside. SD rats were anesthetized and intubated through the oral airway to deliver 2, 4, 8, and 16 μg of the above-mentioned carrier preparation into their nasal cavity. Six rats were set up in each dosage group. Fig.15As shown in B, 48 hours after administration, when the siRNA dose reached 8 μg or more, the mRNA level of β-actin decreased by more than 50%, while the gene silencing effect of the 2 μg and 4 μg dose groups was not significant compared with the blank group.

[0146] Comparison of Example 1 (10% DILs) and Comparative Example 2 (ILs) shows that the addition of lipid material DOTAP can promote the penetration of nucleic acid drugs into cells.

[0147] (12) Gene silencing therapy for allergic rhinitis (AR) in rats

[0148] Forty male SD rats weighing about 200 g were selected and randomly divided into four groups, each with 10 rats, namely, AR blank control group (AR group), non-coding siRNA negative control group (siNC group), KCa3.1 siRNA treatment group (siKCa3.1 group) and healthy group (Healthy group). The rats in the AR, siNC and siKCa3.1 groups were intraperitoneally injected with 0.5 mL / rat of OVA sensitizer, once every two days, for a total of seven times (i.e., days 1, 3, 5, 7, 9, 11, and 13). The rats in the Healthy group were intraperitoneally injected with 0.5 mL / rat of PBS, once every two days, for a total of seven times. Starting from the 21st day of modeling, 10 μL of OVA solution (125 mg / mL) was sensitized to the rats in the AR, siNC and siKCa3.1 groups every day, once a day, for a total of 7 times (i.e., days 21 to 27). Six hours after each intranasal administration of OVA, negative control siRNA (siNC) and siRNA targeting KCa3.1 (siKCa3.1) (prepared according to the method of Example 1) encapsulated in DILs were delivered to the nasal cavity of rats in the siNC and siKCa3.1 groups, respectively. Fig.16 As shown in A.

[0149] On the 28th day of modeling and treatment, the number of nasal scratching in the AR and siNC groups was significantly different from that in the siKCa3.1 and Healthy groups (P<0.0001). Fig.16 B), which preliminarily indicates that delivery of siKCa3.1 by DILs can effectively alleviate AR symptoms. Fig.13 As shown in F-13H, in the AR disease model, the mRNA level of KCa3.1 in the nasal mucosa was approximately 6.2 times that of the Healthy rat group ( Fig.16 F), the mRNA level of mucin MUC5AC was 3.9 times that of the Healthy group ( Fig.16 G), the mRNA level of tryptase was 12.2 times that of the rats in the Healthy group ( Fig.16H). There was no significant difference between the results of the siNC group and the AR group (P>0.05). After treatment with siKCa3.1 delivered by DILs, the mRNA levels of KCa3.1, MUC5AC, and Tryptase in rats were significantly decreased (P<0.0001) ( Fig.16 F-16H). In addition, the ELISA results are as follows Fig.16 As shown in C-16E, the levels of IgE, histamine, and prostaglandin D2 (PGD2) in the serum and nasal lavage fluid of rats in the AR group were significantly increased. After administration of the DILs carrier preparation loaded with siKCa3.1 prepared in Example 1, the levels of the above three were significantly downregulated. The results of H&E staining of nasal mucosal sections showed that ( Fig.16 I), the nasal mucosa of rats in the AR group showed a large number of inflammatory cell infiltrations, disordered arrangement of the epithelial cell layer, mucosal edema, epithelial cell destruction and increased mucosal thickness; the inflammatory cell infiltration in the siKCa3.1 group was reduced, there was no obvious edema, the degree of mucosal tissue destruction was mild, and the mucosal thickness was not significantly different from that in the Healthy group ( Fig.16 J). The results of AB-PAS staining of nasal mucosa sections showed that ( Fig.16 I), goblet cells in the AR group were disordered, acidic mucus secretion increased, and neutral mucus was observed in the basal layer; the mucus secretion in the upper layer of the mucosa in the siKCa3.1 group was significantly reduced, and the cilia were relatively intact. Toluidine blue (TB) staining results of nasal mucosal sections ( Fig.16 I) showed that the mast cell infiltration in the AR group increased significantly, mostly concentrated in the lower layer of the mucosa and around blood vessels; the mast cell infiltration in the siKCa3.1 group was significantly reduced ( Fig.16 K). In addition, Fig.16 M is the result of immunohistochemical staining of nasal mucosal sections, which shows that the siRNA preparation delivered by DILs can achieve a good silencing effect on the target gene KCa3.1 from the protein expression level. In addition, the expression of tight junction protein ZO-1 in the AR group was significantly reduced, similar to that in the siNC group. The expression of ZO-1 in the mucosal epithelial cells of the siKCa3.1 group was significantly increased compared with the AR group, indicating that KCa3.1 gene silencing treatment inhibited mast cell activity to a certain extent and the degree of mucosal barrier damage was relatively mild. At the same time, the immunohistochemical sections of ZO-1 in the siKCa3.1 group showed that the effect of the ionic liquid as a permeation enhancer on tight junction proteins was transient, and continuous administration for one week did not cause irreversible damage to tight junction proteins.

