Nucleic acid transfection reagent, preparation method thereof and nucleic acid delivery composition
The nucleic acid transfection reagent, which combines lipid nanoparticles with buffer solution using microfluidic technology, solves the problems of high cost, high toxicity and complicated operation of existing liposome transfection methods, and achieves efficient and stable nucleic acid transfection results.
Patent Information
- Application Number
- CN202511996088.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-20
AI Technical Summary
Existing liposome transfection methods using commercially available transfection reagents such as Lipo2000 and Lipo3000 have drawbacks such as high cost, limited transfection efficiency, high cytotoxicity, and complex operation.
Lipid nanoparticles self-assembled using microfluidic technology, combined with tromethamine or PBS buffer, simplify the preparation process, shorten the operation time, improve stability, reduce costs, and eliminate the need for Opti-MEM incubation during transfection.
It effectively simplifies the preparation process, improves the stability and transfection efficiency of nucleic acid transfection reagents, reduces operational complexity and cytotoxicity, is suitable for long-term storage, and reduces costs.
Smart Images
Figure CN121700002A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of transfection reagent technology, specifically relating to a nucleic acid transfection reagent, its preparation method, and a nucleic acid delivery composition. Background Technology
[0002] Cell transfection refers to the process of introducing exogenous nucleic acids (such as DNA, RNA, siRNA, miRNA, CRISPR-Cas9 system, etc.) into eukaryotic cells to achieve gene expression regulation, gene editing, functional research or therapeutic applications.
[0003] Among existing transfection methods, liposome transfection is widely used due to its advantages of wide applicability to cell types, good reproducibility, and suitability for in vivo injection. Liposome transfection utilizes the binding of positively charged cationic liposomes to negatively charged nucleic acids, forming stable lipid complexes (Lipoplexes). These particles enter cells via endocytosis and release nucleic acids. However, existing commercially available transfection reagents such as Lipo2000 and Lipo3000 used in liposome transfection have drawbacks including high cost, limited transfection efficiency, significant cytotoxicity, and complex operation. Therefore, there is an urgent need to develop a new, highly efficient transfection reagent. Summary of the Invention
[0004] The purpose of this application is to provide a nucleic acid transfection reagent, its preparation method and application, to solve the shortcomings of existing commercially available transfection reagents such as Lipo2000 and Lipo3000 used in liposome transfection methods, such as high cost, limited transfection efficiency, high cytotoxicity and complicated operation.
[0005] To achieve the above objectives, the first aspect of this application provides a nucleic acid transfection reagent comprising lipid nanoparticles and a buffer solution. The lipid nanoparticles are formed by microfluidic mixing and self-assembly, and comprise cationic lipids, helper phospholipids, and cholesterol. The lipid nanoparticles have a particle size of 30-200 nm and a polydispersity index of less than 0.3. The buffer solution is tromethamine buffer or PBS buffer.
[0006] In one or more embodiments, the molar ratio of cationic lipids, auxiliary phospholipids and cholesterol in the lipid nanoparticles is (50-75):(7.5-25):(0-40).
[0007] In one or more embodiments, the volume ratio of the lipid nanoparticles to the buffer solution is 1:(1~9).
[0008] In one or more embodiments, the pH of the buffer solution is 5.5 to 6.5.
[0009] In one or more embodiments, the concentration of the buffer solution is 10-50 mM.
[0010] In one or more embodiments, the cationic lipid is one or more combinations of DOSPA, DOTAP, DC-Chol, DOTMA, Dlin-DMA-MC3, ALC-0315, SM-102, DOTAP, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, (2,3-dioleoxypropyl)trimethylammonium chloride, C12-200, THP1, 306Oi10, DC-Chol, and 1,2-bisoctadecenoxy-3-methylammonium propane (chloride).
[0011] In one or more embodiments, the auxiliary phospholipid is one or more combinations of DOPE, DOPC, and DSPC.
[0012] To achieve the above objectives, a second aspect of this application provides a method for preparing the nucleic acid transfection reagent according to any of the above embodiments, comprising:
[0013] Weigh cationic lipids, cofactor phospholipids, and cholesterol in molar ratio, dissolve them in a solvent, and obtain a lipid solution;
[0014] The lipid solution and the acidic aqueous solution were mixed using a microfluidic system to obtain a lipid nanoparticle suspension.
