A nucleic acid delivery system containing negatively charged lipids

Abstract

The present invention relates to the field of drug delivery, specifically to the delivery of mRNA by liposome carriers. More specifically, it relates to a liposome nanodelivery system for delivering mRNA to cells, which is composed of cationic liposomes and negatively charged phospholipid POPS. The present invention constructs an mRNA liposome nanodelivery system with cationic liposomes as the main component, and introduces negatively charged phospholipid (PS). On the basis of ensuring transfection efficiency, the introduction of negative charges neutralizes a part of the positive charges carried by cationic lipids, thereby reducing the positive charges of the overall system, increasing the safety of the entire delivery system, and reducing the cytotoxicity of cationic liposomes.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid drug delivery systems. Specifically, it relates to the delivery of mRNA by liposome carriers. More specifically, it relates to a liposome nano-delivery system composed of cationic liposomes and negatively charged phospholipids for delivering mRNA at the cellular level, and the effects of the molar ratio of positive and negative charges, liposome concentration, auxiliary materials, uptake time, transfection time, preparation method, and net charge on the transfection effect, as well as the improvement of the safety of the delivery system by negatively charged phospholipids. Background Art

[0002] Nucleic acid drugs are one of the most rapidly developing and cutting-edge fields at present, and continuous breakthroughs have been made in the treatment of tumors, genetic diseases, metabolic diseases, preventive infectious diseases, etc. As a type of nucleic acid drug, mRNA drugs have the characteristics of strong hydrophilicity, high biological activity, being able to express the corresponding target protein without entering the cell nucleus to play a role, simple process, high efficiency and safety. In theory, they can express all proteins and can treat a variety of diseases.

[0003] However, mRNA drugs currently face various challenges and problems. First of all, mRNA is a single-stranded macromolecule with a negative charge, and such a structure makes it extremely fragile and easily degraded by RNA enzymes in vivo and in vitro. Secondly, both mRNA and cell membranes carry negative charges, and the electrostatic repulsion makes it difficult for mRNA to penetrate the cell membrane and enter the cell to take effect. And the mRNA coding information contains the sequence for ribosome generation, and it must be delivered into the cell to encode proteins. Therefore, mRNA drugs also face huge challenges.

[0004] An effective delivery system can solve various problems faced by mRNA in taking effect. Currently, there are various mRNA delivery systems, such as protamine, cationic nanoemulsions, polysaccharide particles, cationic polymers, liposome nano-delivery systems, etc. Among them, the most mature and widely used is the liposome nano-delivery system, which has the advantages of protecting mRNA from degradation and inactivation, being easy to carry mRNA into cells, high transfection efficiency, and not being restricted by the host as a delivery carrier. However, the liposome nano-delivery system is affected by various conditions such as liposome materials, charges, ratios, and preparation processes, and the core key parameters related to the preparation process are also the key to mRNA delivery.

[0005] There are the following two problems in the current liposome mRNA delivery system. One is that the ratio of liposome nano-delivery materials is complex and the preparation process is precise. Different from small molecule delivery systems, in the mRNA delivery system, a slight change in a parameter will directly affect the presence or absence of the final nucleic acid expression effect.

[0006] Secondly, since mRNA is a negatively charged biological macromolecule and cannot enter target cells through the cytoplasm on its own, positively charged liposomes are generally required to load it through charge attraction for cell delivery. Therefore, cationic liposomes are essential key components for mRNA transfection to take effect. However, due to their polar head structure, cationic liposomes are the main factor causing cytotoxicity during transfection, thus imposing certain limitations on clinical application research.

[0007] Phosphatidylserine (abbreviated as PS) is a phospholipid commonly present in cell membranes, related to a series of cell membrane functions, and is also one of the important components of the cell membrane of the brain, affecting the fluidity and permeability of the cell membrane and activating the metabolism and synthesis of various enzymes. In addition, phosphatidylserine has a net negative charge on the cell membrane, which helps with membrane asymmetry. Therefore, it can neutralize the positive charge carried by cationic liposomes, providing the possibility of reducing the cytotoxicity caused by cationic liposomes.

[0008] Therefore, to explore various parameter factors affecting mRNA delivery and to solve the above-mentioned safety problems of cationic liposome delivery, we constructed an mRNA liposome nanodelivery system mainly composed of cationic liposomes and introduced negatively charged phospholipids (PS). On the basis of ensuring transfection efficiency, the introduction of negative charges neutralizes a part of the positive charge carried by cationic lipids, thereby reducing the overall positive charge of the system, increasing the safety of the entire delivery system, and reducing the cytotoxicity of cationic liposomes. And we investigated the effects of various factors such as liposome materials, charges, ratios, and preparation processes on transfection effects. It is intended to solve many problems faced by mRNA drugs in taking effect and at the same time investigate its safety, making it both effective and safe, and providing treatment plans for various diseases. Summary of the Invention

[0009] One of the purposes of the present invention is to provide a nucleic acid delivery system that uses cationic liposomes and negatively charged phospholipids as the main materials of the delivery carrier. After loading enhanced green fluorescent protein mRNA (EGFP mRNA), the mRNA is delivered to cells and successfully expresses green fluorescent protein. The positive charge carried by cationic liposomes themselves can bind to negatively charged mRNA through electrostatic interaction, which is necessary for carrying mRNA into cells. However, the safety problem caused by the cytotoxicity of cationic liposomes themselves also needs to be solved. Another purpose of the present invention is to introduce negatively charged phospholipids to neutralize part of the positive charge carried by cationic liposomes, improving the safety of the delivery system on the basis of ensuring the transfection efficiency of the overall system. In addition, this patent also provides the effects of various synthesis and preparation conditions such as the main materials of liposomes, the molar ratio of positive and negative charges, liposome concentration, auxiliary materials, uptake time, transfection time, preparation method, and net charge on transfection effects.

[0010] To achieve the above object, a first aspect of the present invention relates to the composition of a nucleic acid delivery system, which uses spherical cationic liposomes and negatively charged phospholipids as the main carrier materials, and the carrier contains ribonucleic acid to be delivered.

[0011] In some embodiments of the first aspect of the present invention, "spherical" refers to spherical or similar to spherical. In some embodiments of the first aspect of the present invention, the cationic lipid material is preferably (2,3-dioleyloxypropyl) trimethyl ammonium chloride (DOTAP), 1,2-dioctadecenoxy-3-methylammonium propane chloride (DOTMA), and further preferably DOTAP; the negatively charged phospholipid material is selected from phosphatidylserine (PS), and further preferably 1-palmitoyl-2-oleoyl phosphatidylserine (POPS); the carrier may further include an auxiliary lipid material, and the auxiliary lipid material is selected from one or more of cholesterol and DSPE-PEG2000.

[0012] In some embodiments of the first aspect of the present invention, the ribonucleic acid to be delivered is selected from messenger RNA (mRNA), and further preferably enhanced green fluorescent protein mRNA (EGFP mRNA).

[0013] In some embodiments of the first aspect of the present invention, the spherical carrier is formed by surrounding with a bilayer, wherein the inner layer is hydrophilic and the outer layer is hydrophobic.

[0014] In some embodiments of the first aspect of the present invention, the molar ratio of the cationic liposome material to the negatively charged phospholipid material in the nucleic acid delivery system is 10:0 - 0:10, such as 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, preferably 9:1 - 6:4, and more preferably 7:3.

[0015] In some embodiments of the first aspect of the present invention, the liposome material in the nucleic acid delivery system is a cationic liposome material, a negatively charged phospholipid material, and an auxiliary lipid material, and the molar ratio of the cationic liposome material, the negatively charged phospholipid material, and the auxiliary lipid material is 7:3:(0 - 21), preferably 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, 7:3:21, and further preferably 7:3:14.

[0016] In some embodiments of the first aspect of the present invention, the mass ratio of total lipid to nucleic acid in the nucleic acid delivery system is 0.37 - 37 g / g, preferably 0.444 - 35.556 g / g, more preferably 0.889 - 26.667 g / g. Among them, the mass ratio of total lipid to nucleic acid (g / g) with the best transfection effect without adding co-lipid is 7.111 g / g, and the mass ratio of total lipid to nucleic acid (g / g) with the best transfection effect under the condition of adding co-lipid is 14.222 g / g.

