WO2024160089A1 - Use of transfection complex containing aescin and/or salt compound thereof in transfection promotion - Google Patents
Use of transfection complex containing aescin and/or salt compound thereof in transfection promotion Download PDFInfo
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- WO2024160089A1 WO2024160089A1 PCT/CN2024/073566 CN2024073566W WO2024160089A1 WO 2024160089 A1 WO2024160089 A1 WO 2024160089A1 CN 2024073566 W CN2024073566 W CN 2024073566W WO 2024160089 A1 WO2024160089 A1 WO 2024160089A1
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- transfection
- aescin
- dna
- nucleic acid
- rna
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present application relates to the field of biomedical technology, and in particular to the application of a transfection complex containing aescin and/or its salt compounds in promoting transfection.
- 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.
- nucleic acid drugs mainly include plasmid DNA, ASON, mRNA, siRNA, miRNA, RNA aptamer, saRNA, SAM, sgRNA, snRNA and other gene therapy drugs.
- nucleic acid delivery and nucleic acid drugs have made great progress.
- the development of nucleic acid drugs also faces huge challenges.
- 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.
- RNA drugs based on non-viral carrier lipid nanoparticles, including Patisiran, an RNAi drug for the treatment of polyneuropathy caused by hereditary thyroxine-mediated amyloidosis, and Tozinameran (trade name: Comirnaty) and Elasomeran (trade name: Spikevax), mRNA vaccines for the prevention of COVID-19; there is also the ZyCoV-D vaccine, which is administered by pressing a needle-free syringe on the skin and is a DNA drug.
- Patisiran an RNAi drug for the treatment of polyneuropathy caused by hereditary thyroxine-mediated amyloidosis
- Tozinameran trade name: Comirnaty
- Elasomeran trade name: Spikevax
- ZyCoV-D vaccine which is administered by pressing a needle-free syringe on the skin and is a DNA drug.
- nucleic acid delivery For nucleic acid delivery, commonly used carriers are usually lipid nanoparticles. The huge patent barriers limit the development of nucleic acid drugs.
- One of the purposes of the present application is to provide a use of aescin compound in gene delivery.
- the present application has conducted a large number of studies to evaluate the effects of the combination of different non-viral vectors and aescin compounds on the transfection of different types of nucleic acids.
- the results show that aescin compounds can improve the transfection effects of different non-viral vectors, can be used for the in vivo and in vitro delivery of nucleic acids, and can be further developed into nucleic acid drugs.
- One of the purposes of the present application is to provide a transfection complex comprising an aescin compound, wherein the aescin compound is an aescin and/or an aescin salt compound.
- the transfection complex further comprises 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.
- the nucleic acid drug includes plasmid DNA (pDNA), antisense oligonucleotide (ASON), messenger RNA (mRNA) encoding therapeutic protein or vaccine antigen gene, mRNA containing nucleotide modification, small interfering RNA (siRNA), microRNA (miRNA), RNA aptamer (RNA aptamer) that regulates 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.
- pDNA plasmid DNA
- ASON antisense oligonucleotide
- mRNA messenger RNA
- mRNA containing nucleotide modification mRNA containing nucleotide modification
- small interfering RNA small interfering RNA
- miRNA microRNA
- RNA aptamer RNA aptamer
- SAM small guide RNA
- the non-viral vector comprises 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- ⁇ -amino esters (PBAE) and its derivatives, and other synthetic polymers.
- PEI polyethyleneimine
- PAMAM dendritic polyamidoamine
- PBAE poly- ⁇ -amino esters
- the aescin salt compound has a structure as shown in formula (II):
- R in formula (II) includes 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 R in formula (II) also includes a metal salt, and the metal salt includes any one of sodium, potassium, zinc, aluminum, iron, zirconium, calcium, manganese, magnesium, copper, lead, nickel and cadmium.
- the molar ratio of aescin compound to nucleic acid in the transfection complex is 0.01-100.
- the molar ratio of aescin compound to nucleic acid is 0.05-50, a further preferred ratio is 0.1-25, a more preferred ratio is 0.15-15, a further preferred ratio is 0.2-8, and the most preferred ratio is 0.4-6.
- One of the purposes of the present application 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 compound can improve the transfection efficiency of non-viral vectors.
- 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.
- One of the purposes of the present application is to provide a nucleic acid delivery system comprising aescin compound, wherein the transfection efficiency is further improved due to the aescin compound.
- the transfection complex provided in the present application 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 application 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.
- 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.
- the present application uses the transfection complex for in vivo transfection, and the results show that aescin and/or aescin salt compounds can still improve the transfection efficiency under complex in vivo environments. It can be seen that aescin and/or aescin salt compounds of the present application 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.
- Figure 1 shows the transfection effect of luciferase-DNA delivered by liposomes containing aescin on Hela cells for 24 hours.
- FIG. 2 shows the transfection effect of luciferase-DNA delivered by liposomes containing sodium aescinate on Hela cells for 24 hours.
- FIG. 3 shows the transfection effect of luciferase-mRNA delivered by liposomes containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
- FIG. 4 shows the transfection effect of GFP-siRNA delivered by liposomes containing aescin on GFP-293T cells with and without transfection resistance for 48 hours.
- FIG. 5 shows the transfection effect of luciferase-mRNA delivered by lipid nanoparticles containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
- Figure 6 shows the transfection effect of luciferase-DNA delivered by nanoemulsion containing aescin on DC 2.4 cells with blood and resistance to transfection for 24 hours.
- FIG. 7 shows the transfection effect of luciferase-DNA delivered by nanoemulsion containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
- FIG. 8 shows the transfection effect of GFP-siRNA delivered by nanoemulsion containing sodium aescinate on GFP-293T cells with or without transfection resistance for 48 hours.
- FIG. 9 shows the transfection effect of luciferase-DNA delivered by protamine containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
- Figure 10 shows the transfection effect of luciferase-DNA delivered by polyethyleneimine 25k (PEI 25k) containing sodium aescinate on transfection-resistant DC 2.4 cells for 24 hours.
- PEI 25k polyethyleneimine 25k
- FIG. 11 shows the results of in vivo imaging of a nanoemulsion containing sodium aescinate delivering luciferase-DNA and intramuscularly injecting it into mice. 72 hours later, firefly luciferase substrate was intraperitoneally injected into the mice.
