Liposome / polymer / nucleic acid composite delivery system with aqueous phase storage stability and high transfection property

Abstract

The invention discloses a liposome / polymer / nucleic acid composite delivery system with aqueous phase storage stability and high transfection property, and belongs to the technical field of biological materials. The liposome / polymer / nucleic acid compound prepared by the method has a cationic lipid layer and a hyperbranched polylysine / nucleic acid compound particle nucleus, and hyperbranched polylysine / mRNA particles have enhanced hydrolysis resistance, enhance the aqueous phase storage stability of the compound, and can maintain high transfection activity as long as three months under a 4 DEG C aqueous phase condition. In addition, the hyperbranched polylysine can promote efficient release of mRNA, so that efficient transfection is realized while the stability of the composite carrier is enhanced. The method provided by the invention is simple to operate and high in repeatability, and has a good application prospect in the field of mRNA vaccines.

Description

Technical Field

[0001] This invention relates to a liposome / polymer / nucleic acid composite delivery system that combines aqueous storage stability with high transfection performance, belonging to the field of biomaterials technology. Background Technology

[0002] mRNA vaccines have gained widespread attention due to their short development cycle and rapid expression, enabling rapid iteration to address constantly evolving viral strains. Compared to DNA vaccines, mRNA vaccines are more efficient, requiring only one-step translation into antigen in the cytoplasm, and do not need to enter the cell nucleus, thus eliminating the risk of insertional mutagenesis. Furthermore, mRNA therapy is based on transient transfection of non-dividing cells, exerting its effects in the cytoplasm, resulting in rapid clearance and a low likelihood of side effects, making it of significant clinical research value and practical importance. However, naked mRNA exhibits extremely low stability under physiological conditions, and its high negative charge density greatly limits cellular uptake. Therefore, it is necessary to develop safe and efficient mRNA delivery systems that protect mRNA from degradation and ensure efficient cellular uptake to achieve effective mRNA therapy.

[0003] Lipid-based nucleic acid carriers, comprising liposomes and nanotechnology-dependent lipid nanoparticles, encapsulate mRNA within their hydrophilic cavities, protecting it from nuclease degradation under complex physiological conditions. Furthermore, the phospholipid bilayer of these carriers exhibits high affinity for cell membranes, enabling efficient uptake of mRNA by cells. Therefore, lipid-based nucleic acid carriers are widely used in mRNA vaccine research. However, they often fail to encapsulate mRNA into a tight solid structure, leading to mRNA degradation during aqueous storage and resulting in poor storage stability and demanding storage conditions. Currently, lyophilization and cryogenic storage are the primary methods for ensuring the effectiveness of lipid-based nucleic acid delivery systems in clinically applied nucleic acid vaccines. However, lyophilization carries the risk of lipid breakage and mRNA leakage, and these stringent storage conditions also incur significant transportation costs. Aqueous lipid-based nucleic acid delivery systems, refrigerated at 4°C, are typically stable only for a few days to a few weeks. Cationic polymers, on the other hand, can compress mRNA through charge interactions, forming a more compact nanostructure. Therefore, combining polymers and lipids to form a composite delivery system is an effective strategy to improve the aqueous storage stability of lipid-based nucleic acid delivery systems. However, when the polymer binds too tightly to mRNA, the release rate of mRNA in cells is reduced, thereby decreasing mRNA utilization and further leading to a decrease in transfection efficiency. Therefore, it is crucial to develop lipid / polymer composite delivery systems that combine aqueous storage stability with high transfection performance. Summary of the Invention

[0004] The purpose of this invention is to provide a liposome / polymer / nucleic acid complex delivery system that combines aqueous storage stability with high transfection performance. The liposome / polymer / nucleic acid complex delivery system prepared using this invention can maintain transfection activity for up to three months in sucrose solution at 4°C, and achieves good transfection results in HEK293T, HeLa, DC2.4, CHO-K1, and MSCs cells.

[0005] The technical solution provided by this invention is as follows:

[0006] A liposome / polymer / nucleic acid complex delivery system exhibiting both aqueous storage stability and high transfectivity, wherein the delivery system is a polymer / nucleic acid complex encapsulated in liposomes, and its preparation method specifically includes the following steps:

[0007] Step 1: Preparation of cationic liposomes;

[0008] Step 2: Mix the polymer diluent and nucleic acid diluent evenly and incubate for at least 30 min to prepare the polymer / nucleic acid complex;

[0009] Step 3: Mix the cationic liposomes prepared in Step 1 with the polymer / nucleic acid complex prepared in Step 2 uniformly, and incubate for at least 40 min to obtain the polymer / nucleic acid complex encapsulated by liposomes.

