Application of transfection complex containing aescin and / or its salt compound in promoting transfection

Abstract

The present invention provides a transfection complex comprising aescin and / or aescin salt compound, wherein the complex is used to deliver nucleic acids to improve transfection efficiency, and the delivered nucleic acids include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). The present invention further uses a transfection complex in combination with aescin and / or aescin salt compounds and other non-viral vectors for transfection. The present invention first discovered that aescin and / or aescin salt compounds can effectively improve the transfection ability of a vector in transfection, and can be used in nucleic acid drugs.

Description

Technical Field

[0001] The present invention relates to the field of gene delivery and / or nucleic acid drugs, and in particular to a transfection complex containing aescin and / or aescin salt compound, wherein the transfection complex comprises a non-viral vector and / or a nucleic acid molecule, wherein the non-viral vector comprises one or more of liposomes, lipid nanoparticles, nanoemulsions, natural polymers, and synthetic polymers, and also relates to a preparation method of the transfection complex and an application of the transfection complex as a nucleic acid drug carrier / drug, and can be used for nucleic acid drugs to treat or prevent human diseases such as cancer, viral infection, and genetic diseases. Background Art

[0002] With the development of the medical industry, gene therapy has gradually emerged. Gene therapy refers to the introduction of normal exogenous genes into target cells to remedy diseases caused by gene defects or abnormalities, thereby achieving the purpose of treatment. Nucleic acids are mainly divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) according to their chemical composition. Nucleic acid drugs provide a new treatment method for the treatment or prevention of human diseases such as cancer, viral infections and genetic diseases. At present, nucleic acid drugs mainly include plasmid DNA, ASON, mRNA, siRNA, miRNA, RNA aptamer, saRNA, SAM, sgRNA, snRNA and other gene therapy drugs.

[0003] In recent years, nucleic acid delivery and nucleic acid drugs have made great progress. However, the development of nucleic acid drugs also faces huge challenges. As negatively charged bioactive macromolecules, nucleic acid drugs have many obstacles in their in vivo delivery. After entering the blood, nucleic acid drugs are easily cleared by nucleases in the blood, resulting in their shortcomings such as short half-life and low bioavailability. Therefore, an efficient and low-toxic delivery carrier is crucial for the application of nucleic acid drugs.

[0004] So far, a number of nucleic acid drugs have been approved for marketing, such as three RNA drugs based on non-viral carrier lipid nanoparticles, including the RNAi drug Patisiran (trade name: ), mRNA vaccines Tozinameran (trade name: Comirnaty) and Elasomeran (trade name: Spikevax) for the prevention of COVID-19; and ZyCoV-D vaccine, which is a DNA drug that is administered by pressing a needle-free syringe on the skin.

[0005] For nucleic acid delivery, commonly used carriers are usually lipid nanoparticles. However, there are huge patent barriers for lipid nanoparticles, which limits the development of nucleic acid drugs.

[0006] Therefore, it is necessary to develop delivery vectors with higher transfection efficiency to construct nucleic acid drug platforms. Summary of the invention

[0007] In order to solve the problems of low transfection efficiency and many patent barriers of existing gene delivery vectors, the present invention has found through creative research that aescin and / or its salt compounds can significantly improve the transfection efficiency of non-viral vectors. One of the purposes of the present invention is to provide the application of aescin and / or aescin salt compounds in gene delivery.

[0008] In order to achieve the above objectives, the inventors of the present invention have conducted a large number of studies and evaluated the effects of different non-viral vectors combined with aescin and / or aescin salt compounds on different types of nucleic acid transfection. The results showed that aescin and / or aescin salt compounds can improve the transfection effects of different non-viral vectors, can be applied to the in vivo and in vitro delivery of nucleic acids, and can be further developed into nucleic acid drugs.