[0015] The solvent is removed from the lipid nanoparticle suspension to obtain the lipid nanoparticles;
[0016] Dissolve tromethorphan or PBS in water, adjust the pH, and obtain the buffer solution;
[0017] The lipid nanoparticles and the buffer solution were packaged separately to obtain the nucleic acid transfection reagent.
[0018] In one or more embodiments, in the step of mixing the lipid solution and the acidic aqueous solution by microfluidics, the flow rate ratio of the lipid solution and the acidic aqueous solution is 1:(1~8).
[0019] In one or more embodiments, the flow rate ratio of the lipid solution to the acidic aqueous solution is 1:(3~5), the particle size of the lipid nanoparticles is 35~40 nm, and the polydispersity index is less than 0.12.
[0020] To achieve the above objectives, a third aspect of this application provides a nucleic acid delivery composition, comprising a nucleic acid molecule and the nucleic acid transfection reagent described in any of the above embodiments; the nucleic acid delivery composition is prepared using the following steps:
[0021] The nucleic acid molecules were diluted with the buffer solution to obtain a nucleic acid dilution solution;
[0022] The nucleic acid diluent is mixed with the lipid nanoparticles to obtain the nucleic acid delivery composition.
[0023] In one or more embodiments, the volume ratio of the lipid nanoparticles to the nucleic acid diluent is 1:1.
[0024] In one or more embodiments, the N / P ratio of the lipid nanoparticles to the nucleic acid molecules is greater than 5.
[0025] The advantages of this application, which differ from existing technologies, are:
[0026] The nucleic acid transfection reagent of this application effectively simplifies the preparation process, shortens the process operation time, improves the stability of the nucleic acid transfection reagent, facilitates long-term storage, and helps reduce costs. In addition, it can be used without Opti-MEM incubation during transfection, and can mix lipid nanoparticles and nucleic acid molecules, which effectively simplifies the transfection operation and improves stability. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 This is a flowchart illustrating one embodiment of the preparation method of the nucleic acid transfection reagent of this application;
[0029] Figure 2 This is a graph showing the correlation between the transfection dosage and the Luciferase detection signal value in Example 5 of this application. Detailed Implementation
[0030] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.
[0031] Existing liposome transfection methods using commercially available transfection reagents such as Lipo2000 and Lipo3000 have drawbacks such as high cost, limited transfection efficiency, high cytotoxicity, and complex operation.
[0032] Specifically, existing transfection reagents prepare lipid films using a "film hydration method," followed by the addition of a hydration solution for further hydration. Finally, a "film extrusion technique" is used to optimize the particle size and uniformity of the nanoparticles. While this method produces BLM particles with controllable size and good stability, it requires equipment such as rotary evaporators and film extruders, resulting in high costs. Furthermore, the process is complex, requiring the control of numerous variable parameters, leading to poor batch-to-batch consistency.
[0033] In addition, existing transfection reagents require dilution with Opti-MEM attenuated serum when mixed with nucleic acids, and need to be left to stand for 5-20 minutes after mixing, which is costly and complicated.
[0034] To address the aforementioned issues, the applicant has developed a novel nucleic acid transfection reagent. This reagent effectively simplifies the preparation process, improves batch-to-batch consistency, and significantly shortens incubation time, thus simplifying the transfection procedure.
[0035] Specifically, the nucleic acid transfection reagent includes lipid nanoparticles and a buffer solution.
[0036] The lipid nanoparticles are formed through microfluidic mixing and self-assembly, and include cationic lipids, accessory phospholipids, and cholesterol. The particle size of the lipid nanoparticles is 30-200 nm, and the polydispersity index is less than 0.3. The buffer is tromethamine buffer or PBS buffer.
[0037] Lipid nanoparticles are obtained by mixing and self-assembling cationic lipids, auxiliary phospholipids and cholesterol based on microfluidic technology. Compared with traditional thin film dispersion and thin film extrusion methods, the operation process is simplified; the process operation is simple, the process operation time is shortened, and the batch-to-batch consistency is good.
[0038] Specifically, in one embodiment, the molar ratio of cationic lipids, auxiliary phospholipids and cholesterol in the lipid nanoparticles can be (50-75):(7.5-25):(0-40).
[0039] Based on this ratio of cationic lipids, auxiliary lipids and cholesterol, lipid nanoparticles, namely blank liposome mixtures (BLM), were prepared. The particle size was between 30-200 nm, the polydispersity index was less than 0.3, and the stability was good. They could be stored for a long time at 2-8℃.