[0150] 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 and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition comprising the following components: (i) deep eutectic solvent; (ii) lipid material; (iii) Nucleic acid drugs.

2. The composition according to claim 1, characterized in that In the composition, (ii) the lipid material and (iii) the nucleic acid drug are dispersed in (i) the deep eutectic solvent; preferably, the homogeneous material is dispersed in (i) the deep eutectic solvent; And / or, the composition is a uniform and stable dispersion system, such as a solution system, a suspension system, or an emulsion system; and / or, the composition is capable of forming lipid nanoformulations (e.g., lipid nanoparticles) in situ; And / or, the composition is administered in a manner selected from nasal administration, oral administration, sublingual administration, skin administration, vaginal administration, local injection, etc.

3. The composition according to claim 1 or 2, characterized in that (iii) Nucleic acid drugs are selected from RNA drugs (such as miRNA drugs, siRNA drugs (such as KCa3.1 siRNA), mRNA drugs, shRNA drugs, lncRNA drugs, ASO drugs, saRNA drugs) or DNA drugs. and / or, (ii) the lipid material is selected from one or more of ionizable lipids, cationic lipids (e.g., DLin-MC3-DMA, SM-102, ALC-0315, DOTAP), auxiliary lipids (e.g., DSPC, DOPE, DGTS), cholesterol, β-sitosterol, oxidized cholesterol or its derivatives, PEG-modified lipids (e.g., DMG-PEG 2000, DSPE-PEG 2000, ALC-0159); And / or, the eutectic solvent is a two-component or three-component eutectic mixture composed of a hydrogen bond acceptor (such as a quaternary ammonium salt, a tertiary amine or other compound) and a hydrogen bond donor (such as a carboxylic acid, a polyol, a sugar or other compound) in a certain stoichiometric ratio; and / or, in the composition, (ii) the lipid material content is 1% to 50% (mass percentage), preferably 5% to 50%, such as 10%, 30%, 50%; And / or, the mass ratio of the nucleic acid drug to (low eutectic solvent + lipid material) is 1:10-5000, preferably 1:100-3000; for example, 1:500, 1:1000, 1:2000.