- Liposomes 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.
- the preparation method of liposomes containing aescin is 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 ultrasonicated at 180w for 5 min under ice bath conditions.
- Lip-Esc-DNA Take an appropriate amount of Lip or Lip-Esc, drop luciferase-DNA solution under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain a complex of liposomes and DNA.
- the molar ratio of aescin to DNA is 0.4.
- the preparation method of liposomes 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
- the preparation method of liposomes containing sodium aescinate is as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, and the organic solvent was removed by rotary evaporation. 3 ml 0.25 mg/ml sodium aescinate aqueous solution was added to hydrate the film, and the probe was ultrasonicated at 180 w for 5 min under ice bath conditions.
- Lip-Aes-DNA 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.
- the molar ratio of sodium aescinate to DNA is 0.2.
- the preparation method of liposomes 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
- the preparation method of liposomes containing sodium aescinate is as follows: 5 mg DOTMA, 10 mg lecithin, 5 mg cholesterol, and 1 mg DSPE-PEG were dissolved in 3 ml methanol, and the organic solvent was removed by rotary evaporation. 3 ml 0.25 mg/ml sodium aescinate aqueous solution was added to hydrate the film, and the probe was ultrasonicated at 180 w for 5 min under ice bath conditions.
- Lip-Aes-mRNA the molar ratio of sodium aescinate to mRNA is 0.2.
- DC2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g of mRNA was given 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. 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 was significantly improved (****, p ⁇ 0.0001).
- aescinate compounds can significantly improve the transfection effect of ordinary non-viral vectors on difficult-to-transfect cells (such as immune cells).
- Lip-mRNA represents liposomes carrying luciferase-mRNA
- Lip-Aes-mRNA represents liposomes co-loaded with luciferase-mRNA and sodium aescinate.
- the preparation method of liposomes 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
- the preparation method of liposomes containing aescin is 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 ultrasonicated at 180w for 5 min under ice bath conditions.
- Lip-Esc-siRNA the molar ratio of aescin to siRNA is 1.6.
- siRNA is a double-stranded small RNA, which is extremely unstable in the environment and body fluids and easily degraded by nucleases.
- the knockout efficiency of Lip-Esc-siRNA was significantly improved (*, p ⁇ 0.05). It can be seen that aescin can significantly improve the transfection efficiency.
- Lip-siRNA represents liposomes loaded with GFP-siRNA
- Lip-Esc-siRNA represents nanoemulsions co-loaded with GFP-siRNA and aescin.
- LNP lipid nanoparticles
- 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 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.
- LNP-Aes lipid nanoparticles containing sodium aescinate
- 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
- concentration ratio of mRNA to sodium aescinate is 0.25.
- 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
- LNP-Aes-mRNA is obtained by rapid mixing
- the molar ratio of sodium aescinate to mRNA is 1.2.
- DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g of mRNA was given 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 show that DC2.4 cells are difficult to transfect cells, 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).
- LNP-mRNA represents lipid nanoparticles carrying luciferase-mRNA
- LNP-Aes-mRNA represents lipid nanoparticles co-loaded with luciferase-mRNA and sodium aescinate.
- the preparation method of nanoemulsion is as follows: 86 mg of squalene and 16 mg of DOTAP are dissolved in an appropriate 100 ml of chloroform was used as the oil phase, and the emulsifier was dissolved in 3 ml of UP water as the water phase. The oil phase was dropped into the water phase, and the probe was ultrasonicated at 150w for 5min under ice bath condition.
- the preparation method of nanoemulsion containing aescin 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.
- NE-Esc-DNA the molar ratio of aescin to DNA is 0.68.
- DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g of DNA was given 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 cells. 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 was significantly improved (*, p ⁇ 0.05). It can be seen that the addition of aescin can significantly improve the transfection effect of ordinary non-viral vectors on difficult-to-transfect cells (such as immune cells).
- NE-DNA represents nanoemulsions carrying luciferase-DNA
- NE-Esc-DNA represents nanoemulsions co-loaded with luciferase-DNA and aescin.
- the preparation method of nanoemulsion is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
- NE-Aes-DNA 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.
- DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g 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 was significantly improved (****, p ⁇ 0.0001), which shows that aescinate compounds can significantly improve the transfection efficiency.
- NE-DNA represents nanoemulsion loaded with luciferase-DNA
- NE-Aes-DNA represents nanoemulsion co-loaded with luciferase-DNA and sodium aescinate.
- the preparation method of nanoemulsion is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
- NE-Aes-siRNA 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.
- 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 was significantly improved (**, p ⁇ 0.01). It can be seen that aescinate compounds can significantly improve transfection efficiency.
- NE-siRNA represents a nanoemulsion loaded with GFP-siRNA
- NE-Aes-siRNA represents a nanoemulsion co-loaded with GFP-siRNA and sodium aescinate.
- 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.
- DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g of DNA was given 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 9. Conventional natural polymers have poor transfection effects on these cells (PRTM-DNA group). Compared with PRTM-DNA without sodium aescinate, the transfection efficiency of PRTM-Aes-DNA 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, PRTM-DNA represents nanoparticles loaded with luciferase-DNA, and PRTM-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.
- Preparation method of synthetic polymer-DNA nanoparticles dissolve PEI 25k in UP water to make 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 PEI 25k solution under vortex conditions, incubate for 30 minutes, and obtain DNA-loaded nanoparticles, PEI 25k-DNA.
- DC 2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 ⁇ g of DNA was given 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 10. The results show 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 PEI25k-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.
- the preparation method of nanoemulsion is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
- NE-Aes-DNA 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.
- NE-DNA or NE-Aes-DNA was injected into the gastrocnemius of mice, and the DNA content injected into each mouse was 25 ⁇ g. After 72 hours, the firefly luciferase substrate was injected intraperitoneally for in vivo imaging, and the results are shown in Figure 11. Compared with NE-DNA without sodium aescinate, NE-Aes-DNA has a higher transfection efficiency in vivo.
- NE-DNA represents a nanoemulsion loaded with luciferase-DNA
- NE-Aes-DNA represents a nanoemulsion co-loaded with luciferase-DNA and sodium aescinate.