[0040] In one embodiment, the volume ratio of lipid nanoparticles to buffer solution can be 1:(1~9). Specifically, in use, the buffer solution can be mixed with nucleic acid molecules for dilution first, and then the lipid nanoparticles can be mixed with the diluent to obtain a nucleic acid delivery composition for delivery.
[0041] In one embodiment, to optimize the reduction of the particle size of the nucleic acid delivery composition, the volume ratio of lipid nanoparticles to buffer can be 1:1.
[0042] In one embodiment, the pH of the buffer solution can be 5.0 to 7.4, preferably 5.5 to 6.5, to facilitate the recombination of lipid nanoparticles with nucleic acid molecules.
[0043] In one embodiment, the concentration of the buffer solution can be 10 to 50 mM.
[0044] In one embodiment, the cationic lipid may be one or more combinations of DOSPA, DOTAP, DC-Chol, DOTMA, Dlin-DMA-MC3, ALC-0315, SM-102, DOTAP, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, (2,3-dioleoxypropyl)trimethylammonium chloride, C12-200, THP1, 306Oi10, DC-Chol, and 1,2-bisoctadecenoxy-3-methylammonium propane (chloride).
[0045] In one embodiment, the auxiliary phospholipid is one or more combinations of DOPE, DOPC, and DSPC.
[0046] The nucleic acid transfection reagents based on the above embodiments effectively simplify the preparation process, shorten the process operation time, improve the stability of the nucleic acid transfection reagents, facilitate long-term storage, and help reduce costs. In addition, during the transfection operation, it can be used without the need for Opti-MEM incubation, and can mix lipid nanoparticles and nucleic acid molecules, which effectively simplifies the transfection operation and improves stability.
[0047] This application also provides a method for preparing the nucleic acid transfection reagents of the above embodiments. Please refer to [link to relevant documentation]. Figure 1 , Figure 1 This is a flowchart illustrating one embodiment of the preparation method of the nucleic acid transfection reagent of this application.
[0048] like Figure 1 As shown, the preparation method includes:
[0049] S100, weigh cationic lipids, cofactor phospholipids and cholesterol in molar ratio, dissolve them in a solvent to obtain a lipid solution.
[0050] In one embodiment, the solvent may be an organic solvent; for example, the solvent may be ethanol.
[0051] S200. The lipid solution and the acidic aqueous solution are mixed by microfluidics to obtain a lipid nanoparticle suspension.
[0052] In one embodiment, the microfluidic channel type can be "T" type, "Y" type, "fishbone" type, etc., all of which can achieve the effect of this embodiment.
[0053] The volume ratio of lipid solution to acidic aqueous solution is related to the particle size and polydispersity index of lipid nanoparticles. In one embodiment, the flow rate ratio of lipid solution to acidic aqueous solution can be 1:(1~8), and correspondingly, the prepared lipid nanoparticles have a particle size of 30-200 nm and a polydispersity index of less than 0.3.
[0054] Preferably, in order to reduce the particle size of lipid nanoparticles and improve uniformity, in one embodiment, the flow rate ratio of lipid solution to acidic aqueous solution can be 1:(3~5), and correspondingly, the prepared lipid nanoparticles have a particle size of 35~40nm and a polydispersity index of less than 0.12.
[0055] S300: Remove the solvent from the lipid nanoparticle suspension to obtain lipid nanoparticles.
[0056] In one embodiment, the solvent can be removed by methods such as dialysis or tangential flow filtration to obtain lipid nanoparticles.
[0057] S400: Dissolve tromethorphan or PBS in water, adjust the pH, and obtain a buffer solution.
[0058] S500: Separately package lipid nanoparticles and buffer solution to obtain nucleic acid transfection reagent.
[0059] The preparation methods described above effectively simplify the preparation process of transfection reagents, shorten the process operation time, improve the stability of nucleic acid transfection reagents, facilitate long-term storage, and help reduce costs.
[0060] This application also provides a nucleic acid delivery composition, comprising nucleic acid molecules and nucleic acid transfection reagents of any of the above embodiments.
[0061] Specifically, the nucleic acid delivery composition is prepared using the following steps:
[0062] Nucleic acid molecules were diluted with buffer solution to obtain a nucleic acid dilution solution;
[0063] The nucleic acid dilution solution was mixed with lipid nanoparticles to obtain a nucleic acid delivery composition.