[0009] One of the purposes of the present invention is to provide a transfection complex comprising aescin and / or aescin salt compound, and also comprising a non-viral vector and / or a nucleic acid molecule. The nucleic acid molecule is a nucleic acid drug, and more preferably DNA and / or RNA. Further, the nucleic acid drug includes plasmid DNA (pDNA), antisense oligonucleotides (ASON), messenger RNA (mRNA) encoding therapeutic proteins or vaccine antigen genes, mRNA containing nucleotide modifications, small interfering RNA (siRNA), microRNA (miRNA), RNA aptamers (RNA aptamers) regulating protein activity, small activating RNA (saRNA), circular RNA, self-amplifying mRNA (SAM), small guide RNA (sgRNA), U1 small nuclear RNA (snRNA) and one or more other gene therapy drugs. The RNA molecule includes one or more of mRNA, siRNA, miRNA, saRNA, circular RNA, SAM, sgRNA, U1 snRNA and other gene therapy drugs containing or not containing nucleotide modifications.

[0010] The non-viral vector includes one or more of liposomes, lipid nanoparticles, nanoemulsions, natural polymers, and synthetic polymers. The natural polymers include one or more of protamine, polylysine, polyarginine, chitosan, and other natural polymers. The synthetic polymers include one or more of polyethyleneimine (PEI) and its derivatives, dendritic polyamidoamine (PAMAM) and its derivatives, poly-β-amino esters (PBAE) and its derivatives, and other synthetic polymers.

[0011] The structure of the aescin is shown in formula (I):

[0012]

[0013] The structure of the aescin salt compound is shown in formula (II), wherein R in formula (II) is one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, and various metal salts, including one of sodium, potassium, zinc, aluminum, iron, zirconium, calcium, manganese, magnesium, copper, lead, nickel and cadmium.

[0014]

[0015] The transfection complex is characterized in that the molar ratio of aescin and / or its salt compound to nucleic acid is 0.01-100.

[0016] Preferably, the molar ratio of aescin and / or its salt compound to nucleic acid is 0.05-50, more preferably 0.1-25, more preferably 0.15-15, further preferably 0.2-8, and most preferably 0.4-6.

[0017] One of the purposes of the present invention is to provide a transfection complex and use it as a nucleic acid drug for use in the preparation of nucleic acid drugs, wherein the aescin and / or aescin salt compound can improve the transfection efficiency of non-viral vectors.

[0018] One of the purposes of the present invention is to provide the use of aescin and / or aescin salt compound in the preparation of a transfection-promoting substance. Preferably, the transfection substance is a non-viral vector, which can be used as a transfection preparation. The transfection substance can promote the transfection effect of nucleic acids in various cells.

[0019] Beneficial technical effects:

[0020] One of the objects of the present invention is to provide a nucleic acid delivery system comprising aescin and / or its salt compound, wherein the transfection efficiency is further improved due to the aescin and / or its salt compound.

[0021] The transfection complex described in the present invention can improve the transfection efficiency of nucleic acids on various cells, such as easily transfected cells represented by 293T cells, general cells represented by Hela cells, and immune cells that are difficult to transfect represented by DC2.4. The present invention surprisingly found that aescin and / or aescin salt compounds are universal in improving the transfection efficiency and are effective for various cells, especially for cells that are difficult to transfect, such as immune cells.

[0022] The present invention creatively finds that aescin and / or aescin salt compounds can also improve the transfection efficiency of the transfection complex on cells that are difficult to transfect, such as immune cells.

[0023] The present invention uses the transfection complex for in vivo transfection, and the results show that aescin and / or aescin salt compound can still improve the transfection efficiency under complex in vivo environment. It can be seen that the aescin and / or aescin salt compound of the present invention can improve the transfection efficiency of nucleic acids both in vivo and in vitro, which is conducive to the wide application of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0025] Figure 1 The luciferase-DNA was delivered by liposomes containing aescin, and the transfection effect of blood was resistant to Hela cells for 24h.

[0026] Figure 2 The luciferase-DNA was delivered by liposomes containing sodium aescinate, and the transfection effect of blood was resistant to Hela cells for 24h.