- aescin compounds can improve the transfection efficiency of nucleic acid delivery systems, and have the effect of improving transfection efficiency for easily transfected cells, general cells and difficultly transfected cells, and at the same time, the above-mentioned effects can be exerted in various non-viral nucleic acid delivery systems, therefore, aescin compounds 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.
- aescin compounds can still improve the transfection efficiency of nucleic acids under complex environments in vivo. It can be seen that the aescin compounds of the present application can improve the transfection efficiency of nucleic acids both in vivo and in vitro, which is conducive to the widespread application of nucleic acid drugs.
- one embodiment means that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present application.
- examples of the term “in one embodiment” here do not necessarily all refer to the same embodiment.
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Abstract
Description
相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求在2023年01月31日提交中国专利局、申请号为202310047356.X、名称为“含七叶皂苷和/或其盐化合物的转染复合物在促转染的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on January 31, 2023, with application number 202310047356.X and titled “Application of transfection complexes containing aescin and/or its salt compounds in promoting transfection”, the entire contents of which are incorporated by reference into this application.
本申请涉及生物医药技术领域，特别是涉及含七叶皂苷和/或其盐化合物的转染复合物在促转染的应用。The present application relates to the field of biomedical technology, and in particular to the application of a transfection complex containing aescin and/or its salt compounds in promoting transfection.
随着医疗行业的发展，基因治疗也逐渐展露锋芒。基因治疗是指将正常的外源基因导入靶细胞，补救基因缺陷或者异常所导致的疾病，从而达到治疗目的。核酸根据化学组成不同主要分为脱氧核糖核酸(DNA)和核糖核酸(RNA)。核酸药物为治疗或预防癌症、病毒感染和遗传疾病等人类疾病提供了新的治疗方式。目前，核酸药物主要包括质粒DNA，ASON，mRNA，siRNA，miRNA，RNA aptamer，saRNA，SAM，sgRNA，snRNA和其他基因治疗药物等。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.
近年来，核酸的递送和核酸药物取得了长足的发展。然而，核酸药物的发展也面临着巨大的挑战。核酸药物作为带负电的生物活性大分子，其体内递送存在诸多障碍。核酸药物入血后，极易被血液中的核酸酶清除，因此导致其具有半衰期短、生物利用度低等缺点。所以，一个高效低毒的递送载体对于核酸药物的应用来说显得至关重要。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.
目前为止，已有多款核酸药物被批准上市，如三种基于非病毒载体脂质纳米颗粒的RNA药物，包括用于治疗遗传性甲状腺素介导的淀粉样变性的多发性神经病RNAi药物Patisiran，用于COVID-19预防的mRNA疫苗Tozinameran(商品名：Comirnaty)和Elasomeran(商品名：Spikevax)；另有利用无针注射器按压于皮肤给药的ZyCoV-D疫苗，其属于DNA药物。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 Patisiran, an RNAi drug for the treatment of polyneuropathy caused by hereditary thyroxine-mediated amyloidosis, and Tozinameran (trade name: Comirnaty) and Elasomeran (trade name: Spikevax), mRNA vaccines for the prevention of COVID-19; there is also the ZyCoV-D vaccine, which is administered by pressing a needle-free syringe on the skin and is a DNA drug.
对于核酸递送，常用的载体通常为脂质纳米颗粒，由于脂质纳米颗粒存 在巨大的专利壁垒，从而限制了核酸药物的发展。For nucleic acid delivery, commonly used carriers are usually lipid nanoparticles. The huge patent barriers limit the development of nucleic acid drugs.
因此，需要开发转染效率更高的递送载体构建核酸药物平台。Therefore, it is necessary to develop delivery vectors with higher transfection efficiency to construct nucleic acid drug platforms.
发明内容Summary of the invention
为了解决现有的基因递送载体转染效率低，专利壁垒多的问题。本申请通过创造性的研究发现，七叶皂苷化合物可明显提高非病毒载体的转染效率。In order to solve the problems of low transfection efficiency and many patent barriers of existing gene delivery vectors, the present application found through creative research that aescin compounds can significantly improve the transfection efficiency of non-viral vectors.
本申请的目的之一是提供七叶皂苷化合物在基因递送中的应用。One of the purposes of the present application is to provide a use of aescin compound in gene delivery.
为了实现上述目的，本申请进行了大量的研究，评价了不同非病毒载体与七叶皂苷化合物组合使用对不同类型核酸转染的影响，结果发现七叶皂苷化合物均能提高不同非病毒载体的转染效果，可以应用于核酸的体内外递送，并可以进一步发展成为核酸药物。In order to achieve the above objectives, the present application has conducted a large number of studies to evaluate the effects of the combination of different non-viral vectors and aescin compounds on the transfection of different types of nucleic acids. The results show that aescin compounds can improve the transfection effects of different non-viral vectors, can be used for the in vivo and in vitro delivery of nucleic acids, and can be further developed into nucleic acid drugs.
本申请的目的之一是提供一种转染复合物，其包含七叶皂苷化合物，所述七叶皂苷化合物为七叶皂苷和/或七叶皂苷盐化合物。One of the purposes of the present application is to provide a transfection complex comprising an aescin compound, wherein the aescin compound is an aescin and/or an aescin salt compound.
可选地，所述转染复合物还包含非病毒载体和/或核酸分子。Optionally, the transfection complex further comprises a non-viral vector and/or a nucleic acid molecule.
可选地，所述核酸分子是核酸药物。进一步优选为DNA和/或RNA。Optionally, the nucleic acid molecule is a nucleic acid drug, and more preferably DNA and/or RNA.
可选地，所述核酸药物包括质粒DNA(plasmid DNA，pDNA)、反义寡核苷酸(antisense oligonucleotide，ASON)、编码治疗性蛋白或疫苗抗原基因的信使RNA(mRNA)、含有核苷酸修饰的mRNA、小干扰RNA(smallinterfering RNA，siRNA)、微小RNA(microRNA，miRNA)、调控蛋白活性的RNA适配体(RNAaptamer)、小激活RNA(saRNA)、环状RNA、自扩增mRNA(SAM)、小向导RNA(sgRNA)、U1小核RNA(snRNA)和其他基因治疗药物的一种或多种。Optionally, the nucleic acid drug includes plasmid DNA (pDNA), antisense oligonucleotide (ASON), messenger RNA (mRNA) encoding therapeutic protein or vaccine antigen gene, mRNA containing nucleotide modification, small interfering RNA (siRNA), microRNA (miRNA), RNA aptamer (RNA aptamer) that regulates 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.