[0064] In one embodiment, in order to reduce the particle size of the nucleic acid delivery composition and improve uniformity, the volume ratio of lipid nanoparticles to nucleic acid diluent can be 1:1.
[0065] In one embodiment, to improve transfection efficiency, the N / P ratio of lipid nanoparticles to nucleic acid molecules is greater than 5, wherein the N / P ratio refers to the ratio of the total positive charge provided by the lipid nanoparticles to the total negative charge carried by the nucleic acid molecules.
[0066] Furthermore, in order to reduce the toxicity of transfection reagents, the amount of lipid nanoparticles used can be reduced as much as possible while ensuring transfection efficiency. Preferably, the N / P ratio of lipid nanoparticles to nucleic acid molecules can be equal to 6.
[0067] The nucleic acid delivery compositions based on the above embodiments can significantly improve transfection efficiency while helping to reduce the toxicity of transfection reagents, resulting in a highly efficient and low-toxicity nucleic acid delivery system.
[0068] The beneficial effects of the technical solution of this application will be further explained in detail below with reference to specific embodiments.
[0069] Example 1:
[0070] A nucleic acid delivery composition is prepared by the following steps:
[0071] Step 1: Preparation of lipid nanoparticles:
[0072] The lipid raw materials were weighed according to the molar ratio of DOTMA : DOPE : Chol = 52.5 : 7.5 : 40, and an organic phase with a total lipid concentration of 20 mg / mL was prepared using ethanol as the organic solvent.
[0073] Using a microfluidic device, the organic phase and the aqueous phase are rapidly mixed at a flow rate ratio of 1:3;
[0074] The lipid nanoparticles were diluted with Tris buffer at pH 6.0 to remove ethanol and the pH was adjusted to 6.0 to obtain lipid nanoparticles, in which the concentration of cationic lipids was 6 mM.
[0075] Step 2: Buffer Preparation
[0076] Tromethamine was dissolved in water and the pH was adjusted to 6.0 to obtain a 10 mM tromethamine buffer solution.
[0077] Step 3:
[0078] Weigh out 2.5 μg of mRNA according to the transfection dosage, and dilute it with tromethamine buffer to obtain the mRNA dilution solution;
[0079] The mRNA dilution buffer and lipid nanoparticles were directly vortexed at a volume ratio of 1:1, with an N / P ratio of 4 for the lipid nanoparticles to mRNA, to obtain the nucleic acid delivery composition.
[0080] Examples 2-4:
[0081] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0082] In microfluidics, the flow rate ratio of the organic phase to the aqueous phase is different, as shown in Example 1.
[0083] Examples 5-7:
[0084] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0085] The volume ratio of lipid nanoparticles to mRNA dilution solution varies, as shown in Example 2.
[0086] Examples 8-10:
[0087] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0088] The concentration of cationic lipids in lipid nanoparticles varies, as shown in Example 3.
[0089] Examples 11-13:
[0090] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0091] The cationic lipid is DOTAP, and the auxiliary phospholipid is DSPC. The concentration of cationic lipid in the lipid nanoparticles varies, as shown in Example 3.
[0092] Examples 14-16:
[0093] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0094] The N / P ratio of lipid nanoparticles differs from that of mRNA; see Example 4 for details.
[0095] Examples 17-20:
[0096] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0097] The dosage of the drug varies depending on the transfection; see Example 5 for specific results.
[0098] Examples 21-23:
[0099] A nucleic acid delivery composition, prepared in a manner essentially the same as in Example 1, except that:
[0100] The molar ratios of DOTMA, DOPE, and Chol are different; see Example 6 for details.
[0101] Example of effect 1:
[0102] To verify the effect of the flow rate ratio of organic phase to aqueous phase in microfluidics on the particle size and uniformity of lipid nanoparticles, the particle size and polydispersity index of lipid nanoparticles prepared in Examples 1 to 4 were measured, and the data are shown in the table below.
[0103]
[0104] As shown in the table above, when the flow rate ratio of the organic phase to the aqueous phase in the microfluidic system is in the range of 1: (1~8), the particle size of the prepared lipid nanoparticles is within the target range.
[0105] Among them, when the flow rate ratio of the organic phase to the aqueous phase in the microfluidic system is 1:3 to 1:5, the PDI is smaller and the prepared lipid nanoparticles are more uniform, which is a preferred embodiment.