[0027] Figure 3 The luciferase-mRNA was delivered by liposomes containing sodium aescinate, and the transfection effect of blood was resistant to transfection of DC2.4 cells for 24h.

[0028] Figure 4 To test the transfection effect of GFP-siRNA delivered by liposomes containing aescin, we investigated the transfection effect of GFP-resistant 293T cells for 48 h.

[0029] Figure 5 The delivery of luciferase-mRNA by lipid nanoparticles containing sodium aescinate showed a transfection effect on anti-transfected DC2.4 cells for 24h.

[0030] Figure 6 The luciferase-DNA delivery by aescin-containing nanoemulsion showed a transfection effect on anti-transfected DC 2.4 cells for 24 h.

[0031] Figure 7 The luciferase-DNA was delivered by nanoemulsion containing sodium aescinate, and the transfection effect of anti-transfected DC 2.4 cells was observed for 24 h.

[0032] Figure 8 The delivery of GFP-siRNA by nanoemulsion containing sodium aescinate showed a transfection effect on GFP-resistant 293T cells for 48 h.

[0033] Fig. 9 The luciferase-DNA was delivered by protamine containing sodium aescinate, and the transfection effect of blood was resistant to transfection of DC2.4 cells for 24h.

[0034] Fig.10 To deliver luciferase-DNA using polyethyleneimine 25k (PEI 25k) containing sodium aescinate, there was a 24h transfection effect on DC 2.4 cells.

[0035] Fig.11 The luciferase-DNA was delivered by nanoemulsion containing sodium aescinate and injected intramuscularly into mice. After 72 hours, firefly luciferase substrate was injected intraperitoneally into the mice for in vivo imaging. DETAILED DESCRIPTION

[0036] The following examples are further explanations of the present invention, but are by no means limitations of the scope of the present invention. The present invention will be further described in detail below with reference to the examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art may carry out equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0037] Example 1

[0038] Liposomes (Lip) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and probe ultrasound was performed at 180 w for 5 min under ice bath conditions.

[0039] Liposomes containing aescin (Lip-Esc) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, 1 mg DSPE-PEG and 0.5 mg aescin were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and the probe was sonicated at 180 w for 5 min under ice bath conditions.

[0040] Take an appropriate amount of Lip or Lip-Esc, drop luciferase-DNA solution into it under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain a complex of liposomes and DNA. In Lip-Esc-DNA, the molar ratio of aescin to DNA is 0.4.

[0041] Hela cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 1 . During DNA transfection, it needs to enter the cell membrane first and then the nuclear pore to achieve the subsequent transcription and translation process. Therefore, the process of DNA transfection is complicated and difficult. Compared with Lip-DNA without aescin, the transfection efficiency of Lip-Esc-DNA is significantly improved (**, p<0.01). It can be seen that aescin can significantly improve the transfection efficiency of non-viral vectors. In the figure, Lip-DNA represents liposomes carrying luciferase-DNA, and Lip-Esc-DNA represents liposomes co-loaded with luciferase-DNA and aescin.

[0042] Example 2

[0043] Liposomes (Lip) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and probe ultrasound was performed at 180 w for 5 min under ice bath conditions.

[0044] The preparation method of liposomes containing sodium aescinate (Lip-Aes) is as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG are dissolved in 3 ml methanol, the organic solvent is removed by rotary evaporation, 3 ml of 0.25 mg / ml sodium aescinate aqueous solution is added to hydrate the film, and the probe is sonicated at 180 w for 5 min under ice bath conditions.

[0045] Take an appropriate amount of Lip or Lip-Aes, drop luciferase-DNA solution under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain a complex of liposomes and DNA. In Lip-Aes-DNA, the molar ratio of sodium aescinate to DNA is 0.2.