4. The composition according to any one of claims 1 to 3, characterized in that The low eutectic solvent is selected from one or more of an ionic liquid, a non-ionic low eutectic solvent, and a low eutectic mixture; preferably, the low eutectic solvent is an ionic liquid, such as a choline ionic liquid; And / or, the preparation method of the low eutectic solvent is: after the hydrogen bond donor compound and the hydrogen bond acceptor compound are uniformly mixed in solid state, heating and melting, to obtain the low eutectic solvent; And / or, the preparation method of the low eutectic solvent is: dissolving the hydrogen bond donor compound and the hydrogen bond acceptor compound in a solvent, mixing and stirring in a certain proportion, and after the reaction is completed, rotary evaporating and vacuum drying to obtain the low eutectic solvent; And / or, the hydrogen bond donor compound is selected from any one or more of the following: geranic acid, sorbic acid, malic acid, citric acid, tartaric acid, oxalic acid, lactic acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, acetic acid, malonic acid, succinic acid, caprylic acid, capric acid, oleic acid, linoleic acid, arachidonic acid, retinoic acid, hibiscus acid or chlorogenic acid; preferably, the hydrogen bond donor compound is selected from any one or more of the following: sorbic acid, malic acid, geranic acid, citric acid, oleic acid, linoleic acid, malonic acid; more preferably, the hydrogen bond donor compound is geranic acid; and / or, the hydrogen bond acceptor compound is selected from any one or more of the following: choline hydroxide, choline bicarbonate, choline chloride, triethanolamine, diethanolamine, betaine, phospholipid or glycerophosphocholine; preferably, the hydrogen bond acceptor compound is selected from any one or more of the following: choline hydroxide, choline bicarbonate, choline chloride, triethanolamine or diethanolamine; more preferably, the hydrogen bond donor compound is choline bicarbonate; and / or, the molar ratio of the hydrogen bond acceptor compound to the hydrogen bond donor compound is 1:0.5 to 3; And / or, the molar ratio of geranic acid to choline bicarbonate is 1:0.5-3, preferably 1:

1.

5. A method for preparing the composition according to any one of claims 1 to 4, comprising the following steps: The composition can be obtained by separately preparing a low eutectic solvent system containing lipid materials and a low eutectic solvent system containing nucleic acid drugs, and then mixing the two. Preferably, the preparation method comprises the following steps: 1) adding lipid material and solvent to a low eutectic solvent, dissolving and mixing, and removing the solvent to obtain a low eutectic solvent system loaded with lipid material; 2) adding the nucleic acid drug and the solvent to the deep eutectic solvent, dissolving and mixing, and removing the solvent to obtain a deep eutectic solvent system containing the nucleic acid drug; 3) mixing the low eutectic solvent system respectively loaded with lipid materials and the low eutectic solvent system loaded with nucleic acid drugs to obtain the composition; Further preferably, in step 1), the solvent used is an organic solvent; preferably one or more selected from methanol, ethanol, isopropanol, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and acetone; Further preferably, in step 1), the method of removing the solvent is: vacuum drying or nitrogen blowing drying; preferably, the drying temperature is: 25°C to 60°C; preferably, the drying time is 20min to 60min; Further preferably, in step 2), the solvent used is enzyme-free water or DEPC water; Further preferably, in step 2), the specific operation of removing the solvent is: freezing at -80°C for 2 to 8 hours, and vacuum drying for 30 minutes to 4 hours; Further preferably, in step 3), the mixing method is water bath ultrasound; preferably, the power of the water bath ultrasound is 90 Hz; preferably, the water bath temperature is 4° C. to 25° C.; preferably, the water bath time is 5 min to 30 min.

6. A pharmaceutical composition comprising the composition according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier; Preferably, the administration method of the pharmaceutical composition is selected from nasal administration, oral administration, sublingual administration, skin administration, vaginal administration, local injection, etc.; Preferably, the dosage form of the pharmaceutical composition is selected from gels (eg, in situ gels), ointments, nasal drops, sprays (nasal sprays), emulsions, suspensions, powders, and the like.

7. Use of the composition according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 6 in the preparation of a product for delivery of nucleic acid drugs; Preferably, the delivery method is selected from nasal delivery, oral delivery, sublingual delivery, skin delivery, vaginal delivery, local injection delivery, etc.; for example, non-invasive nasal delivery.

8. Use of the composition according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 6 in the preparation of drugs for treating and / or preventing diseases.

9. An in-situ preparation method of a lipid nanoformulation, the in-situ preparation method comprising: The composition according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 6 is dispersed in a medium to obtain a lipid nanoformulation (e.g., lipid nanoparticles); preferably, the medium is, for example, water, a buffer system, a physiological system (e.g., in vivo, cavity mucosa, such as nasal mucosa, vaginal mucosa, oral mucosa, skin mucosa, etc.).

10. A lipid nanoformulation (such as lipid nanoparticles) prepared in situ in a medium by the composition according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 6; Preferably, the medium is, for example, water, a buffer system, a physiological system (eg, in vivo, cavity mucosa, such as nasal mucosa, vaginal mucosa, oral mucosa, skin mucosa, etc.).

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