可选地，所述的非病毒载体包括脂质体、脂质纳米颗粒、纳米乳、天然高分子聚合物、合成高分子聚合物的一种或多种。所述天然高分子聚合物包括鱼精蛋白、聚赖氨酸、聚精氨酸、壳聚糖和其他天然高分子聚合物的一种或多种。所述合成高分子聚合物包括聚乙烯亚胺(PEI)及其衍生物、树枝状聚酰胺胺(PAMAM)及其衍生物、聚-β-氨基酯(PBAE)及其衍生物和其他合成高分子聚合物的一种或多种。Optionally, the non-viral vector comprises 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.
可选地，所述七叶皂苷的结构如式(I)所示：
Optionally, the structure of aescin is as shown in formula (I):
可选地，所述七叶皂苷盐化合物，结构如式(II)所示：
Optionally, the aescin salt compound has a structure as shown in formula (II):
其中，式(II)中的R包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天门冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸的一种或多种，式(II)中的R还包括金属盐，所述金属盐包括钠、钾、锌、铝、铁、锆、钙、锰、镁、铜、铅、镍、镉的任意一种。Wherein, R in formula (II) includes 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 R in formula (II) also includes a metal salt, and the metal salt includes any one of sodium, potassium, zinc, aluminum, iron, zirconium, calcium, manganese, magnesium, copper, lead, nickel and cadmium.
可选地，所述转染复合物中七叶皂苷化合物与核酸的摩尔比值为0.01-100。优选的，七叶皂苷化合物与核酸的摩尔比值为0.05-50，进一步优选的比值为0.1-25，更优选的比值为0.15-15，进一步优选的比值为0.2-8，最优选的比值为0.4-6。Optionally, the molar ratio of aescin compound to nucleic acid in the transfection complex is 0.01-100. Preferably, the molar ratio of aescin compound to nucleic acid is 0.05-50, a further preferred ratio is 0.1-25, a more preferred ratio is 0.15-15, a further preferred ratio is 0.2-8, and the most preferred ratio is 0.4-6.
本申请的目的之一，提供一种转染复合物，并将其作为核酸药物，用于在制备核酸药物中的应用。其中，所述七叶皂苷化合物能够提高非病毒载体转染效率。 One of the purposes of the present application 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 compound can improve the transfection efficiency of non-viral vectors.
本申请的目的之一是提供七叶皂苷化合物在制备促进转染物质中的应用。优选的，所述转染物质为非病毒载体，其可作为转染制剂。所述转染物质可以促进核酸在各种细胞中的转染效果。One of the purposes of the present application is to provide an application of aescin 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.
有益的技术效果：Beneficial technical effects:
本申请的目的之一是提供一种包含有七叶皂苷化合物的核酸递送系统，转染效率因会七叶皂苷化合物而进一步提高。One of the purposes of the present application is to provide a nucleic acid delivery system comprising aescin compound, wherein the transfection efficiency is further improved due to the aescin compound.
本申请提供的转染复合物能够提高核酸在各种细胞上的转染效率，例如以293T细胞为代表的易转染细胞、以Hela细胞为代表的一般细胞和以DC2.4为代表的较难转染的免疫细胞，本申请惊奇地发现七叶皂苷和/或七叶皂苷盐化合物对于转染效率的提升是普适性的，对各种细胞均有效，特别是对于难以转染的细胞，例如免疫细胞。The transfection complex provided in the present application 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 application 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.
本申请创造性地发现七叶皂苷和/或七叶皂苷盐化合物在难转染的细胞，如免疫细胞上，也能提高转染复合物的转染效率。The present application creatively discovered 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.
本申请将所述的转染复合物用于体内转染，结果表明七叶皂苷和/或七叶皂苷盐化合物在体内复杂环境下，依然可以提高转染效率。可见，本申请的七叶皂苷和/或七叶皂苷盐化合物在体内和体外均能提高核酸的转染效率，有利于核酸药物的广泛应用。The present application uses the transfection complex for in vivo transfection, and the results show that aescin and/or aescin salt compounds can still improve the transfection efficiency under complex in vivo environments. It can be seen that aescin and/or aescin salt compounds of the present application 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.
上述说明仅是本申请技术方案的概述，为了能够更清楚了解本申请的技术手段，而可依照说明书的内容予以实施，并且为了让本申请的上述和其它目的、特征和优点能够更明显易懂，以下特举本申请的具体实施方式。The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below.
为了更清楚地说明本申请实施例或相关技术中的技术方案，下面将对实施例或相关技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related technologies, the following is a brief introduction to the drawings required for use in the embodiments or the related technical descriptions. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
图1为用含七叶皂苷的脂质体递送luciferase-DNA，有血有抗转染Hela细胞24h的转染效果。Figure 1 shows the transfection effect of luciferase-DNA delivered by liposomes containing aescin on Hela cells for 24 hours.
图2为用含七叶皂苷钠的脂质体递送luciferase-DNA，有血有抗转染Hela细胞24h的转染效果。 FIG. 2 shows the transfection effect of luciferase-DNA delivered by liposomes containing sodium aescinate on Hela cells for 24 hours.
图3为用含七叶皂苷钠的脂质体递送luciferase-mRNA，有血有抗转染DC2.4细胞24h的转染效果。FIG. 3 shows the transfection effect of luciferase-mRNA delivered by liposomes containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
图4为用含七叶皂苷的脂质体递送GFP-siRNA，有血有抗转染GFP-293T细胞48h的转染效果。FIG. 4 shows the transfection effect of GFP-siRNA delivered by liposomes containing aescin on GFP-293T cells with and without transfection resistance for 48 hours.
图5为用含七叶皂苷钠的脂质纳米颗粒递送luciferase-mRNA，有血有抗转染DC2.4细胞24h的转染效果。FIG. 5 shows the transfection effect of luciferase-mRNA delivered by lipid nanoparticles containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
图6为用含七叶皂苷的纳米乳递送luciferase-DNA，有血有抗转染DC 2.4细胞24h的转染效果。Figure 6 shows the transfection effect of luciferase-DNA delivered by nanoemulsion containing aescin on DC 2.4 cells with blood and resistance to transfection for 24 hours.