[0046] Hela cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 2 Compared with Lip-DNA without sodium aescinate, the transfection efficiency of Lip-Aes-DNA was significantly improved (**, p<0.01), indicating that aescinate compounds can significantly improve the transfection efficiency of non-viral vectors. In the figure, Lip-DNA represents liposomes loaded with luciferase-DNA, and Lip-Aes-DNA represents liposomes co-loaded with luciferase-DNA and sodium aescinate.

[0047] Example 3

[0048] Liposomes (Lip) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and probe ultrasound was performed at 180 w for 5 min under ice bath conditions.

[0049] The preparation method of liposomes containing sodium aescinate (Lip-Aes) is as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG are dissolved in 3 ml methanol, the organic solvent is removed by rotary evaporation, 3 ml of 0.25 mg / ml sodium aescinate aqueous solution is added to hydrate the film, and the probe is sonicated at 180 w for 5 min under ice bath conditions.

[0050] Take an appropriate amount of Lip or Lip-Aes, drop the luciferase-mRNA solution into it under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain a complex of liposomes and mRNA. In Lip-Aes-mRNA, the molar ratio of sodium aescinate to mRNA is 0.2.

[0051] DC2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of mRNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 3. mRNA is a single-stranded biological macromolecule and is easily degraded by nucleases in the environment. DC2.4 cells are difficult to transfect, and conventional liposomes have poor transfection effects on these cells (Lip-mRNA group). Compared with Lip-mRNA without sodium aescinate, the transfection efficiency of Lip-Aes-mRNA is significantly improved (****, p<0.0001). It can be seen that aescin salt compounds can significantly improve the transfection effect of ordinary non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, Lip-mRNA represents liposomes carrying luciferase-mRNA, and Lip-Aes-mRNA represents liposomes co-loaded with luciferase-mRNA and sodium aescinate.

[0052] Example 4

[0053] Liposomes (Lip) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and probe ultrasound was performed at 180 w for 5 min under ice bath conditions.

[0054] Liposomes containing aescin (Lip-Esc) were prepared as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, 1 mg DSPE-PEG and 0.5 mg aescin were dissolved in 3 ml methanol, the organic solvent was removed by rotary evaporation, 3 ml UP water was added to hydrate the film, and the probe was sonicated at 180 w for 5 min under ice bath conditions.

[0055] Take an appropriate amount of Lip, drop GFP-siRNA solution under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain Lip-siRNA. In Lip-Esc-siRNA, the molar ratio of aescin to siRNA is 1.6. Take an appropriate amount of Lip-Esc, drop GFP-siRNA solution and sodium aescin solution under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain Lip-Esc-siRNA.

[0056] GFP-293T cells were placed in a 24-well plate. When the cell fusion rate was 50%, 1.5 μg of siRNA was given to each well. After 48 hours of transfection, the cells were washed and resuspended with PBS and detected by flow cytometry. Figure 4. siRNA is a double-stranded small RNA, which is extremely unstable in the environment and body fluids and easily degraded by nucleases. Compared with Lip-siRNA without aescin, the knockdown efficiency of Lip-Esc-siRNA is significantly improved (*, p<0.05), which shows that aescin can significantly improve the transfection efficiency. In the figure, Lip-siRNA represents liposomes loaded with GFP-siRNA, and Lip-Esc-siRNA represents nanoemulsions co-loaded with GFP-siRNA and aescin.

[0057] Example 5

[0058] The preparation method of lipid nanoparticles (LNP) is as follows: ALC-0315, DSPC, cholesterol and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 as an organic phase, and luciferase-mRNA is dissolved in DEPC water as an aqueous phase. A microfluidic device is used to control the total flow rate to 9 ml / min, the flow rate ratio of the aqueous phase to the organic phase to 3:1, and the nitrogen-phosphorus ratio to 6, and LNP-mRNA is obtained by rapid mixing.