[0106] Example of effect 2:
[0107] To verify the effect of the volume ratio of lipid nanoparticles to mRNA dilution on the particle size and uniformity of the nucleic acid delivery composition, the particle size and polydispersity index of the nucleic acid delivery compositions prepared in Examples 1, 5-7 were measured, and the data are shown in the table below.
[0108]
[0109] As shown in the table above, when the volume ratio of lipid nanoparticles to mRNA dilution is in the range of 1: (1~9), the particle size and PDI of the prepared nucleic acid delivery composition are both within the target range.
[0110] Among them, when the volume ratio of lipid nanoparticles to mRNA dilution is 1:1, the prepared nucleic acid delivery composition has the smallest particle size and the lowest PDI, which is a preferred embodiment.
[0111] Example of effect 3:
[0112] To verify the effect of cationic lipid concentration on the particle size and uniformity of lipid nanoparticles, the particle size, polydispersity index and zeta potential of the lipid nanoparticles prepared in Examples 8-13 were measured, and the data are shown in the table below.
[0113]
[0114] As shown in the table above, within the selected lipid range, changing the lipid does not affect the physicochemical properties; in the same lipid nanoparticle formulation, as the molar concentration of cationic lipids increases, the particle size and PDI gradually decrease, while the zeta potential gradually increases.
[0115] Example of effect 4:
[0116] To verify the effect of the N / P ratio on transfection efficiency, transfection experiments were conducted using the nucleic acid delivery compositions prepared in Examples 1 and 14-16, respectively.
[0117] The specific steps of the transfection experiment are as follows:
[0118] (1) Cell plating
[0119] Prepare 1×PBS (for cell culture), trypsin, and DMEM medium containing 10% FBS by equilibrating to room temperature beforehand. Remove a flask containing a confluent monolayer of T75 cells from the CO2 incubator, discard the culture medium, and add 5 mL of 1×PBS (for cell culture) to wash away any residual medium. Add 3 mL of trypsin and digest at room temperature for 2-4 min. Add 5 mL of fresh DMEM medium containing 10% FBS, repeatedly pipetting the cells off the flask wall to break up any clumps. Collect the cell mixture in a 15 mL centrifuge tube and centrifuge at 1200 rpm for 5 min. Discard the supernatant and resuspend the cells in 1 mL of fresh DMEM medium containing 10% FBS. Take 500 μL of the cell suspension and bring the volume to 13 mL with DMEM medium containing 10% FBS. Add 1 mL to each well of a 12-well plate. Gently shake the 12-well plate to evenly distribute the cells, then transfer to a CO2 incubator.
[0120] (2) Transfection
[0121] ① The next day, take out the 12-well plate and observe the cell growth under a microscope. Cell transfection can only be performed when the confluence of cells in each well reaches more than 90% (if the confluence of cells is insufficient, extend the time of this step until the confluence reaches more than 90%).
[0122] ② Remove the 12-well plate from the CO2 incubator and add the nucleic acid transfection composition dropwise to each well (200 μL per well). Then, cover the plate, gently shake it, and label each well with its corresponding name. Place the 12-well cell culture plate in the CO2 incubator for 18-24 h.
[0123] ④ Detection: Detection was performed using a Luciferase kit.
[0124] Specifically, the Luciferase detection signal values for Examples 1, 14-16 are shown in the table below.
[0125]
[0126] As shown in the table above, a transfection efficiency of N / P ratio greater than 4 is generally good. In order to reduce the toxicity of the transfection reagent, the amount of lipid nanoparticles can be reduced as much as possible. Preferably, the N / P ratio can be 6, thereby obtaining a highly efficient and low-toxic transfection reagent.
[0127] Example 5:
[0128] To verify the effect of transfection dosage on transfection efficiency, transfection experiments were conducted using the nucleic acid delivery compositions prepared in Examples 1 and 17-20, respectively. The experimental procedure was the same as in Example 4, and the following data were obtained.
[0129]
[0130] Furthermore, the applicant also measured the Luciferase detection signal values at transfection doses of 7.5 μg and 10.0 μg, and obtained... Figure 2 , Figure 2 This is a graph showing the correlation between the transfection dosage and the Luciferase detection signal value in Example 5 of this application.
[0131] like Figure 2 As shown in the table above, transfection doses of 2.5–5.0 μg all exhibited good effects. Furthermore, even at transfection doses as high as 10 μg, cells maintained good morphology and did not show cytotoxicity, verifying the low cytotoxicity of the nucleic acid transfection reagent presented in this application.