[0017] The second aspect of the present invention relates to a method for synthesizing a nucleic acid delivery system, comprising the following steps:

[0018] (1) Mix cationic liposomes and negatively charged phospholipid materials (or with co-lipids) in different molar ratios, dissolve them in a certain amount of organic solvent, and then rotary evaporate the organic solvent to obtain a uniform lipid film.

[0019] (2) Mix an RNase-free aqueous solution of enhanced green fluorescent protein mRNA and the film at a ratio of 500:(12 - 360) ml / mg (such as 500:12 ml / mg, 500:24 ml / mg, 500:48 ml / mg, 500:96 ml / mg, 500:144 ml / mg, 500:192 ml / mg, 500:240 ml / mg, 500:288 ml / mg, 500:336 ml / mg, 500:360 ml / mg) to obtain a mixture;

[0020] (3) Ultrasonicate the above mixture to obtain a nucleic acid delivery system;

[0021] In some embodiments of the second aspect of the present invention, the method has one or more of the following features A to G:

[0022] A. In step (1), the cationic lipid material is selected from DOTAP, DOTMA, and more preferably (2,3-dioleyloxypropyl) trimethyl ammonium chloride (DOTAP); the negatively charged phospholipid material is selected from phosphatidylserine (PS), and more preferably 1-palmitoyl-2-oleoyl phosphatidylserine (POPS). The co-lipid is selected from cholesterol, DSPE-PEG2000.

[0023] B. In step (1), the organic solvent is selected from at least one of chloroform, acetone, and ethanol, and preferably chloroform;

[0024] C. In step (1), the ratio of the total liposome material to the organic solvent in the solution is 0.01 - 1 mg / ml, such as 0.1 mg / ml, 0.4 mg / ml, 0.8 mg / ml; preferably 0.012 - 0.96 mg / mL, more preferably 0.024 - 0.72 mg / mL.

[0025] D. In step (1), the temperature of rotary evaporation is room temperature;

[0026] E. In step (2), the concentration of the total liposome in the aqueous phase of the mixture is 0.024 - 0.72 mg / ml, such as 0.048 mg / ml, 0.096 mg / ml, 0.192 mg / ml, 0.384 mg / ml, 0.48 mg / ml, 0.672 mg / ml; preferably 0.096 - 0.672 mg / ml.

[0027] F. In step (3), the sonication time is 3 - 10 minutes, such as 3, 4, 5, 6, 7, 8, 9, 10 minutes;

[0028] G. The nucleic acid delivery system is the nucleic acid delivery system described in the first aspect of the present invention.

[0029] In some embodiments of the second aspect of the present invention, room temperature is generally understood as 10°C - 30°C.

[0030] The third aspect of the present invention relates to the influence of the nucleic acid delivery systems formed at different molar ratios of cationic lipids and negatively charged phospholipids under the condition of the same total concentration in the nucleic acid delivery system described in the first aspect of the present invention on the transfection effect on Neuro-2a cells. And the molar ratio at the optimal transfection effect.

[0031] In the embodiments of the third aspect of the present invention, the molar ratio range of the cationic liposome to the negatively charged phospholipid is 10:0 - 0:10, such as 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, preferably 9:1 - 2:8, preferably 9:1 - 6:4, more preferably 7:3.

[0032] In the embodiments of the third aspect of the present invention, the total liposome concentration is 0.48 mg / mL.

[0033] In the embodiments of the third aspect of the present invention, the transfection time is 24 hours.

[0034] The fourth aspect of the present invention relates to the cell viability at different total liposome concentrations when the cationic lipids and negatively charged phospholipids in the nucleic acid delivery system described in the third aspect of the present invention are at the optimal molar ratio, and the cell viability at different total liposome concentrations when the nucleic acid delivery system is a pure cationic liposome or a pure negatively charged phospholipid.

[0035] In the embodiment of the fourth aspect of the present invention, the cationic liposome is DOTAP, the negatively charged phospholipid is POPS, and the optimal molar ratio of the cationic liposome to the negatively charged phospholipid is 7:3.

[0036] In the embodiment of the fourth aspect of the present invention, the concentration gradient ranges of the optimal molar ratio of the cationic liposome to the negatively charged phospholipid, the pure cationic liposome, and the pure negatively charged phospholipid in the CCK-8 cell viability experiment are 0.024 - 0.72 mg / mL, preferably 0.096 - 0.672 mg / mL, more preferably 0.096 - 0.48 mg / mL, such as 0.096 mg / mL, 0.144 mg / mL, 0.192 mg / mL, 0.24 mg / mL, 0.288 mg / mL, 0.336 mg / mL, 0.384 mg / mL, 0.432 mg / mL, 0.48 mg / mL.

[0037] In the embodiment of the fourth aspect of the present invention, the time for treating Neuro-2a cells with different delivery systems is 24 hours.

[0038] The fifth aspect of the present invention relates to the influence of the nucleic acid delivery system formed by different total liposome concentrations on the transfection effect of Neuro-2a cells when the cationic lipid and the negatively charged phospholipid in the nucleic acid delivery system described in the first aspect of the present invention are at the optimal molar ratio. And the total liposome concentration at the optimal transfection effect.

[0039] In the embodiment of the fifth aspect of the present invention, the total concentration range of the cationic liposome and the negatively charged phospholipid at the optimal molar ratio is 0.096 - 0.672 mg / mL, such as 0.672 mg / mL, 0.576 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.288 mg / mL, 0.192 mg / mL, 0.096 mg / mL.

[0040] In the embodiment of the fifth aspect of the present invention, the optimal molar ratio of the cationic liposome to the negatively charged phospholipid is 7:3.

[0041] In the embodiment of the fifth aspect of the present invention, the transfection time is 24 hours.

[0042] The sixth aspect of the present invention relates to the influence of the nucleic acid delivery system formed by adding different molar ratios of co-lipids while ensuring the same total liposome concentration for each group on the transfection effect of Neuro-2a cells when the cationic lipid and the negatively charged phospholipid in the nucleic acid delivery system described in the first aspect of the present invention are at the optimal molar ratio. And the molar ratio at the optimal transfection effect after adding the co-lipid.

[0043] In the embodiment of the sixth aspect of the present invention, the co-lipid added is cholesterol.

[0044] In the embodiment of the sixth aspect of the present invention, the molar ratio of the cationic liposome, the negatively charged phospholipid, and the helper lipid is 7:3:(0 - 21), preferably 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, 7:3:21.

[0045] In the embodiment of the sixth aspect of the present invention, the total concentration of the liposome is 0.48 mg / mL.

[0046] In the embodiment of the sixth aspect of the present invention, the transfection time is 24 hours.

[0047] The seventh aspect of the present invention relates to the influence of the nucleic acid delivery systems formed at different total liposome concentrations on the transfection effect on Neuro-2a cells when the cationic lipid, the negatively charged phospholipid, and the helper lipid in the nucleic acid delivery system described in the first aspect of the present invention are at the optimal molar ratio. And the total liposome concentration at the optimal transfection effect.

[0048] In the embodiment of the seventh aspect of the present invention, the total concentration range of the cationic liposome, the negatively charged phospholipid, and the helper lipid at the optimal molar ratio is 0.024 - 0.72 mg / mL, such as 0.72 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.192 mg / mL, 0.096 mg / mL, 0.048 mg / mL, 0.024 mg / mL.

[0049] In the embodiment of the seventh aspect of the present invention, the optimal molar ratio of the cationic liposome, the negatively charged phospholipid, and the helper lipid is 7:3:14.

[0050] In the embodiment of the seventh aspect of the present invention, the transfection time is 24 hours.

[0051] The eighth aspect of the present invention relates to the optimal uptake time and the optimal transfection time of the nucleic acid delivery system described in the first aspect of the present invention on Neuro-2a cells.

[0052] In some embodiments of the eighth aspect of the present invention, the uptake time is to add the nucleic acid delivery system described in the first aspect of the present invention to a 96-well cell plate pre-seeded with Neuro-2a cells and aspirate it within 0 - 4 hours, then continue to add fresh complete medium and transfect for 24 hours, where 0 - 4 hours is the uptake time, such as 0.5 hour, 1 hour, 2 hours, 4 hours.

