CN118879783A - A cyclic lipopeptide carrier for encapsulating nucleic acid drugs - Google Patents

Abstract

The invention discloses a cyclic lipopeptide carrier for wrapping nucleic acid medicaments, which comprises a cyclic lactone structure formed by heptapeptide R ₁R₂R₂R₃R₁R₂R₂ and beta-hydroxy fatty acid with a carbon chain length of 6-44, and the structural general formula is as follows: Wherein R ₁ is a basic amino acid comprising one of histidine His, lysine Lys or arginine Arg, R ₂ is one of hydrophobic amino acids leucine Leu, isoleucine Ile or valine Val, and R ₃ is one of any amino acid; n is an integer of 2 to 40; the positively charged basic amino acid of the cyclic lipopeptide is combined with the negatively charged nucleic acid drug by electrostatic interaction to form a cyclic lipopeptide-nucleic acid particle. The cyclic lipopeptide carrier for wrapping the nucleic acid medicine has high safety, can effectively protect the nucleic acid medicine, improves the stability of the nucleic acid medicine, has good transfection efficiency on various cells, and has wide application prospect in the field of nucleic acid medicine delivery.

Description

A cyclic lipopeptide carrier for encapsulating nucleic acid drugs

Technical Field

The present invention relates to the technical field of biopharmaceuticals, and in particular to a cyclic lipopeptide carrier for encapsulating nucleic acid drugs.

Background Art

Gene therapy and nucleic acid vaccines are the introduction of exogenous genetic material into cells. However, the effective delivery of genetic material and its effective transport to target cells is hindered by many extracellular and intracellular barriers. The negative charge of nucleic acids and the difficulty of hydrophilic substances to penetrate the cell membrane, as well as the ease of nucleic acid degradation by nucleases after entering the cell make gene carriers particularly important. Currently, the most commonly used nanoliposomes are those with cationic lipids as the core, but the cationic lipids currently on the market for nucleic acid delivery are almost all patented by foreign pharmaceutical companies.

The uptake of non-viral vectors by cells is mainly through endocytosis, most commonly with the use of nanoliposomes. Nucleic acids are combined with cationic lipids to form nanoliposomes (LNPs) that enter cells, and the cell surface is then internalized by them and releases the nucleic acids that enter the cells (Dries et al 2012). Since cell membranes are usually negatively charged, and the nanoparticle lipids used for nucleic acid delivery are positively charged, electrostatic interactions promote the adsorption of LNPs to cell membranes and fusion with cell membranes; their attraction drives membrane fusion and endocytosis. After entering the cell, the nucleic acid is released from the complex with cationic lipids. The anionic lipids of the cell may help release the nucleic acid from LNPs by neutralizing the charge of its cationic lipid carriers and destroying the electrostatic interaction between the lipid carrier and the nucleic acid (Tarahovsky et al 2000, Tarahovsky et al 2004).

Lipopeptides are compounds of lipids and amino acids. They were first isolated in the 1950s and 1960s. Bacillus is the most important microorganism producing lipopeptides. As a type of biosurfactant, lipopeptides can be used in biological control, drug delivery and other fields (Raju et al 2023).

Lipids (such as nanoliposomes) and peptides (such as polylysine) can be used as carriers for nucleic acid delivery to cells, but they all have unstable defects. For example, polylysine will present irregular coils at a solution pH of 7, and this structure has an important effect on bioactive substances. Several studies have found that the introduction of fatty acids into peptides can increase the stability of the complex and enhance the activity of membrane decomposition (He et al 2020), thereby enhancing the delivery of nucleic acids into cells. There are already literatures stating that lipopeptides can be used for drug delivery, such as using diacylated lysine as a delivery carrier for the local anesthetic lipopeptide ropivacaine (Eixeira et al 2014).