[0010] In the above technical solution, further, the cationic liposomes in step 1 are specifically prepared by dissolving cationic lipids, phospholipids, cholesterol and PEG lipids in anhydrous ethanol at a molar ratio of (36-40):(10-11):(48-50):(1-3) parts in anhydrous ethanol (more preferably 40:10:48:2 parts), then injecting 3-6 times the volume of 50-58℃ DEPC water (water treated with diethyl pyrocarbonate), stirring to obtain a lipid droplet dispersion, then rotary evaporating the dispersion under reduced pressure to remove ethanol, and finally treating it with an ultrasonic cell disruptor to obtain a cationic liposome dispersion.

[0011] Furthermore, the polymer in step 2 is hyperbranched polylysine with a molecular weight of 2000-3000 g / mol, and the diluent solvent is DEPC water.

[0012] Furthermore, the mass ratio of the liposomes, hyperbranched polylysine, and nucleic acid is (5.8-7.5):(0.3-1.5):1, more preferably 6.54:1:1.

[0013] Furthermore, the liposome-encapsulated polymer / nucleic acid complex has a particle size of 100-300 nm and a zeta potential of 3-5 mV.

[0014] Furthermore, the cationic lipid is dimethyl trifluoroacetate-2,3-difluoroacetate. One or more of the following: diolenoyloxypropyl-2-(2-speramidoformamido)ethylammonium (DOSPA), trimethyl-2,3-diolenoyloxypropylammonium chloride (DOTMA), 1,2-dioleoyl-3-trimethylpropylammonium chloride (DOTAP), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), and methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester (DLin-MC3-DMA). The phospholipid is one or more of dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), and dioleoylphosphatidylethanolamine (DOPE). The PEG lipid is one or more of methoxy polyethylene glycol bis(tetradecylacetamide) (ALC-0159) and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000).

[0015] Furthermore, the nucleic acid is at least one of DNA and mRNA.

[0016] The delivery system, after being stored in aqueous phase at 4°C for up to three months, maintains a transfection efficiency of ≤15% when co-cultured with cells. The cells are one of HEK293T, HeLa, DC2.4, CHO-K1, and MSCs.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The liposome-encapsulated polymer / nucleic acid complex prepared in this invention has a lipid layer and a polymer / nucleic acid complex particle core, which can enhance the ability of mRNA to resist hydrolysis and maintain transfection activity for up to three months under aqueous conditions at 4°C.

[0019] (2) The liposome / polymer / nucleic acid complex prepared by the present invention has good mRNA utilization and maintains efficient mRNA release during storage. It can enhance the stability of the complex while achieving high transfection efficiency for a variety of cells.

[0020] (3) The preparation method of the present invention is simple and highly reproducible, and has a good application prospect in the field of nucleic acid vaccines. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the particle size, potential, and transmission electron microscopy of a polymer / nucleic acid complex encapsulated in liposomes.

[0022] Figure 2 The transfection efficiency of the liposome-encapsulated polymer / nucleic acid complex in Example 1 is shown.

[0023] Figure 3 The transfection activity of the liposome-encapsulated polymer / nucleic acid complex under aqueous conditions at 4°C changes over time.

[0024] Figure 4 The transfection efficiency of the liposome-encapsulated polymer / nucleic acid complexes in Examples 2 and 3 is shown.

[0025] Figure 5 This is a schematic diagram of the particle size, potential, and transmission electron microscopy of the liposome / nucleic acid complex in Comparative Example 1.

[0026] Figure 6 As a comparative example, the change in transfection activity of the liposome / nucleic acid complex over time.

[0027] Figure 7 The transfection efficiency of the polymer / nucleic acid complex encapsulated in liposomes (Formulation 2) in Comparative Example 2 is shown.

[0028] Figure 8 Transfection efficiency of polymer / nucleic acid complexes encapsulated in liposomes at different mass ratios.

[0029] Figure 9 This is a comparison of the transfection efficiency of HEK293T cells using liposome-encapsulated polymer / nucleic acid complexes in Examples 1, 4, and 5.