[0053] In some embodiments of the eighth aspect of the present invention, the transfection time is the time from adding the nucleic acid delivery system described in the first aspect of the present invention to a 96-well cell plate pre-seeded with Neuro-2a cells until staining and photographing, such as 0 h, 3 h, 6 h, 12 h, 24 h, 48 h.

[0054] In some embodiments of the eighth aspect of the present invention, the nucleic acid delivery system described in the first aspect of the present invention is prepared from a cationic lipid and a negatively charged phospholipid under the condition that the optimal molar ratio is 7:3 as described in the third aspect of the present invention, and the total lipid concentration is 0.192 mg / mL as described in the fifth aspect of the present invention.

[0055] The ninth aspect of the present invention relates to the influence of various preparation methods including the second aspect of the present invention on the transfection effect of the nucleic acid delivery system described in the first aspect of the present invention.

[0056] In some embodiments of the ninth aspect of the present invention, the thin film dispersion method is the method described in the second aspect of the present invention.

[0057] The preparation method by extrusion involved in the ninth aspect of the present invention is as follows: on the basis of the liposome / mRNA complex prepared by the thin film dispersion method, at a temperature not lower than the phase transition temperature of the phosphatidylserine-based material, the ultrasonicated mixture is reciprocally extruded through a liposome extruder 5 - 40 times (preferably 10 - 30 times, such as 11 times, 13 times, 15 times, 16 times, 17 times, 18 times, 20 times, 22 times, 25 times, 28 times, 35 times) to obtain the nucleic acid delivery system; wherein, the pore size of the polycarbonate membrane in the liposome extruder is 80 - 200 nm (preferably 100 - 200 nm, such as 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm).

[0058] The preparation method by mixing involved in the ninth aspect of the present invention includes the following steps:

[0059] (1) Mix the cationic liposome and the negatively charged phospholipid in an optimal molar ratio and dissolve them in a certain amount of chloroform, and rotary evaporate with a rotary evaporator until a uniform thin film is formed on the pear-shaped flask.

[0060] (2) Add RNase-free water and sonicate to obtain an aqueous solution of empty liposomes.

[0061] (3) Add the EGFP mRNA solution (1 mg / mL) and mix with it to obtain a liposome / mRNA complex solution.

[0062] In some embodiments of the ninth aspect of the present invention, the total volume of the RNase-free water added in step (2) and the EGFP mRNA solution in step (3) is 500 μL.

[0063] In some embodiments of the ninth aspect of the present invention, the mixing time in step (3) is 5 - 15 min, such as 6 min, 7 min, 8 min, 9 min, 10 min, preferably 10 min.

[0064] The ninth aspect of the present invention relates to a preparation method by ethanol mixing, which includes the following steps:

[0065] (1) Mix cationic liposomes and negatively charged phospholipids in an optimal molar ratio and dissolve them in ethanol. Take EGFP mRNA (1 mg / mL) and dissolve it in RNase-free water and place it in a cardiocentesis flask.

[0066] (2) Place a magnetic stir bar and turn on the magnetic stirrer.

[0067] (3) Slowly add the ethanol solution of liposomes to the aqueous mRNA solution using a pipette.

[0068] (4) After the addition is complete, remove the ethanol by rotary evaporation using a rotary evaporator.

[0069] In some embodiments of the ninth aspect of the present invention, the total volume of RNase-free water and EGFP mRNA solution added in step (1) is 500 μL, the volume of ethanol is 1 / 3 of the total volume of the aqueous phase, and the final concentration of the total liposomes in the aqueous phase is 0.192 mg / mL.

[0070] In some embodiments of the ninth aspect of the present invention, the rotation speed of the magnetic stirrer in step (2) is 500 - 1000 rpm, such as 600 rpm, 700 rpm, 800 rpm, 900 rpm.

[0071] The tenth aspect of the present invention relates to the transfection effect of liposome / mRNA complexes with different molar ratios formed by cationic lipids and negatively charged phospholipids prepared by the mixing method of the ninth aspect of the present invention on Neuro-2a cells.

[0072] In some embodiments of the tenth aspect of the present invention, the absolute concentration of the cationic liposome DOTAP is always kept consistent at 0.1328 mg / mL.

[0073] In some embodiments of the tenth aspect of the present invention, the molar ratio range of cationic liposomes to negatively charged phospholipids is 7:0 - 7:14, such as 7:0, 7:3, 7:7, 7:14.

[0074] In the embodiments of the tenth aspect of the present invention, the transfection time is 24 hours.

[0075] The eleventh aspect of the present invention relates to the synthesis method of the nucleic acid delivery system described in the second aspect of the present invention.

[0076] The eleventh aspect of the present invention relates to the influence of nucleic acid delivery systems formed at different molar ratios of ionizable lipids and negatively charged phospholipids under the condition of the same total concentration on the transfection effect on Neuro-2a cells.

[0077] In an embodiment of the eleventh aspect of the present invention, the cationic liposome DOTAP is replaced by an ionizable liposome, and the ionizable lipid material is preferably DODAP, DODMA, DLin-MC3-DMA, and further preferably DODAP. The negatively charged phospholipid material is selected from phosphatidylserine (PS), and further preferably 1-palmitoyl-2-oleoyl phosphatidylserine (POPS).

[0078] In an embodiment of the eleventh aspect of the present invention, the molar ratio of the ionizable liposome to the negatively charged phospholipid ranges from 10:0 to 0:10, preferably 9:1 to 2:8, and further preferably 8:2 to 3:7, such as 8:2, 6:4, 5:5, 4:6, 3:7.

[0079] In an embodiment of the eleventh aspect of the present invention, the total concentration of the liposome is 0.48 mg / mL.

[0080] In an embodiment of the eleventh aspect of the present invention, the transfection time is 24 hours.

[0081] The twelfth aspect of the present invention relates to a synthesis method of the nucleic acid delivery system described in the second aspect of the present invention. The liposome used therein is the ionizable liposome DODAP, and no negatively charged phospholipid is added. The mRNA solution system is RNase-free water or citric acid-sodium citrate buffer with a pH of 4.

[0082] In an embodiment of the twelfth aspect of the present invention, the total concentration of the liposome is 0.384 mg / mL.

[0083] In an embodiment of the twelfth aspect of the present invention, the transfection time is 24 hours.

[0084] In the present invention, unless otherwise specified:

[0085] The term "mRNA" refers to messenger ribonucleic acid, which is transcribed from one strand of DNA as a template and is a type of single-stranded ribonucleic acid that carries genetic information and can guide protein synthesis.

[0086] The term "MEM" refers to minimum essential medium.

[0087] The term "EGFP mRNA" is mRNA that can express enhanced green fluorescent protein.

[0088] The term "PBS" refers to phosphate buffered solution.

[0089] The term "Hoechst 33342" is a blue fluorescent dye used for nuclear staining.

[0090] The term "POPS" refers to 1-palmitoyl-2-oleoyl phosphatidylserine.

[0091] The term "DOTAP" refers to (2,3-dioleoyl-propyl)-trimethylammonium chloride.

[0092] The term "DODAP" refers to 1,2-dioleoyloxy-3-(dimethylamino)propane.

[0093] The term "cck-8" refers to a rapid, highly sensitive, non-radioactive colorimetric detection kit based on WST-8 and widely used for cell proliferation and cytotoxicity.

[0094] The term "Neuro-2a cells" refers to mouse neuroblastoma cells.

[0095] Beneficial effects achieved by the present invention:

[0096] 1. The present invention reveals the effects of various conditional parameters such as the main materials of liposomes, the molar ratio of positive and negative charges, liposome concentration, auxiliary materials, uptake time, transfection time, preparation method, and net charge on the transfection effect of EGFP mRNA on neuro-2a cells.