The present invention designs a non-viral gene delivery vector based on the natural lipopeptide of Bacillus, and confirms that it can efficiently transfect cells, opening up a new research direction for the development of novel nucleic acid delivery systems.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is to provide a cyclic lipopeptide carrier for encapsulating nucleic acid drugs. The cyclic lipopeptide carrier has high safety, can effectively protect nucleic acid drugs, improve their stability, and has good transfection efficiency for a variety of cells. It has broad application prospects in the field of nucleic acid drug delivery.

The technical solution adopted by the present invention to solve the technical problem is: a cyclic lipopeptide carrier for encapsulating nucleic acid drugs, the cyclic lipopeptide carrier comprises a cyclic _lactone structure formed by a _{heptapeptide R1R2R2R3R1R2R2} and a _β - _hydroxy fatty _acid with a carbon chain length of 6 to 44, _and its general structural formula is as follows:

In the formula, _R1 is a basic amino acid, including one of histidine His, lysine Lys or arginine Arg; _R2 is one of the hydrophobic amino acids leucine Leu, isoleucine Ile or valine Val; _R3 is one of any amino acids; n is an integer of 2 to 40;

The positively charged basic amino acids in the cyclic lipopeptide and the negatively charged nucleic acid drugs are combined through electrostatic interaction to form cyclic lipopeptide-nucleic acid particles.

Furthermore, in the cyclic lipopeptide, the carbon chain length of the β-hydroxy fatty acid is 13-19, and n is an integer of 9-15.

Furthermore, the nucleic acid drug includes mRNA, plasmid DNA, double-stranded DNA fragments or single-stranded DNA fragments.

Furthermore, the molar ratio of the cyclic lipopeptide to mRNA is 5-20:1; the mass ratio of the cyclic lipopeptide to plasmid DNA is 20-30:1; the mass ratio of the cyclic lipopeptide to double-stranded DNA is 20-30:1; and the mass ratio of the cyclic lipopeptide to single-stranded DNA is 20-30:1.

The preparation method of the cyclic lipopeptide complex solution encapsulating nucleic acid drugs is as follows: firstly dissolving the cyclic lipopeptide in ethanol to prepare a solution of 5-10 mg/mL, dissolving the nucleic acid drug in a 50 mM sodium acetate solution with a pH of 4-7 to obtain a nucleic acid solution with a concentration of 0.13-1.0 mg/mL; mixing the cyclic lipopeptide and the nucleic acid according to a certain molar ratio or mass ratio, the mixing volume ratio of the cyclic lipopeptide and the nucleic acid is 1:1-1:3, the mixing flow rate ratio of the cyclic lipopeptide and the nucleic acid is 1:3, and after the mixing is completed, standing for 5-30 minutes to obtain the cyclic lipopeptide complex solution encapsulating the nucleic acid drug.

Furthermore, the cyclic lipopeptide and the nucleic acid are mixed by manual mixing or microfluidic mixing.

Furthermore, the pH response range of the cyclic lipopeptide complex solution encapsulating the nucleic acid drug is 6 to 9, and when the pH is 6 to 9, the encapsulated nucleic acid is released.

The cyclic lipopeptide vector is mainly used in mRNA vaccines, mRNA drugs, DNA vaccines, DNA drugs or nucleic acid transfection. It can transfect a variety of animal cells, such as breast cancer Hela cells, 293T cells, DC2.4 cells, C2C12 cells, and Raw264.7 cells.

The beneficial effects of the present invention are as follows: compared with the prior art, the cyclic lipopeptide carrier for encapsulating nucleic acid drugs provided by the present invention can be tightly combined with nucleic acid drugs, protect nucleic acid drugs from degradation by DNase I, enter cells through endocytosis, and release the encapsulated nucleic acid drugs in response to changes in pH, thereby achieving the effect of intracellular release. The cyclic lipopeptide in the present invention has low hemolytic activity, and through cytotoxicity experiments, it is found that its cytotoxicity is low.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an electrophoretic diagram of a mixture of lipopeptides and nucleic acids at different concentrations;