[0059] The preparation method of lipid nanoparticles (LNP-Aes) containing sodium aescinate is as follows: ALC-0315, DSPC, cholesterol and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 as an organic phase, luciferase-mRNA is dissolved in a sodium aescinate aqueous solution as an aqueous phase, and the concentration ratio of mRNA to sodium aescinate is 0.25. Using a microfluidic device, the total flow rate is controlled to be 9 ml / min, the flow rate ratio of the aqueous phase to the organic phase is 3:1, the nitrogen-phosphorus ratio is 6, and LNP-Aes-mRNA is obtained by rapid mixing, and the molar ratio of sodium aescinate to mRNA is 1.2.

[0060] DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of mRNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 5 The results showed that DC2.4 cells are difficult to transfect, and conventional lipid nanoparticles have poor transfection effects on these cells (LNP-mRNA group). Compared with LNP-mRNA without sodium aescinate, the transfection efficiency of LNP-Aes-mRNA was significantly improved (**, p<0.01). It can be seen that the addition of aescinate compounds can significantly improve the transfection effect of common non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, LNP-mRNA represents lipid nanoparticles carrying luciferase-mRNA, and LNP-Aes-mRNA represents lipid nanoparticles co-loaded with luciferase-mRNA and sodium aescinate.

[0061] Example 6

[0062] The preparation method of nanoemulsion (NE) is as follows: 86 mg squalene and 16 mg DOTAP are dissolved in an appropriate amount of chloroform as the oil phase, the emulsifier is dissolved in 3 ml UP water as the water phase, the oil phase is dropped into the water phase, and the probe is ultrasonicated at 150w for 5min under ice bath conditions.

[0063] The preparation method of nanoemulsion containing aescin (NE-Esc) is as follows: 86 mg squalene and 16 mg DOTAP are dissolved in an appropriate amount of chloroform as the oil phase, the emulsifier is dissolved in 3 ml UP water as the water phase, the oil phase is dropped into the water phase, and the probe is ultrasonicated at 150w for 5 minutes under ice bath conditions. Aescin is dissolved in methanol, an appropriate amount of aescin methanol solution is taken and rotary evaporated to form a film, and an appropriate amount of nanoemulsion is used to hydrate the film so that the concentration of aescin is 0.7 mg / ml.

[0064] Take an appropriate amount of NE or NE-Esc, drop luciferase-DNA solution into it under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain a complex of nanoemulsion and DNA. In NE-Esc-DNA, the molar ratio of aescin to DNA is 0.68.

[0065] DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 6 DC2.4 cells are difficult to transfect, and conventional nanoemulsions have poor transfection effects on these cells (NE-DNA group). Compared with NE-DNA without aescin, the transfection efficiency of NE-Esc-DNA is significantly improved (*, p<0.05). It can be seen that the addition of aescin can significantly improve the transfection effect of common non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, NE-DNA represents a nanoemulsion loaded with luciferase-DNA, and NE-Esc-DNA represents a nanoemulsion co-loaded with luciferase-DNA and aescin.

[0066] Example 7

[0067] The preparation method of nanoemulsion (NE) is as follows: 86 mg squalene and 16 mg DOTAP are dissolved in an appropriate amount of chloroform as the oil phase, the emulsifier is dissolved in 3 ml UP water as the water phase, the oil phase is dropped into the water phase, and the probe is ultrasonicated at 150w for 5min under ice bath conditions.

[0068] Take an appropriate amount of NE, add the luciferase-DNA solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-DNA.

[0069] Take an appropriate amount of NE, add luciferase-DNA solution and 0.5 mg / ml sodium aescinate solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-Aes-DNA. At this time, the molar ratio of sodium aescinate to DNA is 1.2.

[0070] DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Figure 7 DC2.4 cells are difficult to transfect, and conventional nanoemulsions have poor transfection effects on these cells (NE-DNA group). Compared with NE-DNA without sodium aescinate, the transfection efficiency of NE-Aes-DNA is significantly improved (****, p<0.0001), indicating that aescinate compounds can significantly improve transfection efficiency. In the figure, NE-DNA represents nanoemulsions loaded with luciferase-DNA, and NE-Aes-DNA represents nanoemulsions co-loaded with luciferase-DNA and sodium aescinate.