[0132] Example of effect 6:
[0133] Furthermore, in order to verify the effect of the molar ratio of each component in the lipid nanoparticles on the transfection efficiency, transfection experiments were conducted using the nucleic acid delivery compositions prepared in Examples 1 and 21-23, respectively. The experimental procedure was the same as in Example 4, and the following data were obtained.
[0134]
[0135] As shown in the table above, the lipid nanoparticles of Examples 1 and 21-23 all have excellent transfection efficiency. Among them, Example 1 has the highest transfection efficiency and is therefore the preferred example.
[0136] Furthermore, the applicant tested the particle size and PDI of the lipid nanoparticles and nucleic acid delivery compositions prepared in Examples 1, 21-23, and obtained the data in the table below.
[0137]
[0138] As shown in the table above, the particle size and PDI of lipid nanoparticles and nucleic acid delivery compositions prepared with different lipid ratio formulations are not significantly different. They all have small particle size and excellent uniformity, which helps to improve transfection stability.
[0139] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this disclosure. No reference numerals in the claims should be construed as limiting the scope of the claims.
[0140] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. A nucleic acid transfection reagent, characterized in that, The product comprises lipid nanoparticles and a buffer solution. The lipid nanoparticles are formed by microfluidic mixing and self-assembly, and include cationic lipids, helper phospholipids and cholesterol. The lipid nanoparticles have a particle size of 30-200 nm and a polydispersity index of less than 0.
3. The buffer solution is tromethamine buffer or PBS buffer.
2. The nucleic acid transfection reagent according to claim 1, characterized in that, The molar ratio of cationic lipids, auxiliary phospholipids and cholesterol in the lipid nanoparticles is (50-75):(7.5-25):(0-40).
3. The nucleic acid transfection reagent according to claim 1, characterized in that, The volume ratio of the lipid nanoparticles to the buffer solution is 1:(1~9).
4. The nucleic acid transfection reagent according to claim 1, characterized in that, The pH value of the buffer solution is 5.5~6.5; and / or, The concentration of the buffer solution is 10~50 mM.
5. The nucleic acid transfection reagent according to claim 1, characterized in that, The cationic lipid is one or more combinations of DOSPA, DOTAP, DC-Chol, DOTMA, Dlin-DMA-MC3, ALC-0315, SM-102, DOTAP, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, (2,3-dioleoxypropyl)trimethylammonium chloride, C12-200, THP1, 306Oi10, DC-Chol, and 1,2-bis(octadecenoxy-3-methylammonium propane) (chloride); and / or, The auxiliary phospholipid is one or more combinations of DOPE, DOPC and DSPC.
6. A method for preparing the nucleic acid transfection reagent according to any one of claims 1 to 5, characterized in that, include: Weigh cationic lipids, cofactor phospholipids, and cholesterol in molar ratio, dissolve them in a solvent, and obtain a lipid solution; The lipid solution and the acidic aqueous solution were mixed using a microfluidic system to obtain a lipid nanoparticle suspension. The solvent is removed from the lipid nanoparticle suspension to obtain the lipid nanoparticles; Dissolve tromethorphan or PBS in water, adjust the pH, and obtain the buffer solution; The lipid nanoparticles and the buffer solution were packaged separately to obtain the nucleic acid transfection reagent.
7. The preparation method according to claim 6, characterized in that, In the step of mixing the lipid solution and the acidic aqueous solution via microfluidics, the flow rate ratio of the lipid solution to the acidic aqueous solution is 1:(1~8).
8. The preparation method according to claim 7, characterized in that, The flow rate ratio of the lipid solution to the acidic aqueous solution is 1:(3~5), the particle size of the lipid nanoparticles is 35~40 nm, and the polydispersity index is less than 0.
12.
9. A nucleic acid delivery composition, characterized in that, The nucleic acid delivery composition includes nucleic acid molecules and the nucleic acid transfection reagent according to any one of claims 1 to 5; the nucleic acid delivery composition is prepared by the following steps: The nucleic acid molecules were diluted with the buffer solution to obtain a nucleic acid dilution solution; The nucleic acid diluent is mixed with the lipid nanoparticles to obtain the nucleic acid delivery composition.
10. The nucleic acid delivery composition according to claim 9, characterized in that, The volume ratio of the lipid nanoparticles to the nucleic acid diluent is 1:1; and / or, The N / P ratio of the lipid nanoparticles to the nucleic acid molecules is greater than 5.