[0030] Figure 10 This is a comparison of the transfection efficiency of MSC cells using liposome-encapsulated polymer / nucleic acid complexes in Example 1 and Comparative Example 6. Detailed Implementation

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. In the following embodiments of the present invention, the cationic lipid used is (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), the phospholipid used is distearylphosphatidylcholine (DSPC), the cholesterol used is cholesterol (Chol), and the PEG lipid used is 1,2-dimyristic-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000). In other embodiments of the present invention, the cationic lipid may also be trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTMA), the phospholipid may be dioleoylphosphatidylethanolamine (DOPE), and the PEG lipid may be methoxypolyethylene glycol bis(tetradecylacetamide) (ALC-0159).

[0032] Example 1: Preparation of liposome-encapsulated polymer / nucleic acid complexes and their characterization during storage at 4°C

[0033] (1) Preparation of polymer / nucleic acid complexes encapsulated in liposomes

[0034] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0035] Preparation of the polymer / nucleic acid complex: First, a hyperbranched polylysine solution with a concentration of 100 μg / mL was prepared using DEPC-coated water as the solvent, with a number-average molecular weight of 2000 g / mol. Then, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water. Equal volumes of the hyperbranched polylysine solution and the mRNA dilution buffer were mixed, vortexed for 30 s, and allowed to stand for 30 min to prepare the hyperbranched polylysine / mRNA complex.

[0036] Preparation of liposome-encapsulated polymer / nucleic acid complex: First, prepare a liposome dilution solution with a concentration of 327 μg / mL using DEPC water as the solvent. Mix the above hyperbranched polylysine / mRNA complex dispersion with the liposome dilution solution in equal volumes and incubate for 40 min.

[0037] Preparation of liposome-encapsulated polymer / nucleic acid complex storage solution: Add a 30% sucrose solution to the above complex dispersion using DEPC water as the solvent, so that the sucrose concentration in the final liposome-encapsulated hyperbranched polylysine / mRNA complex storage solution is 10%.

[0038] (2) Performance characterization of liposome-encapsulated polymer / nucleic acid complexes

[0039] The particle size and zeta potential of the composite nanoparticles were measured using a nanoparticle size potentiometry instrument, and the morphology of liposomes / hyperbranched polylysine / mRNA was imaged using transmission electron microscopy. Figure 1 As shown in (a), the hydrated particle size of the liposome / hyperbranched polylysine / mRNA complex is between 100 nm and 300 nm. Figure 1 In (b), the zeta potential of the liposome / hyperbranched polylysine / mRNA complex is 3-5 mV, exhibiting a near-neutral charge. Figure 1(c) shows that the liposome / hyperbranched polylysine / mRNA complex consists of a liposome shell and a hyperbranched polylysine / mRNA complex core.

[0040] Transfection with liposome-encapsulated polymer / nucleic acid complexes: Cells were seeded in 24-well plates and cultured in a sterile incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of the complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The cell types involved included HEK293T cells, HeLa cells, CHO-K1 cells, DC2.4 cells, and MSCs, with seeding densities of 2 × 10⁻⁶ cells. 5 cells / well, 5×10 4 cells / well, 8×10 4 cells / well, 2×10 5 cells / well, 5×10 4 cells / pores.

[0041] Cell transfection efficiency determination: First, the green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Then, the cell culture plate was removed, the medium containing the transfection complex was discarded, the cells were washed twice with PBS, and 200 μL of trypsin was added to digest the cells for 1-4 min. Then, 400 μL of complete medium was added to terminate the digestion. The cell suspension was then transferred to centrifuge tubes, centrifuged at 1000 r / min for 5 min, the supernatant was removed, and the cells were resuspended in 500 μL of PBS. The cell transfection rate was determined by flow cytometry. Results are as follows: Figure 2 As shown, the liposome / hyperbranched polylysine / mRNA complex achieved intracellular mRNA delivery and protein expression in HEK293T cells, HeLa cells, CHO-K1 cells, DC2.4 cells, and MSCs, with transfection efficiency exceeding 90% in HEK293T and HeLa cells. This is comparable to the transfection effect of the commercially available transfection reagent Lipo2000.