图7为用含七叶皂苷钠的纳米乳递送luciferase-DNA，有血有抗转染DC2.4细胞24h的转染效果。FIG. 7 shows the transfection effect of luciferase-DNA delivered by nanoemulsion containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
图8为用含七叶皂苷钠的纳米乳递送GFP-siRNA，有血有抗转染GFP-293T细胞48h的转染效果。FIG. 8 shows the transfection effect of GFP-siRNA delivered by nanoemulsion containing sodium aescinate on GFP-293T cells with or without transfection resistance for 48 hours.
图9为用含七叶皂苷钠的鱼精蛋白递送luciferase-DNA，有血有抗转染DC2.4细胞24h的转染效果。FIG. 9 shows the transfection effect of luciferase-DNA delivered by protamine containing sodium aescinate on DC2.4 cells with and without transfection resistance for 24 hours.
图10为用含七叶皂苷钠的聚乙烯亚胺25k(PEI 25k)递送luciferase-DNA，有血有抗转染DC 2.4细胞24h的转染效果。Figure 10 shows the transfection effect of luciferase-DNA delivered by polyethyleneimine 25k (PEI 25k) containing sodium aescinate on transfection-resistant DC 2.4 cells for 24 hours.
图11为含七叶皂苷钠的纳米乳递送luciferase-DNA，肌肉注射于小鼠体内，72h后，于小鼠腹腔注射萤火虫荧光素酶底物，进行活体成像的结果。FIG. 11 shows the results of in vivo imaging of a nanoemulsion containing sodium aescinate delivering luciferase-DNA and intramuscularly injecting it into mice. 72 hours later, firefly luciferase substrate was intraperitoneally injected into the mice.
为使本申请实施例的目的、技术方案和优点更加清楚，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.
实施例1Example 1
脂质体(Lip)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。 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.
含七叶皂苷的脂质体(Lip-Esc)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG和0.5mg七叶皂苷溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。The preparation method of liposomes containing aescin (Lip-Esc) is 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 ultrasonicated at 180w for 5 min under ice bath conditions.
取适量Lip或Lip-Esc，在涡旋条件下滴入luciferase-DNA溶液，涡旋15s后孵育30min，得到脂质体和DNA的复合物。在Lip-Esc-DNA中，七叶皂苷和DNA的摩尔比值为0.4。Take an appropriate amount of Lip or Lip-Esc, 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-Esc-DNA, the molar ratio of aescin to DNA is 0.4.
将Hela细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图1。DNA转染时，需要先进入细胞膜，后进入核孔，才能实现后续的转录和翻译过程。因此DNA转染的过程复杂，难度大。与不含七叶皂苷的Lip-DNA相比，Lip-Esc-DNA的转染效率显著提高(**，p<0.01)，可见，七叶皂苷能够显著提高非病毒载体的转染效率。图中，Lip-DNA表示载luciferase-DNA的脂质体，Lip-Esc-DNA表示共载luciferase-DNA和七叶皂苷的脂质体。Hela cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of DNA was given 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 is necessary to enter the cell membrane first and then the nuclear pore to achieve the subsequent transcription and translation process. Therefore, the DNA transfection process is complicated and difficult. Compared with Lip-DNA without aescin, the transfection efficiency of Lip-Esc-DNA was 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.
实施例2Example 2
脂质体(Lip)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。The preparation method of liposomes (Lip) 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
含七叶皂苷钠的脂质体(Lip-Aes)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml0.25mg/ml七叶皂苷钠水溶液水化薄膜，冰浴条件下探头超声180w，5min。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 were dissolved in 3 ml methanol, and the organic solvent was removed by rotary evaporation. 3 ml 0.25 mg/ml sodium aescinate aqueous solution was added to hydrate the film, and the probe was ultrasonicated at 180 w for 5 min under ice bath conditions.
取适量Lip或Lip-Aes，在涡旋条件下滴入luciferase-DNA溶液，涡旋15s后孵育30min，得到脂质体和DNA的复合物。在Lip-Aes-DNA中，七叶皂苷钠和DNA的摩尔比值为0.2。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.
将Hela细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图2。与不含七叶皂苷钠的Lip-DNA相比，Lip-Aes-DNA的转染效率显著提高(**，p<0.01)，可见，七叶皂苷盐化合物能够显著提高非病毒载体的转染效率。图中，Lip-DNA表示载luciferase-DNA的脂质体，Lip-Aes-DNA表示共载 luciferase-DNA和七叶皂苷钠的脂质体。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). It can be seen 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 co-loaded Liposomes containing luciferase-DNA and sodium aescinate.
实施例3Example 3
脂质体(Lip)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。The preparation method of liposomes (Lip) 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
含七叶皂苷钠的脂质体(Lip-Aes)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml0.25mg/ml七叶皂苷钠水溶液水化薄膜，冰浴条件下探头超声180w，5min。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 were dissolved in 3 ml methanol, and the organic solvent was removed by rotary evaporation. 3 ml 0.25 mg/ml sodium aescinate aqueous solution was added to hydrate the film, and the probe was ultrasonicated at 180 w for 5 min under ice bath conditions.
取适量Lip或Lip-Aes，在涡旋条件下滴入luciferase-mRNA溶液，涡旋15s后孵育30min，得到脂质体和mRNA的复合物。在Lip-Aes-mRNA中，七叶皂苷钠和mRNA的摩尔比值为0.2。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.
将DC2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给mRNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图3。mRNA为单链的生物大分子，极容易被环境中的核酸酶降解。DC2.4细胞为难转染细胞，常规脂质体在该细胞上的转染效果不佳(Lip-mRNA组)，与不含七叶皂苷钠的Lip-mRNA相比，Lip-Aes-mRNA的转染效率显著提高(****，p<0.0001)，可见，七叶皂苷盐化合物能够显著提高普通非病毒载体在难转染细胞(例如免疫细胞)上的转染效果。图中，Lip-mRNA表示载luciferase-mRNA的脂质体，Lip-Aes-mRNA表示共载luciferase-mRNA和七叶皂苷钠的脂质体。DC2.4 cells were placed in a 24-well plate. When the cell fusion rate was 70-80%, 1.5 μg of mRNA was given 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. 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 was significantly improved (****, p<0.0001). It can be seen that 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, Lip-mRNA represents liposomes carrying luciferase-mRNA, and Lip-Aes-mRNA represents liposomes co-loaded with luciferase-mRNA and sodium aescinate.