FIG2 is an electrophoretic diagram of a mixture of ethanol and pDNA to investigate the protective ability of lipopeptides on DNA;

FIG3 is an electrophoretic diagram of a mixture of lipopeptides and pDNA to investigate the protective ability of lipopeptides on DNA;

FIG4 is an electrophoretic diagram showing the response of the lipopeptide nucleic acid complex to pH changes;

FIG5 is a bar graph showing the relative hemolytic activity of lipopeptides;

FIG6 is a bar graph showing the cytotoxicity of lipopeptides;

FIG7 shows the transfection efficiency of lipopeptide mixed with pDNA at different volume ratios in Hela cells;

FIG8 shows the transfection efficiency of lipopeptide and pDNA mixed at different mass ratios in Hela cells;

Figures 9-11 show the transfection efficiency of Hela cells after lipopeptides were mixed with different nucleic acids under different pH solvent conditions;

FIG12 shows the transfection efficiency of different nucleic acids in Hela cells under different mixing modes;

FIG13 is the transfection efficiency of lipopeptides in C2C12 cells;

FIG14 is the transfection efficiency of lipopeptides in 293T cells;

FIG15 is a graph showing the transfection efficiency of lipopeptides in DC2.4 cells;

FIG. 16 shows the transfection efficiency of lipopeptides in Raw264.7 cells.

DETAILED DESCRIPTION

The present invention is further described below by specific examples, but these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

The meanings of the English abbreviations involved in the following embodiments are shown in Table 1.

Table 1

English abbreviation Chinese meaning English abbreviation Chinese meaning DNA DNA mRNA mRNA DNase I Deoxyribonuclease 1 pDNA Plasmid DNA PBS Phosphate buffered saline KM Kunming LDH Lactate dehydrogenase NAD ⁺ Nicotinamide adenine dinucleotide ssDNA ssDNA dsDNA dsDNA

The lipopeptides used in the following examples were synthesized by themselves. The synthesis method was as follows: 3-acetoxytetradecanoic acid was added to a 500 ml three-necked flask, nitrogen was introduced, the temperature was lowered to 3-6°C, 250 ml of DMF was added, stirred, 2.06 g (0.01 mol) of DCC was added, 1.15 g of HOSU was added, stirred for 30 min, 3.98 g (0.01 mol) of Trt-histidine was added, stirred, and reacted for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.17 g (0.01 mol) of valine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.17 g (0.01 mol) of valine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 3.98 g (0.01 mol) of Trt-histidine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

Add 2.06 g (0.01 mol) of DCC, add 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

Add sodium hydroxide solution, adjust the pH to 10-10.5, stir for 30 min, adjust the pH to neutral, and precipitate solid.

The solid was added into a 500 ml three-necked flask, and 250 ml of DMF, 2.06 g (0.01 mol) of DCC, and 1.15 g of HOSU were added. The mixture was stirred and reacted for 2 h.

Hydrochloric acid solution was added to adjust the pH to 2 and stirred for 1 hour. The solid was precipitated and slurried with methyl tert-butyl ether and dried to obtain 7.47 g of the target product with a yield of 71%.

The specific reaction equation is:

Example 1

Study on the binding ability of different concentrations of lipopeptides to plasmid DNA

After manually mixing lipopeptides at different concentrations of 0, 2, 4, 6, 8, and 10 mg/mL with equal volumes of 0.13 mg/mL DNA, the mixture was allowed to stand at room temperature for 3 hours. The changes in the brightness of the nucleic acid bands at different ratios were observed by agarose gel electrophoresis to determine whether cationic liposomes can effectively encapsulate nucleic acids and select the optimal ratio of nucleic acid-lipopeptide complexes. The principle of the gel retardation experiment is that the single plasmid DNA is easily migrated through the gel due to its negative charge; when the plasmid DNA and lipopeptide are not bound or not completely bound, it is easy to migrate through the gel and the displayed fluorescence intensity is strong; when the plasmid DNA-lipopeptide complex contains positive charge and is larger in size, their migration through the gel is hindered, so the free plasmid DNA detected in the agarose gel will decrease and the displayed fluorescence intensity will weaken; when the plasmid DNA-lipopeptide complex is fully bound, no free plasmid DNA can be detected in the agarose gel and the fluorescence is not displayed.