[0097] 2. The present invention introduces negatively charged phospholipid materials, which not only ensure the effectiveness of the transfection effect but also ensure the safety of the delivery system. When the total liposome concentration in this experiment was 0.48 mg / mL, the cell viability of the pure DOTAP group was only about 10%. While the cell viability of the DOTAP:POPS molar ratio of 7:3 group was above 80%, indicating the improvement of the safety of the mRNA delivery system by introducing negatively charged phospholipid materials. Brief description of the drawings

[0098] To make the content of the present invention easier to understand clearly, the following further detailed description of the present invention is made according to the specific embodiments of the present invention and in combination with the drawings, where

[0099] Figure 1 is the process of preparing liposome / EGFP mRNA complex by thin film sonication method in Example 1 and the structural schematic diagram of the complex;

[0100] Figure 2 are high-content fluorescence photographs taken at a 10-fold ratio of transfecting Neuro-2a cells with different molar ratios of DOTAP:POPS when the total liposome concentration in Example 1 is 0.48 mg / mL;

[0101] Figure 3 is the fluorescence quantitative statistical chart of transfecting Neuro-2a cells with different molar ratios of DOTAP:POPS when the total liposome concentration in Example 1 is 0.48 mg / mL;

[0102] Figure 4Cell viability statistical charts of treating Neuro-2a cells with pure DOTAP, pure POPS, and a molar ratio of DOTAP:POPS = 7:3 for 24 hours in Example 2;

[0103] Figure 5 Structures and flowcharts of preparing liposome / EGFP mRNA complexes by thin-film ultrasound method in Example 3;

[0104] Figure 6 High-content fluorescence photographs of transfecting Neuro-2a cells at a 10-fold magnification when the molar ratio of DOTAP:POPS is 7:3 and the total liposome concentrations are 0.672 mg / mL, 0.576 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.288 mg / mL, 0.192 mg / mL, and 0.096 mg / mL respectively in Example 3;

[0105] Figure 7 For Example 3 and Figure 6 Fluorescence quantitative statistical charts of transfecting Neuro-2a cells under the same conditions in Example 3;

[0106] Figure 8 Structures and flowcharts of preparing liposome / EGFP mRNA complexes by thin-film ultrasound method in Example 4;

[0107] Figure 9 High-content photographs of the transfection effects of Neuro-2a cells when the molar ratios of DOTAP:POPS:cholesterol are 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, 7:3:21 respectively in Example 4, and the total liposome concentration in each group is 0.48 mg / mL and the EGFP mRNA concentration is 0.027 mg / mL;

[0108] Figure 10 For Example 4 and Figure 9 Fluorescence quantitative statistical charts of transfecting Neuro-2a cells under the same conditions in Example 4;

[0109] Figure 11 High-content fluorescence photographs of transfecting Neuro-2a cells at a 10-fold magnification when the molar ratio of DOTAP:POPS:cholesterol is 7:3:14 and the total liposome concentrations are 0.672 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.192 mg / mL, 0.096 mg / mL, 0.048 mg / mL, and 0.024 mg / mL respectively in Example 5;

[0110] Figure 12 For Example 5 and Figure 11Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the same conditions;

[0111] Figure 13 Flow chart of the uptake experiment in Example 6;

[0112] Figure 14 Neuro-2a cell transfection effect pictures taken by high-content imaging at 0.5 h, 1 h, 2 h, and 4 h of uptake in Example 6;

[0113] Figure 15 In Example 6 and Figure 14 Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the same conditions;

[0114] Figure 16 Flow chart of the transfection experiment at different times in Example 7;

[0115] Figure 17 Neuro-2a cell transfection effect pictures taken by high-content imaging at 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h of transfection in Example 7;

[0116] Figure 18 In Example 7 and Figure 17 Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the same conditions;

[0117] Figure 19 Preparation process of liposome / EGFP mRNA complexes with different methods in Example 8;

[0118] Figure 20 Neuro-2a cell transfection effect pictures taken by high-content imaging at 24 h of transfection under four different preparation methods in Example 8;

[0119] Figure 21 In Example 8 and Figure 20 Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the same conditions;

[0120] Figure 22 Preparation flow chart (taking pure DOTAP as an example) of liposome / EGFP mRNA complexes prepared by the mixing method and schematic diagram of the formed complex structure in Example 9;

[0121] Figure 23 Neuro-2a cell transfection effect pictures taken by high-content imaging under four different ratios in Example 9;

[0122] Figure 24 In Example 9 and Figure 23 Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the same conditions;

[0123] Figure 25 It is the flow chart for preparing liposome / EGFP mRNA complex by thin film ultrasound method in Example 10.

[0124] Figure 26 It is the high-content fluorescence photograph taken at 10-fold magnification of Neuro-2a cells transfected with different molar ratios of DODAP:POPS when the total liposome concentration is 0.48 mg / mL in Example 10.

[0125] Figure 27 It is the fluorescence quantitative statistical chart of Neuro-2a cells transfected with different molar ratios of DODAP:POPS when the total liposome concentration is 0.48 mg / mL in Example 10.

[0126] Figure 28 It is the flow chart for preparing liposome / EGFP mRNA complex by thin film ultrasound method in Example 11.

[0127] Figure 29 It is the high-content fluorescence photograph taken at 10-fold magnification of Neuro-2a cells transfected with liposome / EGFP mRNA complex prepared with pure DODAP in different solution systems when the total liposome concentration is 0.48 mg / mL in Example 11.

[0128] Figure 30 It is the fluorescence quantitative statistical chart of Neuro-2a cells transfected with liposome / EGFP mRNA complex prepared with pure DODAP in different solution systems when the total liposome concentration is 0.48 mg / mL in Example 11. Detailed implementation manners

[0129] The embodiments of the present invention will be clearly and completely described below in conjunction with the examples. Obviously, the described examples are only a part of the embodiments of the present invention, rather than all of the embodiments. The description of at least one exemplary embodiment below is actually only illustrative and in no way restrictive of the present invention and its application or use. All other examples obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0130] Example 1: Influence of different molar ratios of DOTAP / POPS on the transfection effect of EGFP mRNA (thin film ultrasound method)

[0131] (1) Cell culture: Seed Neuro-2a cells at 3×10 5Inoculate cells at a density of cells / mL into a 96-well plate, with 100 μL of digested cell suspension in each well. Culture in a MEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody in a 37 °C CO₂ cell incubator for 24 hours. After 24 hours, change the medium, with 100 μL of fresh medium in each well.

[0132] (2) Experimental grouping: There are 12 groups with DOTAP:POPS molar ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, and a blank group. The total concentration of liposomes is 0.48 mg / mL for all groups.

[0133] (3) Preparation of liposome / EGFP mRNA complexes by the thin film ultrasonic method: Weigh DOTAP and POPS and mix them in molar ratios from 10:0 to 0:10. The total mass of liposomes in each group is 240 μg, and they are dissolved in a certain amount of chloroform respectively. Use a rotary evaporator to evaporate until a uniform thin film forms on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the pear-shaped flask that has been rotary evaporated. Ultrasonic in an ultrasonic instrument for 5 minutes to make the total concentration of liposomes at each molar ratio 0.48 mg / mL, and finally form 11 groups of liposome / mRNA complexes with different ratios. The preparation process and the schematic diagram of the structure of the formed liposome / mRNA complexes are as Figure 1 shown.

[0134] (4) Cell transfection: Add the liposome / mRNA complexes to the cells cultured in (1). Set 3 replicate wells for each group, with 150 μL in each well. The blank group adds an equal volume of PBS solution. Continue to place in the cell incubator for 24 hours. Stain the cell nuclei with Hoechst33342, use an IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10× microscope. The fluorescence pictures and quantitative analysis of each group are as Figure 2 、 Figure 3 shown.

[0135] (5) Experimental results:

[0136] Figure 1 a is the flow chart for preparing liposome / EGFP mRNA complexes by the thin film ultrasonic method. Taking the preparation process of pure DOTAP / EGFP mRNA as an example, during the ultrasonic treatment of the liposome thin film on the pear-shaped flask, the liposome thin film detaches from the wall of the pear-shaped flask and arranges into a spherical bilayer structure, encapsulating the EGFP mRNA in the aqueous solution inside the spherical structure. Figure 1 b is the schematic diagram of the structure of the complex prepared when the DOTAP:POPS molar ratio is 10:0; Figure 1c is a schematic diagram of the structure of the complex prepared when the molar ratio of DOTAP:POPS is 9:1, 8:2, 7:3, and 6:4; Figure 1 d is a schematic diagram of the structure of the complex prepared when the molar ratio of DOTAP:POPS is 5:5; Figure 1 e is a schematic diagram of the structure of the complex prepared when the molar ratio of DOTAP:POPS is 4:6, 3:7, 2:8, and 1:9; Figure 1 f is a schematic diagram of the structure of the complex prepared when the molar ratio of DOTAP:POPS is 0:10.