[0042] Determination of transfection activity over time: A batch of liposome / hyperbranched polylysine / mRNA complex stock solution containing sucrose stabilizer was prepared and stored at 4°C. At 1 week, 2 weeks, 1 month, 2 months, and 3 months after preparation, 0.25 μg of the complex stock solution containing mRNA was taken and diluted to 100 μL with serum-free medium. The mRNA was then analyzed using 2.5 × 10⁻⁶ mRNA-containing media. 4HEK293T cells were seeded in 96-well plates and cultured at 37°C in a sterile incubator with 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 100 μL of the aforementioned complex dilution buffer was added. The cells were then co-cultured for another 24 hours. After 24 hours, the cell culture plate was removed from the incubator, the medium containing the transfection complex was removed, the cells were washed twice with PBS, and 80 μL of trypsin was added to digest the cells for 1 min. Then, 160 μL of complete culture medium was added to stop the digestion. The cell suspension was then transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL of PBS. The transfection rate was determined by flow cytometry. Results are as follows: Figure 3 As shown, the liposome / hyperbranched polylysine / mRNA complex storage solution, stored at 4°C for up to two months, still maintained a high transfection efficiency of 88.40% for HEK293T cells. Compared with the transfection efficiency of freshly prepared liposome / hyperbranched polylysine / mRNA complex (90.53%), the transfection efficiency decreased by only 2.35%. After three months of storage at 4°C, the transfection efficiency for HEK293T cells was 78.73%, a decrease of 13% compared with the transfection efficiency of freshly prepared transfection complex, demonstrating good storage stability in aqueous phase at 4°C.

[0043] Example 2: Preparation and performance testing of liposome-encapsulated polymer / nucleic acid complexes

[0044] The specific implementation steps are the same as in Example 1, except that the number-average molecular weight of the hyperbranched polylysine used is 2500 g / mol.

[0045] Determination of cell transfection efficiency: using 2×10⁻⁶ cells / day. 5 HEK293T cells were seeded in 24-well plates and cultured at a density of cells / well. The cells were then incubated in a sterile incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of each complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Figure 4 As shown, the liposome / hyperbranched polylysine / mRNA complex achieved effective delivery to a variety of cells.

[0046] Example 3: Preparation and performance testing of liposome-encapsulated polymer / nucleic acid complexes

[0047] The specific implementation steps are the same as in Example 1, except that the number-average molecular weight of the hyperbranched polylysine used is 3000 g / mol.

[0048] The determination of cell transfection efficiency was the same as in Example 2, such as... Figure 4 As shown, the liposome / hyperbranched polylysine / mRNA complex achieved effective green fluorescent protein expression in a variety of cell types.

[0049] Comparative Example 1: Preparation and Characterization of Liposome / Nucleic Acid Complexes

[0050] (1) Preparation of liposome / nucleic acid complex

[0051] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0052] Preparation of liposome / nucleic acid complex: First, a liposome solution with a concentration of 654 μg / mL was prepared using DEPC-coated water as the solvent. Then, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water as the solvent. The mRNA dilution buffer and liposome dispersion were mixed in equal volumes and incubated for 30 min.

[0053] Preparation of liposome / mRNA complex storage solution: Add a 30% sucrose solution to the above complex dispersion using DEPC water as the solvent, so that the final sucrose concentration in the liposome / mRNA complex storage solution is 10%.

[0054] (2) Performance characterization of liposome / mRNA complex

[0055] The particle size and zeta potential of the composite nanoparticles were measured using a nanoparticle size potentiometry instrument, and the morphology of the liposome / mRNA complex was imaged using transmission electron microscopy. Figure 5 As shown.

[0056] Determination of transfection activity over time: A batch of liposome / mRNA complex stock solution containing sucrose stabilizer was prepared and stored at 4°C. At 1 week, 2 weeks, 1 month, 2 months, and 3 months after preparation, 0.25 μg of the complex stock solution containing mRNA was taken out and diluted to 100 μL with serum-free medium. The transfection activity was then measured at 2.5 × 10⁻⁶ mRNA / mRNA. 4HEK293T cells were seeded in 96-well plates and cultured at 37°C in a sterile incubator with 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 100 μL of the aforementioned complex dilution buffer was added. The cells were then co-cultured for another 24 hours. After 24 hours, the cell culture plate was removed from the incubator, the medium containing the transfection complex was removed, the cells were washed twice with PBS, and 80 μL of trypsin was added to digest the cells for 1 min. Then, 160 μL of complete culture medium was added to stop the digestion. The cell suspension was then transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL of PBS. The transfection rate was determined by flow cytometry. Results are as follows: Figure 6 As shown, the liposome / mRNA complex storage solution was stored at 4°C. After two months of storage, its average transfection efficiency on HEK293T cells was 75.23%, which was about 20.20% lower than that of freshly prepared liposome / mRNA complex (94.27%). After three months of storage at 4°C, its transfection efficiency on HEK293T cells was only about 29.93%, which was 68.25% lower than that of freshly prepared transfection complex. Under the storage conditions of 4°C in aqueous phase, it easily loses its transfection activity and exhibits weak storage stability.