[0071] Example 8

[0072] The preparation method of nanoemulsion (NE) is as follows: 86 mg squalene and 16 mg DOTAP are dissolved in an appropriate amount of chloroform as the oil phase, the emulsifier is dissolved in 3 ml UP water as the water phase, the oil phase is dropped into the water phase, and the probe is ultrasonicated at 150w for 5min under ice bath conditions.

[0073] Take an appropriate amount of NE, add GFP-siRNA solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-siRNA.

[0074] Take an appropriate amount of NE, add GFP-siRNA solution and 0.5 mg / ml sodium aescinate solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-Aes-siRNA. At this time, the molar ratio of sodium aescinate to siRNA is 2.4.

[0075] GFP-293T cells were placed in a 24-well plate. When the cell fusion rate was 50%, 1.5 μg of siRNA was given to each well. After 48 hours of transfection, the cells were washed and resuspended with PBS and detected by flow cytometry. Figure 8. siRNA is a double-stranded small RNA, which is extremely unstable in the environment and body fluids and easily degraded by nucleases. Compared with NE-siRNA without sodium aescinate, the knockout efficiency of NE-Aes-siRNA is significantly improved (**, p<0.01), which shows that aescinate compounds can significantly improve transfection efficiency. In the figure, NE-siRNA represents nanoemulsion loaded with GFP-siRNA, and NE-Aes-siRNA represents nanoemulsion co-loaded with GFP-siRNA and sodium aescinate.

[0076] Example 9

[0077] Preparation method of natural high molecular polymer-DNA nanoparticles: dissolve protamine in 5% glucose solution to prepare a 0.1 mg / ml solution, prepare luciferase-DNA into a 0.1 mg / ml solution, add an equal volume of luciferase-DNA solution to the protamine solution under vortex conditions, incubate for 30 minutes, and obtain DNA-loaded nanoparticles, PRTM-DNA.

[0078] Dissolve protamine in 5% glucose solution to prepare a 0.1 mg / ml solution, prepare luciferase-DNA into a 0.1 mg / ml solution, add equal volumes of luciferase-DNA solution and 0.5 mg / ml sodium aescinate solution to the protamine solution under vortex conditions, incubate for 30 minutes, and obtain nanoparticles co-loaded with sodium aescinate and DNA, PRTM-Aes-DNA, at which the molar ratio of sodium aescinate to DNA is 2.4.

[0079] DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Fig. 9 . Conventional natural polymers have poor transfection effects on the cells (PRTM-DNA group). Compared with PRTM-DNA without sodium aescinate, the transfection efficiency of PRTM-Aes-DNA is significantly improved (**, p<0.01). It can be seen that the addition of aescinate compounds can significantly improve the transfection effect of common non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, PRTM-DNA represents nanoparticles loaded with luciferase-DNA, and PRTM-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.

[0080] Example 10

[0081] Preparation method of synthetic polymer-DNA nanoparticles: dissolve PEI 25k in UP water to make a solution of 0.1 mg / ml, prepare luciferase-DNA into a solution of 0.1 mg / ml, add an equal volume of luciferase-DNA solution to PEI 25k solution under vortex conditions, incubate for 30 minutes, and obtain DNA-loaded nanoparticles, PEI 25k-DNA.

[0082] PEI 25k was dissolved in UP water to prepare a 0.1 mg / ml solution, luciferase-DNA was prepared into a 0.1 mg / ml solution, equal volumes of luciferase-DNA solution and 0.5 mg / ml sodium aescinate solution were added to the PEI25k solution under vortex conditions, and incubated for 30 minutes to obtain nanoparticles co-loaded with sodium aescinate and DNA, PEI25k-Aes-DNA, at which the molar ratio of sodium aescinate to DNA was 5.