[0137] Figure 2 These are high-content fluorescence photographs taken at a 10-fold magnification of Neuro-2a cells transfected with different molar ratios of DOTAP:POPS when the total lipid concentration is 0.48 mg / mL.

[0138] Figure 3 This is a fluorescence quantitative statistical chart of Neuro-2a cells transfected with different molar ratios of DOTAP:POPS when the total lipid concentration is 0.48 mg / mL.

[0139] It can be seen from Figure 2 that effective transfection of mRNA can be achieved when the net charge of the delivery system is positive or zero, that is, when the molar ratio of DOTAP to POPS is greater than or equal to 1, EGFP mRNA can be successfully delivered into cells and the green fluorescent protein can be successfully expressed.

[0140] It can be seen from Figure 3 that under the condition of the same total lipid concentration, the transfection effect of the pure DOTAP group is the highest, and it is significantly higher than other groups with a molar ratio of DOTAP:POPS greater than or equal to 1. The transfection effect of the DOTAP:POPS molar ratio of 7:3 is significantly higher than that of the 6:4 and 5:5 groups, but there is no significant difference compared with the 9:1 and 8:2 groups, only a tendency to be slightly higher.

[0141] Example 2: Investigation of the cell viability of DOTAP, POPS, and DOTAP:POPS = 7:3.

[0142] (1) Cell culture is as described in Example 1(1)

[0143] (2) Cell viability assay: Weigh an appropriate amount of liposomes, and rotary evaporate them in a pear-shaped flask with an appropriate amount of chloroform. Then, ultrasonicate them with RNase-free water to make the concentration of liposomes 2.9 mg / mL. Use a small liposome extruder to extrude and push them through a polycarbonate membrane with a pore size of 0.1 μm, and repeat the extrusion 10 times. Dilute the extruded liposomes 10-fold with MEM medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics to obtain a diluted solution with a concentration of 0.29 mg / mL. Subsequently, dilute this diluted solution to 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 times the original concentration with the above medium in turn. Aspirate the original medium in the above cells, and then add the 9-concentration liposome diluted solutions and blank medium to the above cells in ascending order of concentration from left to right. Set 6 replicates for each concentration, 250 μL per well, and continue to place them in a cell culture incubator for 24 hours. After 24 hours, aspirate the liquid in the wells, prepare a mixed solution of medium and cck-8 reagent at a volume ratio of 10:1, 110 μL per well, put it back into the cell culture incubator and continue to incubate for 1 hour, and then measure the absorbance value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The cell viability of different concentrations of DOTAP, POPS, and DOTAP:POPS = 7:3 liposomes is as Figure 4 shown.

[0144] (3) Experimental results:

[0145] Figure 4 From left to right are the cell viability statistical charts of Neuro-2a cells treated with pure DOTAP, pure POPS, and DOTAP:POPS = 7:3 molar ratio for 24 hours respectively. The total concentration gradients of each liposome are set as: 0.096 mg / mL, 0.144 mg / mL, 0.192 mg / mL, 0.24 mg / mL, 0.288 mg / mL, 0.336 mg / mL, 0.384 mg / mL, 0.432 mg / mL, 0.48 mg / mL.

[0146] By Figure 4It can be seen that the CCK-8 cell experiment verified the influence of positive charges on the system. Although high positive charges are beneficial to the transfection of the system, their strong interference with the cell membrane leads to a low cell survival rate. In this experiment, when the total liposome concentration was 0.48 mg / mL, the cell viability of the pure DOTAP group was only about 10%. The cell viability of the DOTAP:POPS molar ratio of 7:3 group was above 80%. When the concentration of pure DOTAP reached 0.288 mg / mL, about 65% of the cells apoptosed, while the negatively charged material under the same concentration did not show this phenomenon. When the total liposome concentration of DOTAP decreased to half of the highest concentration, 0.24 mg / mL, the cell activity was only about 70%, indicating its high toxicity. Therefore, based on the transfection data, it can be known that through the adjustment of the system charge, under the condition of a DOTAP molar ratio of 70%, mRNA can achieve effective transfection, and at the same time, the cell survival rate is above 90%. While ensuring the effectiveness of the transfection effect, the safety of the delivery system is also guaranteed. Therefore, the molar ratio of DOTAP and POPS of 7:3 is the best condition to meet the effectiveness and safety of the delivery system. The appropriate addition of negatively charged materials does not affect the transfection efficiency and can improve the safety of the delivery system.

[0147] Example 3: Effect of the total concentration of DOTAP / POPS optimal molar ratio liposomes on the transfection effect of EGFP mRNA (thin film ultrasonic method)

[0148] (1) Cell culture was carried out as described in Example 1(1)

[0149] (2) Experimental grouping: The total concentrations of DOTAP and POPS liposomes were 0.672 mg / mL, 0.576 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.288 mg / mL, 0.192 mg / mL, and 0.096 mg / mL, respectively, for a total of 7 groups. The molar ratio of DOTAP and POPS was 7:3.

[0150] (3) Preparation of liposome / EGFP mRNA complexes by thin-film ultrasound method: Weigh DOTAP and POPS and mix them at a molar ratio of 7:3. Set the total mass of liposomes to be 336 μg, 288 μg, 240 μg, 192 μg, 144 μg, 96 μg, and 48 μg respectively, and dissolve them in a certain amount of chloroform. Use a rotary evaporator to evaporate until a uniform thin film is formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the pear-shaped flask that has been evaporated. Sonicate in an ultrasonic bath for 5 min to make the total concentration of liposomes in each group 0.672 mg / mL, 0.576 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.288 mg / mL, 0.192 mg / mL, and 0.096 mg / mL respectively, and finally form 7 groups of liposome / mRNA complexes with different concentrations. The preparation process is as Figure 5 shown.

[0151] (4) Cell transfection: Add the liposome / mRNA complexes to the cells cultured above. Set 3 replicate wells in each group, with 150 μL in each well, and continue to place them in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, use an IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10× objective lens. The fluorescence photos and quantitative analysis of each group are as Figure 6 、 Figure 7 shown.

[0152] (5) Experimental results:

[0153] Figure 5 is the structure and flowchart of the preparation of liposome / EGFP mRNA complexes by thin-film ultrasound method.

[0154] Figure 6 are high-content fluorescence photos taken at a 10× magnification of Neuro-2a cells transfected when the molar ratio of DOTAP:POPS is 7:3 and the total concentration of liposomes is 0.672 mg / mL, 0.576 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.288 mg / mL, 0.192 mg / mL, and 0.096 mg / mL respectively.

[0155] Figure 7 is the fluorescence quantitative statistical chart of Neuro-2a cells transfected under the above conditions.

[0156] From Figure 6 、 Figure 7It can be seen that the total concentration of liposomes is not the higher the better. Too high a concentration may affect the release and transfection of mRNA. Without adding cholesterol components, when the total concentration of the two main lipids, DOTAP and POPS, which determine the charge (molar ratio of 7:3), is below 0.48 mg / mL, effective transfection of EGFP mRNA can also be achieved. The effect is the best when the concentration is 0.192 mg / mL, and it is significantly higher than other groups.

[0157] Example 4: Effect of different proportions of the auxiliary lipid cholesterol on the transfection effect based on the optimal molar ratio of DOTAP / POPS (thin film ultrasound method)

[0158] (1) Cell culture was carried out as described in Example 1(1).

[0159] (2) Experimental grouping: There were 6 groups with the molar ratio of DOTAP:POPS:cholesterol being 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, and 7:3:21, and the total concentration of liposomes was 0.48 mg / mL for all groups.