[0057] Comparative Example 2: Transfection efficiency of polymer / nucleic acid complexes encapsulated in liposomes (Formula 2)

[0058] (1) Preparation of polymer / nucleic acid complex encapsulated by liposomes (Formula 2)

[0059] A molar ratio of 40:10:48 for cationic lipids, phospholipids, and cholesterol was thoroughly dissolved in one volume of anhydrous ethanol. Then, the solution was injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0060] Preparation of the polymer / nucleic acid complex: First, a hyperbranched polylysine solution with a concentration of 100 μg / mL was prepared using DEPC-coated water. Then, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water. The hyperbranched polylysine solution and mRNA dilution buffer were mixed in equal volumes, vortexed for 30 s, and allowed to stand for 30 min to prepare the hyperbranched polylysine / mRNA complex dispersion.

[0061] Preparation of liposome-encapsulated polymer / nucleic acid complex: First, prepare a liposome dilution solution with a concentration of 327 μg / mL using DEPC water as the solvent. Mix the above hyperbranched polylysine / mRNA complex dispersion with the liposome dispersion in equal volumes and incubate for 40 min.

[0062] (2) Characterization of the transfection performance of the polymer / nucleic acid complex encapsulated in liposomes (Formula 2)

[0063] Transfection with liposome-encapsulated polymer / nucleic acid complexes: at 2 × 10 5 HEK293T cells were seeded in 24-well plates and cultured at a sterile incubator at 37°C and 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Results are as follows: Figure 7 As shown, it can be seen that, compared with formulation 2 which does not contain PEG lipids, the liposome / polymer / mRNA complex prepared by the formulation of Example 1 has significantly higher green fluorescent protein expression.

[0064] Comparative Example 3: Transfection efficiency of liposome-encapsulated polymer / nucleic acid complexes with different mass ratios

[0065] (1) Preparation of polymer / nucleic acid complexes encapsulated in liposomes with different mass ratios

[0066] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0067] Preparation of polymer / nucleic acid complexes: First, hyperbranched polylysine solutions with concentrations of 100 μg / mL, 200 μg / mL, and 500 μg / mL were prepared using DEPC-coated water as the solvent. The hyperbranched polylysine solutions were then prepared by vortexing. Next, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water as the solvent. Equal volumes of the hyperbranched polylysine solution and the mRNA dilution buffer were mixed, vortexed for 30 s, and allowed to stand for 30 min to prepare hyperbranched polylysine / mRNA complexes with mass ratios of 1:1, 2:1, and 5:1, respectively.

[0068] Preparation of liposome-encapsulated polymer / nucleic acid complexes: First, liposome dilutions with concentrations of 327 μg / mL and 490.5 μg / mL were prepared using DEPC water as the solvent. The polylysine / mRNA complex dispersion was mixed with an equal volume of liposome dispersion and incubated for 40 min to prepare liposome / hyperbranched polylysine / mRNA complexes with mass ratios of 6.54:1:1, 6.54:2:1, 6.54:5:1, 9.81:1:1, 9.81:2:1, and 9.81:5:1.

[0069] (2) Characterization of transfection performance of liposome-encapsulated polymer / nucleic acid complexes with different mass ratios

[0070] Transfection with liposome-encapsulated polymer / nucleic acid complexes: at 2 × 10 5 HEK293T cells were seeded in 24-well plates and cultured at a sterile incubator at 37°C and 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of each complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Results are as follows: Figure 8 As shown, the liposome / polymer / mRNA complex with a mass ratio of 6.54:1:1 exhibited significantly high transfection efficiency. When the mass ratio of hyperbranched polylysine / mRNA and the content of cationic liposomes increased, the transfection efficiency of the liposome / polymer / mRNA complex decreased significantly. This may be due to the mutual repulsion between the surface charge of the hyperbranched polylysine / mRNA complex and the cationic liposomes, resulting in incomplete construction of the liposome / polymer / mRNA complex.

[0071] Comparative Example 4: Transfection efficiency of polymer / nucleic acid complexes encapsulated in liposomes containing high molecular weight hyperbranched polylysine.