实施例4Example 4
脂质体(Lip)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。The preparation method of liposomes (Lip) 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 UP water is added to hydrate the film, and the probe is ultrasonicated at 180w for 5 min under ice bath conditions.
含七叶皂苷的脂质体(Lip-Esc)的制备方式如下：将5mg DOTMA、10mg卵磷脂、5mg胆固醇、1mg DSPE-PEG和0.5mg七叶皂苷溶解于3ml甲醇中，通过旋蒸蒸发法除去有机溶剂，加入3ml UP水水化薄膜，冰浴条件下探头超声180w，5min。The preparation method of liposomes containing aescin (Lip-Esc) is 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 ultrasonicated at 180w for 5 min under ice bath conditions.
取适量Lip，在涡旋条件下滴入GFP-siRNA溶液，涡旋15s后孵育30min，得到Lip-siRNA。在Lip-Esc-siRNA中，七叶皂苷和siRNA的摩尔比值为1.6。 取适量Lip-Esc，在涡旋条件下滴入GFP-siRNA溶液和七叶皂苷钠溶液，涡旋15s后孵育30min，得到Lip-Esc-siRNA。Take an appropriate amount of Lip, drop GFP-siRNA solution into it 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, add GFP-siRNA solution and sodium aescinate solution dropwise under vortexing conditions, vortex for 15 seconds and incubate for 30 minutes to obtain Lip-Esc-siRNA.
将GFP-293T细胞置于24孔板，当细胞融合率为50％时，每孔给siRNA1.5μg，转染48h后，用PBS清洗并重悬细胞，用细胞流式仪检测。结果参见图4。siRNA为双链的小RNA，在环境中体液中极不稳定，易被核酸酶降解，与不含七叶皂苷的Lip-siRNA相比，Lip-Esc-siRNA的敲除效率显著提高(*，p<0.05)，可见，七叶皂苷能够显著提高转染效率。图中，Lip-siRNA表示载GFP-siRNA的脂质体，Lip-Esc-siRNA表示共载GFP-siRNA和七叶皂苷的纳米乳。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. The results are shown in 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 knockout efficiency of Lip-Esc-siRNA was significantly improved (*, p<0.05). It can be seen 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.
实施例5Example 5
脂质纳米颗粒(LNP)的制备方法如下：将ALC-0315、DSPC、胆固醇和DMG-PEG2000以摩尔比50:10:38.5:1.5溶解于乙醇中作为有机相，将luciferase-mRNA溶解于DEPC水中作为水相，用微流控装置，控制总流速为9ml/min，水相和有机相流速比为3:1，氮磷比为6，快速混合得到LNP-mRNA。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.
含七叶皂苷钠的脂质纳米颗粒(LNP-Aes)的制备方法如下：将ALC-0315、DSPC、胆固醇和DMG-PEG2000以摩尔比50:10:38.5:1.5溶解于乙醇中作为有机相，将luciferase-mRNA溶解于七叶皂苷钠水溶液中作为水相，mRNA和七叶皂苷钠的浓度比值为0.25。用微流控装置，控制总流速为9ml/min，水相和有机相流速比为3:1，氮磷比为6，快速混合得到LNP-Aes-mRNA，此时七叶皂苷钠和mRNA的摩尔比值为1.2。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.
将DC 2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给mRNA1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图5，其结果显示，DC2.4细胞为难转染细胞，常规的脂质纳米颗粒在该细胞上的转染效果不佳(LNP-mRNA组)，与不含七叶皂苷钠的LNP-mRNA相比，LNP-Aes-mRNA的转染效率显著提高(**，p<0.01)，可见，加入七叶皂苷盐化合物能够显著提高普通非病毒载体在难转染细胞(例如免疫细胞)上的转染效果。图中，LNP-mRNA表示载luciferase-mRNA的脂质纳米颗粒，LNP-Aes-mRNA表示共载luciferase-mRNA和七叶皂苷钠的脂质纳米颗粒。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 given 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 show that DC2.4 cells are difficult to transfect cells, 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 ordinary 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.
实施例6Example 6
纳米乳(NE)的制备方法如下：将86mg角鲨烯和16mg DOTAP溶解于适 量氯仿作为油相，将乳化剂溶解于3ml UP水中作为水相，将油相滴入水相，冰浴条件下探头超声150w，5min。The preparation method of nanoemulsion (NE) is as follows: 86 mg of squalene and 16 mg of DOTAP are dissolved in an appropriate 100 ml of chloroform was used as the oil phase, and the emulsifier was dissolved in 3 ml of UP water as the water phase. The oil phase was dropped into the water phase, and the probe was ultrasonicated at 150w for 5min under ice bath condition.
含七叶皂苷的纳米乳(NE-Esc)的制备方法如下：将86mg角鲨烯和16mg DOTAP溶解于适量氯仿作为油相，将乳化剂溶解于3ml UP水中作为水相，将油相滴入水相，冰浴条件下探头超声150w，5min。将七叶皂苷溶解于甲醇，取适量七叶皂苷甲醇溶液旋蒸形成薄膜，用适量的纳米乳水化薄膜，使七叶皂苷的浓度为0.7mg/ml。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.
取适量NE或NE-Esc，在涡旋条件下滴入luciferase-DNA溶液，涡旋15s后孵育30min，得到纳米乳和DNA的复合物。在NE-Esc-DNA中，七叶皂苷和DNA的摩尔比值为0.68。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.