[0160] (3) Preparation of liposome / EGFP mRNA complexes by the thin film ultrasound method: Weigh DOTAP, POPS, and cholesterol and mix them in the molar ratios of 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, and 7:3:21 (a total of 6 groups). The total mass of liposomes in each group was 240 μg, and they were dissolved in a certain amount of chloroform respectively. Then, use a rotary evaporator to evaporate until a uniform thin film was formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary-evaporated pear-shaped flask and ultrasonicate in an ultrasonic cleaner for 5 min to make the total concentration of liposomes of each molar ratio 0.48 mg / mL, finally forming 6 groups of liposome / mRNA complexes with different ratios. The preparation process is as Figure 8 shown.

[0161] (4) Cell transfection: Add the liposome / mRNA complexes to the above-cultured cells. Set 3 replicate wells for each group, with 150 μL in each well, and continue to place them in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, use the IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10× objective lens. The fluorescence photos and quantitative analysis of each group are as Figure 9 、 Figure 10 shown.

[0162] (5) Experimental results:

[0163] Figure 8 The structure and flow chart of the preparation of liposome / EGFP mRNA complexes by the thin film ultrasound method

[0164] Figure 9 High-content images of the transfection efficiency of Neuro-2a cells when the molar ratios of DOTAP:POPS:cholesterol are 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, 7:3:21, and the total liposome concentration in each group is 0.48 mg / mL and the EGFP mRNA concentration is 0.027 mg / mL.

[0165] Figure 10 Fluorescence quantitative statistical chart of Neuro-2a cells transfected under the above conditions.

[0166] From Figure 9 and Figure 10 it can be seen that the transfection efficiency of the 7:3:14 group is the best, significantly higher than that of other groups with added cholesterol, and there is no significant difference compared with the group without added cholesterol. From the above results, it can be seen that under the condition of maintaining the charge ratio of liposomes, the addition of cholesterol can stabilize the structure of liposomes. When the molar ratio of the system reaches about 14 / 24, the best transfection efficiency can be achieved. The addition of too much cholesterol is not conducive to the overall transfection efficiency because it dilutes the content of the main liposomes (DOTAP and POPS) in the overall system. In addition, the content of the main liposomes (DOTAP and POPS) in the system has no decisive influence on the overall transfection efficiency, leaving room for the addition of other functional components subsequently.

[0167] Example 5: Effect of different total liposome concentrations on the transfection efficiency of EGFP mRNA with the optimal molar ratio of DOTAP / POPS / cholesterol (thin film sonication method)

[0168] (1) Cell culture is as described in Example 1(1)

[0169] (2) Experimental grouping: The total concentrations of DOTAP, POPS, and cholesterol liposomes are 0.72 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.192 mg / mL, 0.096 mg / mL, 0.048 mg / mL, 0.024 mg / mL, a total of 7 groups, and the molar ratio of DOTAP:POPS:cholesterol is 7:3:14.

[0170] (3) Preparation of liposome / EGFP mRNA complexes by thin film ultrasound method: Weigh DOTAP, POPS and cholesterol and mix them in a molar ratio of 7:3:14. Set the total mass of liposomes to be 360 μg, 240 μg, 192 μg, 96 μg, 48 μg, 24 μg, and 12 μg respectively, and dissolve them in a certain amount of chloroform. Rotate and evaporate with a rotary evaporator until a uniform thin film is formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary-evaporated pear-shaped flask and ultrasonicate it in an ultrasonic instrument for 5 minutes, so that the total concentration of liposomes in each group is 0.72 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.192 mg / mL, 0.096 mg / mL, 0.048 mg / mL, and 0.024 mg / mL respectively, and finally form 7 groups of liposome / mRNA complexes with different concentrations. The preparation process is as Figure 8 shown.

[0171] (4) Cell transfection: Add the liposome / mRNA complexes to the above-mentioned cultured cells. Set 3 replicate wells in each group, with 150 μL in each well, and continue to place them in a cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, use an IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10-fold microscope. The fluorescence pictures and quantitative analysis of each group are as Figure 11 、 Figure 12 shown.

[0172] (5) Experimental results:

[0173] Figure 11 are high-content fluorescence pictures taken at a 10-fold ratio of transfected Neuro-2a cells when the molar ratio of DOTAP:POPS:cholesterol is 7:3:14 and the total concentration of liposomes is 0.72 mg / mL, 0.48 mg / mL, 0.384 mg / mL, 0.192 mg / mL, 0.096 mg / mL, 0.048 mg / mL, and 0.024 mg / mL respectively.

[0174] Figure 12 is the fluorescence quantitative statistical chart of transfected Neuro-2a cells under the above conditions.

[0175] From Figure 11 、 Figure 12It can be seen that when further investigating the effect after adding cholesterol, when the best ratio of 7:3:14 is selected, the best effect is achieved when the concentration is 0.384 mg / mL. In this delivery system, the liposome content accounts for 10 / 24, and the actual liposome concentration is 0.16 mg / mL, which is close to the best transfection concentration of liposome (0.192 mg / mL) without cholesterol addition, proving that in the delivery system, the core influencing factors are the charged DOTAP and POPS components.

[0176] Example 6: DOTAP / POPS Optimal Molar Ratio - EGFP mRNA Uptake Experiment at Different Times (Thin Film Sonication Method)

[0177] (1) Cell culture was carried out as described in Example 1(1).

[0178] (2) Experimental grouping: 0.5 h, 1 h, 2 h, and 4 h uptake groups.

[0179] (3) Preparation of liposome / EGFP mRNA complex by thin film sonication method: Weigh DOTAP and POPS and mix them in a molar ratio of 7:3, and make the total mass of liposomes 96 μg. Dissolve them in a certain amount of chloroform and rotary evaporate until a uniform thin film is formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary-evaporated pear-shaped flask and sonicate in an ultrasonic bath for 5 min to make the total concentration of each group of liposomes 0.192 mg / mL, finally forming 4 groups of identical liposome / mRNA complexes. The experimental procedure is as Figure 13 shown.

[0180] (4) Cell transfection: Add the liposome / mRNA complex to the above-cultured cells. Set 3 replicate wells for each group, with 150 μL in each well. After continuing to place them in the cell culture incubator for 0.5 hour, 1 hour, 2 hours, and 4 hours respectively, aspirate all the liquid in each well, wash three times with PBS, and add fresh medium to continue culturing until 24 hours. Stain the cell nuclei with Hoechst33342, use the IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10× microscope. The fluorescence pictures and quantitative analysis of each group are as Figure 14 、 Figure 15 shown.

[0181] (5) Experimental results:

[0182] Figure 13 is the flowchart of the uptake experiment.

[0183] Figure 14 are the pictures of the transfection effect of Neuro-2a cells taken by high-content imaging at 0.5 h, 1 h, 2 h, and 4 h of uptake.

[0184] Figure 15 These are the fluorescence quantitative results of the above experiments.

[0185] Figure 14 、 Figure 15 It shows that within 0.5 h and 1 h, very little EGFP mRNA is taken up by cells. It takes at least 2 h for more EGFP mRNA to be taken up by cells, and a large amount can be taken up at 4 h.

[0186] Example 7: Effect of different times of DOTAP / POPS optimal molar ratio - EGFP mRNA on transfection efficiency (thin film sonication method)

[0187] (1) Cell culture is as described in Example 1(1)

[0188] (2) Experimental grouping: transfection groups at 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h.

[0189] (3) Preparation of liposome - EGFP mRNA complex by thin film sonication method: Weigh DOTAP and POPS and mix them at a molar ratio of 7:3, and make the total mass of the liposome 96 μg. Dissolve it in a certain amount of chloroform and rotary evaporate until a uniform thin film is formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary evaporated pear-shaped flask and sonicate in an ultrasonic instrument for 5 min to make the total concentration of each group of liposomes 0.192 mg / mL, finally forming 6 groups of completely identical liposome / mRNA complexes. The experimental process is as Figure 16 shown.

[0190] (4) Cell transfection: Add the liposome / mRNA complex to the above-mentioned cultured cells. Set 3 replicate wells for each group, with 150 μL in each well, and continue to place them in the cell culture incubator for treatment at 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h respectively. Stain the cell nuclei with Hoechst 33342, use the IXM-C type high-content imaging analysis system to observe the expression of EGFP mRNA and take pictures with a 10× objective lens. The fluorescence pictures and quantitative analysis of each group are as Figure 18 、 Figure 19 shown.

[0191] (5) Experimental results:

[0192] Figure 16 This is the flow chart of the transfection experiment at different times.