[0072] (1) Preparation of polymer / nucleic acid complexes encapsulated in liposomes containing high molecular weight hyperbranched polylysine

[0073] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0074] Preparation of the polymer / nucleic acid complex: A hyperbranched polylysine solution with a number-average molecular weight of 6500 g / mol was prepared to a concentration of 100 μg / mL using DEPC-coated water as the solvent. The hyperbranched polylysine solution was then prepared by vortexing. Subsequently, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water. Equal volumes of the hyperbranched polylysine solution and the mRNA dilution buffer were mixed, vortexed for 30 s, and allowed to stand for 30 min to prepare the hyperbranched polylysine / mRNA complex.

[0075] Preparation of liposome-encapsulated polymer / nucleic acid complex: First, prepare a liposome dilution solution with a concentration of 327 μg / mL using DEPC water as the solvent. Mix the above hyperbranched polylysine / mRNA complex dispersion with the liposome dispersion in equal volumes and incubate for 40 min.

[0076] (2) Characterization of transfection performance of polymer / nucleic acid complexes encapsulated in liposomes containing high molecular weight hyperbranched polylysine

[0077] Transfection with liposome-encapsulated polymer / nucleic acid complexes: at 2 × 10 5 HEK293T cells were seeded in 24-well plates and cultured at a sterile incubator at 37°C and 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of each complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Results are as follows: Figure 9As shown, compared to the examples, the liposome / polymer / mRNA complex prepared using high molecular weight hyperbranched polylysine has a significantly lower transfection efficiency. The high molecular weight hyperbranched polylysine has a relatively stronger binding force on mRNA, which results in the mRNA not being able to fully dissociate from the complex in the cytoplasm.

[0078] Comparative Example 5: Transfection efficiency of polymer / nucleic acid complexes encapsulated in liposomes containing linear polylysine.

[0079] (1) Preparation of polymer / nucleic acid complexes encapsulated in liposomes containing linear polylysine

[0080] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0081] Preparation of the polymer / nucleic acid complex: A linear polylysine solution with a number-average molecular weight of 3200 g / mol was prepared to a concentration of 100 μg / mL using DEPC-coated water as the solvent. The linear polylysine solution was then prepared by vortexing. Next, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water. Equal volumes of the linear polylysine solution and the mRNA dilution buffer were mixed, vortexed for 30 s, and allowed to stand for 30 min to prepare the linear polylysine / mRNA complex.

[0082] Preparation of liposome-encapsulated polymer / nucleic acid complex: First, prepare a liposome dilution solution with a concentration of 327 μg / mL using DEPC water as the solvent. Mix the above linear polylysine / mRNA complex dispersion with the liposome dispersion in equal volumes and incubate for 40 min.

[0083] (2) Characterization of transfection performance of polymer / nucleic acid complexes encapsulated in liposomes containing linear polylysine

[0084] Transfection with liposome-encapsulated polymer / nucleic acid complexes: at 2 × 10 5HEK293T cells were seeded in 24-well plates and cultured at a sterile incubator at 37°C and 5% CO2 for 24 hours. Afterward, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of each complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Results are as follows: Figure 9 As shown, compared to the examples, the liposome / polymer / mRNA complex prepared using linear polylysine has a significantly lower transfection efficiency. It can be seen that, compared to hyperbranched polylysine, linear polylysine causes the mRNA to not be fully dissociated from the complex in the cytoplasm.

[0085] Comparative Example 6: Transfection efficiency of polymer / liposome / nucleic acid complex delivery system

[0086] (1) Preparation of polymer / liposome / nucleic acid complex delivery system

[0087] A molar ratio of 40:10:48:2 for cationic lipids, phospholipids, cholesterol, and PEG lipids was thoroughly dissolved in one volume of anhydrous ethanol. The solution was then injected into four volumes of DEPC water at 55°C using a syringe at a constant rate of 10 μL / s. The solution was stirred at 700 rpm for 60 min to obtain a lipid droplet dispersion. The dispersion was then subjected to rotary evaporation under reduced pressure at 37°C and 0.06 MPa to remove the ethanol. Finally, the solution was sonicated for 3 min using an ultrasonic probe at 200 W with a 5-second start-up and 5-second pause to obtain a cationic liposome dispersion.