将DC 2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图6。DC2.4细胞为难转染细胞，常规的纳米乳在该细胞上的转染效果不佳(NE-DNA组)，与不含七叶皂苷的NE-DNA相比，NE-Esc-DNA的转染效率显著提高(*，p<0.05)，可见，加入七叶皂苷能够显著提高普通非病毒载体在难转染细胞(例如免疫细胞)上的转染效果。图中，NE-DNA表示载luciferase-DNA的纳米乳，NE-Esc-DNA表示共载luciferase-DNA和七叶皂苷的纳米乳。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 given 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 cells. 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 was significantly improved (*, p<0.05). It can be seen that the addition of aescin can significantly improve the transfection effect of ordinary non-viral vectors on difficult-to-transfect cells (such as immune cells). In the figure, NE-DNA represents nanoemulsions carrying luciferase-DNA, and NE-Esc-DNA represents nanoemulsions co-loaded with luciferase-DNA and aescin.
实施例7Example 7
纳米乳(NE)的制备方法如下：将86mg角鲨烯和16mg DOTAP溶解于适量氯仿作为油相，将乳化剂溶解于3ml UP水中作为水相，将油相滴入水相，冰浴条件下探头超声150w，5min。The preparation method of nanoemulsion (NE) is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
取适量NE，在涡旋条件下滴入luciferase-DNA溶液，涡旋15s后孵育30min，得到NE-DNA。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.
取适量NE，在涡旋条件下滴入luciferase-DNA溶液和0.5mg/ml七叶皂苷钠溶液，涡旋15s后孵育30min，得到NE-Aes-DNA，此时七叶皂苷钠和DNA的摩尔比值为1.2。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.
将DC 2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图7。DC2.4细胞为难转染细胞，常规的纳米乳在该细胞上的转染效果不佳 (NE-DNA组)，与不含七叶皂苷钠的NE-DNA相比，NE-Aes-DNA的转染效率显著提高(****，p<0.0001)，可见，七叶皂苷盐化合物能够显著提高转染效率。图中，NE-DNA表示载luciferase-DNA的纳米乳，NE-Aes-DNA表示共载luciferase-DNA和七叶皂苷钠的纳米乳。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 was significantly improved (****, p<0.0001), which shows that aescinate compounds can significantly improve the transfection efficiency. In the figure, NE-DNA represents nanoemulsion loaded with luciferase-DNA, and NE-Aes-DNA represents nanoemulsion co-loaded with luciferase-DNA and sodium aescinate.
实施例8Example 8
纳米乳(NE)的制备方法如下：将86mg角鲨烯和16mg DOTAP溶解于适量氯仿作为油相，将乳化剂溶解于3ml UP水中作为水相，将油相滴入水相，冰浴条件下探头超声150w，5min。The preparation method of nanoemulsion (NE) is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
取适量NE，在涡旋条件下滴入GFP-siRNA溶液，涡旋15s后孵育30min，得到NE-siRNA。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.
取适量NE，在涡旋条件下滴入GFP-siRNA溶液和0.5mg/ml七叶皂苷钠溶液，涡旋15s后孵育30min，得到NE-Aes-siRNA，此时七叶皂苷钠和siRNA的摩尔比值为2.4。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.
将GFP-293T细胞置于24孔板，当细胞融合率为50％时，每孔给siRNA1.5μg，转染48h后，用PBS清洗并重悬细胞，用细胞流式仪检测。结果参见图8。siRNA为双链的小RNA，在环境中体液中极不稳定，易被核酸酶降解，与不含七叶皂苷钠的NE-siRNA相比，NE-Aes-siRNA的敲除效率显著提高(**，p<0.01)，可见，七叶皂苷盐化合物能够显著提高转染效率。图中，NE-siRNA表示载GFP-siRNA的纳米乳，NE-Aes-siRNA表示共载GFP-siRNA和七叶皂苷钠的纳米乳。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. The results are shown in 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 was significantly improved (**, p<0.01). It can be seen that aescinate compounds can significantly improve transfection efficiency. In the figure, NE-siRNA represents a nanoemulsion loaded with GFP-siRNA, and NE-Aes-siRNA represents a nanoemulsion co-loaded with GFP-siRNA and sodium aescinate.
实施例9Embodiment 9
天然高分子聚合物-DNA纳米粒制备方法：用5％的葡萄糖溶液溶解鱼精蛋白为0.1mg/ml的溶液，将luciferase-DNA配制成0.1mg/ml的溶液，将等体积的luciferase-DNA溶液在涡旋条件下加入鱼精蛋白溶液中，孵育30min，得到载有DNA的纳米粒，PRTM-DNA。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.
用5％的葡萄糖溶液溶解鱼精蛋白为0.1mg/ml的溶液，将luciferase-DNA配制成0.1mg/ml的溶液，将等体积的luciferase-DNA溶液和0.5mg/ml七叶皂苷钠溶液在涡旋条件下加入鱼精蛋白溶液中，孵育30min，得到共载七叶皂苷钠和DNA的纳米粒,PRTM-Aes-DNA，此时七叶皂苷钠和DNA的摩尔比值为2.4。 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.
将DC 2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图9。常规的天然高分子聚合物在该细胞上的转染效果不佳(PRTM-DNA组)与不含七叶皂苷钠的PRTM-DNA相比，PRTM-Aes-DNA的转染效率显著提高(**，p<0.01)，可见，加入七叶皂苷盐化合物能够显著提高普通非病毒载体在难转染细胞(例如免疫细胞)上的转染效果。图中，PRTM-DNA表示载luciferase-DNA的纳米粒，PRTM-Aes-DNA表示共载luciferase-DNA和七叶皂苷钠的纳米粒。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 given 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 9. Conventional natural polymers have poor transfection effects on these cells (PRTM-DNA group). Compared with PRTM-DNA without sodium aescinate, the transfection efficiency of PRTM-Aes-DNA 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, PRTM-DNA represents nanoparticles loaded with luciferase-DNA, and PRTM-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.
实施例10Example 10
合成高分子聚合物-DNA纳米粒制备方法：用UP水溶解PEI 25k为0.1mg/ml的溶液，将luciferase-DNA配制成0.1mg/ml的溶液，将等体积的luciferase-DNA溶液在涡旋条件下加入PEI 25k溶液中，孵育30min，得到载有DNA的纳米粒，PEI 25k-DNA。Preparation method of synthetic polymer-DNA nanoparticles: dissolve PEI 25k in UP water to make 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 PEI 25k solution under vortex conditions, incubate for 30 minutes, and obtain DNA-loaded nanoparticles, PEI 25k-DNA.