[0193] Figure 17 These are the pictures of the transfection effect of Neuro-2a cells taken by high-content imaging at transfection times of 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h.

[0194] Figure 18 For the fluorescence quantitative results of the above experiments.

[0195] Figure 17 、 Figure 18 It shows that EGFP mRNA requires at least 12 h to achieve effective transfection. Green fluorescent protein cannot be expressed within 6 h or less. The transfection effect is optimal at 24 h and significantly higher than that of other time groups. After 48 h, the transfection effect decreases compared to 24 h, probably because the cell viability deteriorates and the green fluorescent protein degrades.

[0196] Example 8: Investigation of different preparation methods of DOTAP / POPS-EGFP mRNA

[0197] (1) Cell culture is as described in Example 1(1)

[0198] (2) Experimental grouping: thin film dispersion method, extrusion method, mixing method, ethanol mixing method group.

[0199] (3) Preparation of liposome / mRNA complexes by different methods: (1) Thin film dispersion method: Weigh DOTAP and POPS and mix them in a molar ratio of 7:3, and make the total mass of liposomes 96 μg. Dissolve them in a certain amount of chloroform and rotary evaporate until a uniform thin film is formed on the round-bottom flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary-evaporated round-bottom flask and sonicate in an ultrasonic bath for 5 min to make the total concentration of each group of liposomes 0.192 mg / mL, finally forming the prepared liposome / mRNA complex. (2) Extrusion method: On the basis of the liposome / mRNA complex prepared by the thin film dispersion method, extrude it through a polycarbonate membrane with a pore size of 0.1 μm using a small liposome extruder and extrude it repeatedly 10 times. (3) Mixing method: Weigh DOTAP and POPS and mix them in a molar ratio of 7:3, and make the total mass of liposomes 96 μg. Dissolve them in a certain amount of chloroform and rotary evaporate until a uniform thin film is formed on the round-bottom flask. Add 486.5 μL of RNase-free water and sonicate for 5 min to obtain an aqueous solution of empty liposomes. Subsequently, add 13.5 μL of EGFP mRNA (1 mg / mL) and mix it with the above solution for 10 min to obtain a liposome / mRNA complex solution. (4) Ethanol mixing method: Mix DOTAP and POPS in a molar ratio of 7:3, and dissolve 96 μg of liposomes in 166 μL of ethanol. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water and place it in a round-bottom flask. Put a magnetic stir bar in it, turn on the magnetic stirrer to make the rotation speed 1000 rmp, and slowly drip the ethanol solution of liposomes into the aqueous solution of mRNA with a pipette. After the dripping is completed, rotary evaporate to remove ethanol with a rotary evaporator.

[0200] (4) Cell transfection: Add the liposome / mRNA complexes prepared by the above four methods into the cells cultured above. Set 3 replicate wells for each group, with 150 μL in each well, and continue to place them in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, observe the expression of EGFP mRNA using an IXM-C type high-content imaging analysis system, and take pictures with a 10x microscope.

[0201] (5) Experimental results

[0202] Figure 19 is the preparation process of liposome / EGFP mRNA complexes by different methods.

[0203] Figure 20 are the pictures of the transfection effect of Neuro-2a cells taken by high-content imaging at 24 h of transfection under four different preparation methods.

[0204] Figure 21 are the fluorescence quantitative results of the above experiments.

[0205] Figure 20 、 Figure 21 It shows that the thin film dispersion method and the mixing method can achieve effective transfection of EGFP mRNA, while the extrusion method, which adds the extrusion step on the basis of the thin film dispersion method, hardly has the expression of green fluorescent protein. This indicates that the extrusion process can destroy the formed liposome / mRNA complexes and may also retain the mRNA on the polycarbonate membrane. The ethanol mixing method is similar to the microfluidic synthesis method. The failure to transfect green fluorescent protein may be due to inappropriate parameters such as rotation speed or incomplete removal of residual ethanol, which causes damage to the cells, resulting in transfection failure. Both the thin film dispersion method and the mixing method can achieve good transfection effects. Since the liposome forms a phospholipid bilayer structure, the charged head can either be exposed on the surface of the spherical shell or aggregated inside the spherical liposome. These two preparation methods result in negatively charged mRNA being encapsulated inside the liposome and adhering to the surface of the spherical liposome shell, indicating that in a simple in vitro environment without various complex in vivo environments, even if the mRNA is not encapsulated and only adheres to the surface of the liposome shell, it can still achieve efficient transfection of cells.

[0206] Example 9: Effect of the net charge of DOTAP / POPS on the transfection effect of EGFP mRNA (mixing method)

[0207] (1) Cell culture is the same as in Example 1(1)

[0208] (2) Experimental grouping: There are 4 groups with the molar ratio of DOTAP:POPS being 7:0, 7:3, 7:7, and 7:14. The concentration of DOTAP in each group is 0.1328 mg / mL.

[0209] (3) Preparation of liposome / mRNA complexes by the mixing method: Keeping the total amount of DOTAP liposomes unchanged, gradually increase the proportion of POPS in the liposome system. Weigh DOTAP and POPS and mix them at molar ratios of 7:0, 7:3, 7:7, and 7:14, with the mass of DOTAP fixed at 66.4 μg in each group. Dissolve them in a certain amount of chloroform, and rotary evaporate until a uniform thin film is formed on the pear-shaped flask. Add 486.5 μL of RNase-free water and ultrasonicate for 5 min to obtain an aqueous solution of empty liposomes. Subsequently, add 13.5 μL of EGFP mRNA (1 mg / mL) and mix with it for 10 min to obtain a liposome / mRNA complex solution.

[0210] (4) Cell transfection: Add the four groups of prepared liposome / mRNA complexes to the above-mentioned cultured cells. Set 3 replicate wells for each group, with 150 μL in each well, and continue to place them in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, observe the expression of EGFP mRNA using an IXM-C type high-content imaging analysis system, and take pictures with a 10× objective lens.

[0211] (5) Experimental results

[0212] Figure 22 It is a preparation flow chart for preparing liposome / EGFP mRNA complexes by the mixing method (taking pure DOTAP as an example). Figure 22 b - e are respectively schematic diagrams of the structures of the prepared liposome / mRNA complexes when the molar ratio of DOTAP:POPS is 7:0, 7:3, 7:7, and 7:14.

[0213] Figure 23 They are pictures of the transfection effects of Neuro-2a cells taken by high-content imaging at four different ratios.

[0214] Figure 24 They are the fluorescence quantitative results of the above experiments.

[0215] Figure 23 、 Figure 24The results showed that as the proportion of POPS in the system increased, even when the amount of DOTAP remained unchanged, the transfection efficiency gradually decreased. Compared with the 7:3 group, pure DOTAP had a very high transfection efficiency. However, when the ratio of the two reached 7:7, that is, when the net charge was 0, the transfection efficiency decreased significantly, only a little. And when the ratio was 7:14, the net charge in the whole system was negative, and effective transfection of mRNA could not be achieved. By fixing the dosage of the positively charged liposome in the core and adjusting the dosage of the negatively charged one, it was shown that the factor affecting the mRNA transfection effect was the net charge of the total liposome rather than the amount of positively charged lipid in the total liposome, proving that in the liposome delivery system, what determines the transfection effect is the proportion of the core material in the whole liposome system rather than its single dosage.

[0216] Example 10: Effect of different molar ratios of DODAP / POPS on the transfection effect of EGFP mRNA (thin film dispersion method)

[0217] (1) Cell culture was carried out as described in Example 1(1).

[0218] (2) Experimental grouping: Five groups with molar ratios of DODAP:POPS being 8:2, 6:4, 5:5, 4:6, and 3:7 were set up, and the total liposome concentration in each group was 0.48 mg / mL.

[0219] (3) Preparation of liposome / EGFP mRNA complexes by thin film sonication method: Weigh DODAP and POPS and mix them at molar ratios of 8:2, 6:4, 5:5, 4:6, and 3:7 respectively. The total mass of liposomes in each group was 240 μg, which were dissolved in a certain amount of chloroform and rotary evaporated until a uniform thin film was formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water. After mixing, add it to the rotary evaporated pear-shaped flask and sonicate in an ultrasonic instrument for 5 min to make the total liposome concentration of each molar ratio 0.48 mg / mL, finally forming five groups of liposome / mRNA complexes with different ratios. The preparation process and the schematic diagram of the structure of the formed liposome / mRNA complexes are as Figure 25 shown.