[0083] DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was added to each well. After 24 hours of transfection, the transfection efficiency was determined using a firefly luciferase substrate kit. The results are shown in Fig.10 . The results showed that DC2.4 cells are difficult to transfect cells, and conventional synthetic polymers have poor transfection effects on these cells (PEI 25k-DNA group). Compared with PEI 25k-DNA without sodium aescinate, the transfection efficiency of PEI25k-Aes-DNA was significantly improved (****, p<0.0001). It can be seen that the addition of aescinate compounds can significantly improve the transfection effect of ordinary non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, PEI 25k-DNA represents nanoparticles loaded with luciferase-DNA, and PEI 25k-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.

[0084] Embodiment 11

[0085] The preparation method of nanoemulsion (NE) is as follows: 86 mg squalene and 16 mg DOTAP are dissolved in an appropriate amount of chloroform as the oil phase, the emulsifier is dissolved in 3 ml UP water as the water phase, the oil phase is dropped into the water phase, and the probe is ultrasonicated at 150w for 5min under ice bath conditions.

[0086] Take an appropriate amount of NE, add the luciferase-DNA solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-DNA.

[0087] Take an appropriate amount of NE, add luciferase-DNA solution and 0.25 mg / ml sodium aescinate solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain NE-Aes-DNA. At this time, the molar ratio of sodium aescinate to DNA is 0.25.

[0088] NE-DNA or NE-Aes-DNA was injected into the gastrocnemius muscle of mice, and the DNA content injected into each mouse was 25 μg. After 72 hours, the firefly luciferase substrate was injected intraperitoneally for in vivo imaging. The results are shown in Fig.11 . Compared with NE-DNA without sodium aescinate, NE-Aes-DNA has a higher transfection efficiency in vivo. No fluorescence was detected in vivo after NE-DNA administration, which shows that ordinary nanoemulsions make it difficult to express DNA in vivo. After adding sodium aescinate (NE-Aes-DNA group), obvious fluorescence can be detected in vivo, which shows that sodium aescinate can significantly improve the transfection efficiency of non-viral vectors in vivo. In the figure, NE-DNA represents a nanoemulsion loaded with luciferase-DNA, and NE-Aes-DNA represents a nanoemulsion co-loaded with luciferase-DNA and sodium aescinate.

[0089] In summary, the present invention finds that aescin and / or its salt compound can improve the transfection efficiency of nucleic acid delivery system, and has the effect of improving transfection efficiency for easily transfected cells, general cells and difficultly transfected cells, and at the same time, can play the above-mentioned effect in various non-viral nucleic acid delivery systems, therefore, aescin and / or aescin salt compound are universal for the improvement of transfection efficiency, and are effective for various non-viral nucleic acid delivery systems and various cells, especially for cells that are difficult to transfect, such as immune cells. In addition, aescin and / or aescin salt compound can still improve the transfection efficiency of nucleic acid under complex environment in vivo. It can be seen that aescin and / or aescin salt compound of the present invention can improve the transfection efficiency of nucleic acid both in vivo and in vitro, which is conducive to the wide application of nucleic acid drugs.

[0090] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments of equivalent changes without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of aescin and / or aescin salt in the preparation of a substance for promoting non-viral vector transfection, wherein the structure of the aescin is shown in formula (I):

2. The use according to claim 1, characterized in that: The non-viral vector includes one or more of liposomes, lipid nanoparticles, nanoemulsions, natural high molecular polymers, and synthetic high molecular polymers.

3. The use according to claim 2, characterized in that: The natural high molecular polymer includes one or more of protamine, polylysine, polyarginine and chitosan.

4. The use according to claim 2, characterized in that: The synthetic high molecular polymer includes one or more of polyethyleneimine PEI and its derivatives, dendritic polyamidoamine PAMAM and its derivatives, poly-β-amino ester PBAE and its derivatives.

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