[0088] Preparation of liposome / nucleic acid complex: First, a liposome solution with a concentration of 654 μg / mL was prepared using DEPC-coated water as the solvent. Then, an eGFP mRNA dilution buffer with a concentration of 100 μg / mL was prepared using DEPC-coated water as the solvent. The mRNA dilution buffer and the liposome solution were mixed in equal volumes and incubated for 30 min.

[0089] Preparation of the polymer / liposome / nucleic acid complex: First, a hyperbranched polylysine solution with a concentration of 50 μg / mL was prepared using DEPC-treated water by vortexing. The hyperbranched polylysine solution was then mixed with an equal volume of the liposome / mRNA complex dispersion and incubated for 30 min.

[0090] (2) Characterization of the transfection performance of polymer / liposome / nucleic acid complex

[0091] Transfection of polymer / liposome / nucleic acid complexes: at 5 × 10 4Cells / well density: MSCs were seeded in 24-well plates and cultured in a sterile incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the complete culture medium was removed, the cells were washed twice with PBS, and 440 μL of serum-free culture medium and 60 μL of each complex solution were added. The mixture was gently shaken and co-cultured with the cells for another 24 hours. The green fluorescence of eGFP-positive cells was observed using a fluorescence microscope. Results are as follows: Figure 10 As shown, the order of addition of lipids and polymers is crucial to the delivery efficiency of the composite vector. Compared with the example, the composite delivery vector prepared by adding polymers later has a significantly lower transfection efficiency in MSCs cells.

[0092] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A liposome / polymer / nucleic acid complex delivery system that combines aqueous storage stability and high transfectivity, characterized in that, The delivery system is a polymer / nucleic acid complex encapsulated in liposomes, and its preparation method specifically includes the following steps: Step 1: Preparation of cationic liposomes; Step 2: Mix the polymer diluent and nucleic acid diluent evenly and incubate for at least 30 min to prepare the polymer / nucleic acid complex; Step 3: Mix the cationic liposomes prepared in Step 1 with the polymer / nucleic acid complex prepared in Step 2 uniformly, and incubate for at least 40 min to obtain the polymer / nucleic acid complex encapsulated by liposomes.

2. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 1, characterized in that, The preparation method of cationic liposomes in step 1 is as follows: cationic lipids, phospholipids, cholesterol and PEG lipids in a molar ratio of (36-40):(10-11):(48-50):(1-3) are fully dissolved in anhydrous ethanol, and then injected into 3-6 times the volume of DEPC water at 50-58℃. The mixture is stirred to obtain a lipid droplet dispersion. The dispersion is then evaporated under reduced pressure to remove ethanol. Finally, the dispersion is treated with an ultrasonic cell disruptor to obtain a cationic liposome dispersion.

3. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 1, characterized in that, The polymer mentioned in step 2 is hyperbranched polylysine with a molecular weight of 2000-3000 g / mol, and the solvent of the diluent is DEPC water.

4. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 3, characterized in that, In the liposome / polymer / nucleic acid composite delivery system, the mass ratio of liposomes, hyperbranched polylysine, and nucleic acid is (5.8-7.5):(0.3-1.5):

1.

5. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 1, characterized in that, The liposome-encapsulated polymer / nucleic acid complex has a particle size of 100-300 nm and a zeta potential of 3-5 mV.

6. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 1, characterized in that, The aqueous storage stability is such that the liposome / polymer / nucleic acid complex delivery system can be stably stored for up to three months in a 10-15% sucrose solution at 4°C.

7. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 2, characterized in that, The cationic lipid is one or more of dimethyl-2,3-diolenooxypropyl-2-(2-speramidoamino)ethylammonium trifluoroacetate, trimethyl-2,3-diolenooxypropylammonium chloride, 1,2-dioleoyl-3-trimethylpropylammonium chloride, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester; the phospholipid is one or more of dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine; and the PEG lipid is one or more of methoxy polyethylene glycol bis(tetradecyl acetamide) and dimyristoyl glycerol-polyethylene glycol 2000.

8. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 1, characterized in that, The nucleic acid mentioned is at least one of DNA and mRNA.

9. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 6, characterized in that, The delivery system, after being stored in aqueous phase at 4°C for up to three months, can maintain a change of ≤15% in transfection efficiency when co-cultured with cells.

10. The liposome / polymer / nucleic acid complex delivery system with both aqueous storage stability and high transfectivity according to claim 9, characterized in that, The liposome / polymer / nucleic acid complex delivery system was used to transfect HEK293T, HeLa, DC2.4, CHO-K1, and MSCs cells.

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