[0220] (4) Cell transfection: Add the liposome / mRNA complexes to the cells cultured in (1). Set 3 replicate wells for each group, with 150 μL in each well. The blank group was added with an equal volume of PBS solution. Continue to place it in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst33342, observe the expression of EGFP mRNA using an IXM-C type high-content imaging analysis system, and take pictures with a 10× objective lens. The fluorescence pictures and quantitative analysis of each group are as Figure 2 、 Figure 3 shown.

[0221] (5) Experimental results:

[0222] Figure 25 Structural and flowchart of preparing liposome / EGFP mRNA complex by thin film ultrasound method

[0223] Figure 26 High-content fluorescence photographs taken at a 10-fold ratio of transfecting Neuro-2a cells with different molar ratios of DODAP:POPS when the total liposome concentration is 0.48 mg / mL

[0224] Figure 27 Fluorescence quantitative statistical chart of transfecting Neuro-2a cells with different molar ratios of DODAP:POPS when the total liposome concentration is 0.48 mg / mL

[0225] From Figure 26 Figure 27 it can be seen that when the cationic liposome DOTAP is replaced with the ionizable liposome DODAP and still prepared by the thin film dispersion method, effective transfection of mRNA cannot be achieved regardless of the molar ratio. The reason may be that the ionizable liposome itself has no net charge and needs the hydrogen ions in the citric acid-sodium citrate acidic buffer to ionize it to carry a positive charge to play the role of a cationic liposome and combine with the negatively charged mRNA, while only using neutral water as a solvent cannot electrostatically attract the negatively charged mRNA

[0226] Example 11: Influence of different solution systems of DODAP on the transfection effect of EGFP mRNA (thin film dispersion method)

[0227] (1) Cell culture is shown in Example 1(1)

[0228] (2) Experimental grouping: pure DODAP RNase-free water group, pure DODAP citric acid-sodium citrate buffer group. The total liposome concentration in each group is 0.384 mg / mL

[0229] (3) Preparation of liposome / EGFP mRNA complex by thin film ultrasound method: Weigh pure DODAP, and the total mass of liposomes in each group is 192 μg, which are respectively dissolved in a certain amount of chloroform and rotary evaporated until a uniform thin film is formed on the pear-shaped flask. Take 13.5 μL (1 mg / mL) of EGFP mRNA and dissolve it in 486.5 μL of RNase-free water or citric acid-sodium citrate buffer with pH = 4. After mixing, add it to the rotary evaporated pear-shaped flask and ultrasonicate for 5 min in an ultrasonic instrument to make the total liposome concentration of each molar ratio 0.384 mg / mL, and finally form liposome / mRNA complexes with 2 different solution systems. The preparation process and the structural schematic diagram of the formed liposome / mRNA complexes are as Figure 28 shown

[0230] (4) Cell transfection: Add the liposome / mRNA complex to the cultured cells in (1). Set 3 replicate wells for each group, with 150 μL in each well. Add an equal volume of PBS solution to the blank group. Continue to place it in the cell culture incubator for 24 hours. Stain the cell nuclei with Hoechst 33342, observe the expression of EGFP mRNA using an IXM-C type high-content imaging analysis system, and take pictures with a 10× objective lens. The fluorescence pictures and quantitative analysis of each group are as Figure 2 、 Figure 3 shown.

[0231] (5) Experimental results:

[0232] Figure 28 are the structure and flow chart of the preparation of liposome / EGFP mRNA complex by the thin film ultrasound method.

[0233] Figure 29 are high-content fluorescence pictures of liposome / mRNA complexes prepared in different solution systems transfected into Neuro-2a cells at a 10× magnification when the total liposome concentration is 0.384 mg / mL.

[0234] Figure 30 are the fluorescence quantitative statistical charts of liposome / mRNA complexes prepared in different solution systems transfected into Neuro-2a cells when the total liposome concentration is 0.384 mg / mL.

[0235] It can be seen from Figure 29 Figure 30 that the pure cationic liposome DODAP cannot achieve effective transfection of mRNA whether in a neutral RNase-free buffer system or in an acidic citrate-sodium citrate buffer system with pH = 4. This indicates that the preparation method of the thin film ultrasound method may not be applicable to ionizable liposomes.

[0236] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A nucleic acid delivery system, characterized in that, The nucleic acid delivery system includes a carrier and ribonucleic acid delivered within the carrier. The carrier is a spherical liposome, and the liposome includes a cationic lipid DOTAP and a negatively charged phospholipid 1-palmitoyl-2-oleoyl phosphatidylserine (POPS); the carrier further includes a co-lipid, and the co-lipid is selected from one or more of cholesterol and DSPE-PEG2000; The molar ratio of the cationic lipid, negatively charged phospholipid, and co-lipid is 7:3:(0-21); The ribonucleic acid to be delivered is enhanced green fluorescent protein mRNA (EGFP mRNA); The cationic liposome is prepared by a thin film dispersion method or a mixing method.

2. The nucleic acid delivery system according to claim 1, wherein The molar ratio of the cationic lipid, negatively charged phospholipid, and co-lipid is 7:3:0, 7:3:1, 7:3:3, 7:3:7, 7:3:14, or 7:3:

21.

3. The nucleic acid delivery system according to any one of claims 1-2, characterized in that, In the nucleic acid delivery system: (1) Under the condition of not adding the co-lipid cholesterol, the mass ratio of the total liposome material to the nucleic acid (g / g) is 3.556 - 24.889 g / g; (2) Under the condition of adding the co-lipid cholesterol, the mass ratio of the total liposome material to the nucleic acid (g / g) is 0.889 - 26.667 g / g.

4. The nucleic acid delivery system according to claim 3, wherein In the nucleic acid delivery system: (1) Under the condition of not adding a co-lipid, the mass ratio of the total liposome material to the nucleic acid (g / g) is 7.111 g / g; (2) Under the condition of adding a co-lipid, the mass ratio of the total liposome material to the nucleic acid (g / g) is 14.222 g / g.

5. The preparation method of the nucleic acid delivery system according to claim 1, comprising the following steps: (1) Select several of the cationic lipid, negatively charged phospholipid, and co-lipid as raw materials according to the product structure, weigh the selected raw materials according to the molar ratio, mix them and dissolve them together in a certain amount of organic solvent, and obtain a uniform lipid film after rotary evaporation; (2) Mix the ribonuclease-free aqueous solution system of ribonucleic acid mRNA with the lipid film at a ratio of 500:(12 - 360) ml / mg to obtain a mixture; (3) Ultrasonicate the above mixture to form a liposome / mRNA complex, which is the nucleic acid delivery system.

6. The preparation method of the nucleic acid delivery system according to claim 1, comprising the following steps: (1) Select several of the cationic lipid, negatively charged phospholipid, and co-lipid as raw materials according to the product structure, weigh the selected raw materials according to the molar ratio, mix them and dissolve them together in a certain amount of organic solvent, and obtain a uniform lipid film after rotary evaporation; (2) Add ribonuclease-free water and ultrasonicate to obtain an empty liposome solution; (3) Mix the ribonucleic acid mRNA with the empty liposome solution to obtain a liposome / mRNA complex solution, which is the nucleic acid delivery system.

7. The preparation method of the nucleic acid delivery system according to any one of claims 5-6, characterized in that, In the nucleic acid delivery system: (1) Under the condition of not adding a co-lipid, the total lipid concentration of the total lipid material in the final aqueous solution is 0.096 - 0.672 mg / mL; (2) Under the condition of adding the co-lipid cholesterol, the total lipid concentration of the total lipid material in the final aqueous solution is 0.024 - 0.72 mg / mL.

8. Use of the nucleic acid delivery system according to any one of claims 1-2 in the preparation of a medicament, characterized in that, The nucleic acid delivery system can achieve effective transfection on cells.

9. The use according to claim 8, wherein The cells include one or more of Neuro-2a cells, 293T cells, PC-12 cells, and Hela cells.

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