用UP水溶解PEI 25k为0.1mg/ml的溶液，将luciferase-DNA配制成0.1mg/ml的溶液，将等体积的luciferase-DNA溶液和0.5mg/ml七叶皂苷钠溶液在涡旋条件下加入PEI25k溶液中，孵育30min，得到共载七叶皂苷钠和DNA的纳米粒,PEI25k-Aes-DNA，此时七叶皂苷钠和DNA的摩尔比值为5。Dissolve PEI 25k in UP water 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 into the PEI25k solution under vortex conditions, incubate for 30 min, and obtain nanoparticles co-loaded with sodium aescinate and DNA, PEI25k-Aes-DNA, at which the molar ratio of sodium aescinate to DNA is 5.
将DC 2.4细胞置于24孔板，当细胞融合率为70-80％时，每孔给DNA 1.5μg，转染24h后，用萤火虫荧光素酶底物试剂盒测定转染效率，结果参见图10。其结果显示，DC2.4细胞为难转染细胞，常规的合成聚合物高分子在该细胞上的转染效果不佳(PEI 25k-DNA组)，与不含七叶皂苷钠的PEI 25k-DNA相比，PEI25k-Aes-DNA的转染效率显著提高(****，p<0.0001)，可见，加入七叶皂苷盐化合物能够显著提高普通非病毒载体在难转染细胞(例如免疫细胞)上的转染效果。图中，PEI 25k-DNA表示载luciferase-DNA的纳米粒，PEI25k-Aes-DNA表示共载luciferase-DNA和七叶皂苷钠的纳米粒。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 given 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 10. The results show 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 PEI25k-Aes-DNA represents nanoparticles co-loaded with luciferase-DNA and sodium aescinate.
实施例11Embodiment 11
纳米乳(NE)的制备方法如下：将86mg角鲨烯和16mg DOTAP溶解于适量氯仿作为油相，将乳化剂溶解于3ml UP水中作为水相，将油相滴入水相，冰浴条件下探头超声150w，5min。The preparation method of nanoemulsion (NE) is as follows: dissolve 86 mg squalene and 16 mg DOTAP in an appropriate amount of chloroform as the oil phase, dissolve the emulsifier in 3 ml UP water as the water phase, drop the oil phase into the water phase, and use the probe ultrasound at 150w for 5 minutes under ice bath conditions.
取适量NE，在涡旋条件下滴入luciferase-DNA溶液，涡旋15s后孵育 30min，得到NE-DNA。Take an appropriate amount of NE, add the luciferase-DNA solution under vortexing conditions, vortex for 15 seconds and incubate 30min, NE-DNA was obtained.
取适量NE，在涡旋条件下滴入luciferase-DNA溶液和0.25mg/ml七叶皂苷钠溶液，涡旋15s后孵育30min，得到NE-Aes-DNA，此时七叶皂苷钠和DNA的摩尔比值为0.25。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.
肌肉注射小鼠腓肠肌NE-DNA或NE-Aes-DNA，每只小鼠所注射的DNA含量均为25μg。72h后，腹腔注射萤火虫荧光素酶体内底物活体成像，结果参见图11。与不含七叶皂苷钠的NE-DNA相比，NE-Aes-DNA在体内的转染效率更高，NE-DNA给药后并未在活体检测到荧光，可见，普通纳米乳难以使DNA在体内表达，而加入七叶皂苷钠后(NE-Aes-DNA组)，在活体上能检测到明显的荧光，可见，七叶皂苷钠能够显著非病毒载体在体内的转染效率。图中，NE-DNA表示载luciferase-DNA的纳米乳，NE-Aes-DNA表示共载luciferase-DNA和七叶皂苷钠的纳米乳。NE-DNA or NE-Aes-DNA was injected into the gastrocnemius 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, and the results are shown in Figure 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 are difficult to express DNA in vivo, and after adding sodium aescinate (NE-Aes-DNA group), obvious fluorescence can be detected in vivo, which shows that sodium aescinate can significantly increase 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.
综上可知，本申请发现七叶皂苷化合物能够提高核酸递送系统的转染效率，且对易转染细胞、一般细胞和难转染细胞均有提高转染效率的效果，同时，在各种非病毒载核酸传递系统中均能发挥上述效果，因此，七叶皂苷化合物对于转染效率的提升是普适性的，对各种非病毒核酸递送系统以及各种不同的细胞均有效，特别是对于难以转染的细胞，例如免疫细胞。此外，七叶皂苷化合物在体内复杂环境下，依然可以提高核酸的转染效率。可见，本申请的七叶皂苷化合物在体内和体外均能提高核酸的转染效率，有利于核酸药物的广泛应用。In summary, the present application finds that aescin compounds can improve the transfection efficiency of nucleic acid delivery systems, and have the effect of improving transfection efficiency for easily transfected cells, general cells and difficultly transfected cells, and at the same time, the above-mentioned effects can be exerted in various non-viral nucleic acid delivery systems, therefore, aescin compounds 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 compounds can still improve the transfection efficiency of nucleic acids under complex environments in vivo. It can be seen that the aescin compounds of the present application can improve the transfection efficiency of nucleic acids both in vivo and in vitro, which is conducive to the widespread application of nucleic acid drugs.
本文中所称的“一个实施例”、“实施例”或者“一个或者多个实施例”意味着，结合实施例描述的特定特征、结构或者特性包括在本申请的至少一个实施例中。此外，请注意，这里“在一个实施例中”的词语例子不一定全指同一个实施例。The term "one embodiment", "embodiment" or "one or more embodiments" herein means that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present application. In addition, please note that the examples of the term "in one embodiment" here do not necessarily all refer to the same embodiment.
在此处所提供的说明书中，说明了大量具体细节。然而，能够理解，本申请的实施例可以在没有这些具体细节的情况下被实践。在一些实例中，并未详细示出公知的方法、结构和技术，以便不模糊对本说明书的理解。In the description provided herein, a large number of specific details are described. However, it is understood that the embodiments of the present application can be practiced without these specific details. In some instances, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this description.
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或 者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of this application.
Claims (14)
The transfection complex according to any one of claims 1 to 2, characterized in that the structure of the aescin is as shown in formula (I):
The transfection complex according to any one of claims 1 to 2, characterized in that the aescin salt compound has a structure as shown in formula (II):
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