As shown in Figure 1, lipopeptides can bind to DNA. After DNA binds to lipopeptides, the molecular weight increases and it cannot enter the gel and is retained in the loading well. The gel wells show green color due to the binding of nucleic acid and dye. When the concentration of lipopeptides increases, its binding to DNA will become tighter and the molecular weight will increase. When the concentration of lipopeptides is 8 mg/mL, lipopeptides are completely bound to DNA.

Example 2

Study on the protective ability of lipopeptides on DNA

Taking advantage of the property of DNase I that can degrade DNA, we explored whether the binding of lipopeptides to DNA can resist the degradation of DNase I. Each lipopeptide was mixed with 10 μL of 0.13 mg/mL pDNA at the highest concentration (10 mg/mL) by manual pipetting until uniform. After standing at room temperature, a tube was taken and DNase I was added to it and placed in a 37°C water bath for 30 minutes. Then an equal volume of DNA extract was added. The components of the DNA extract are phenol, chloroform and isoamyl alcohol, which can inactivate DNase I and destroy the binding of lipopeptides to nucleic acids, so that the nucleic acids no longer stay in the gel holes in the agarose gel and run out of the swimming lane.

As shown in Figure 2, lane a is a system of lipopeptide or its solvent, anhydrous ethanol, and pDNA; lane b is a system of directly using a DNA extract to destroy the binding of lipopeptide and nucleic acid, thereby releasing the nucleic acid; lane c is a system of extracting nucleic acid from a lipopeptide and nucleic acid binding solution after being treated with DNase I and then extracted with a DNA extract. Naked DNA will be degraded by DNase I and present diffuse bands in agarose gel, such as the control ethanol mixed with pDNA, which will be degraded by DNase I and present diffuse bands.

As shown in Figure 3, lane c has an intact plasmid band, indicating that the lipopeptide can protect DNA from DNase I degradation.

Example 3

Investigation of the ability of nucleic acid-lipopeptide complexes to release nucleic acids in response to pH changes

Take 40 μL of 10 mg/mL lipopeptide and 40 μL of 0.13 mg/mL DNA fragment dissolved in 50 mM sodium acetate solution at pH 4 and mix them manually and blow repeatedly to mix. The mixture is even and not layered, which means the mixing is complete. Incubate on ice for 2 hours. Divide the mixture evenly into 5 tubes, 10 μL per tube. One tube does not adjust the pH, and the remaining tubes are adjusted to pH 3-4, 6-7, 7-8 and 8-9 with hydrochloric acid and sodium hydroxide, respectively. Because the system contains ethanol, take 6× DNA loading buffer and glycerol in a volume ratio of 1:2, take 10 μL and mix with 10 μL of lipopeptide nucleic acid mixture and add it to the well, and check by agarose gel electrophoresis.

As shown in Figure 4, when the pH of the system is not adjusted, the lipopeptide nucleic acid particles will be retained in the gel pores and the gel pores are red. When the pH of the system is adjusted to 3-4 and 5-6, there is no release of nucleic acid; when the pH of the system is adjusted to 6-7, 7-8 and 8-9, the nucleic acid is gradually released; when the pH of the system is adjusted to 8-9, most of the nucleic acid has been released. The theoretical isoelectric point of the polypeptide part of the lipopeptide is 6.92. When the pH of the solution is less than the isoelectric point, the lipopeptide is positively charged and can bind to the negatively charged nucleic acid. When the pH of the solution is greater than the isoelectric point, the positive charge of the lipopeptide decreases, and the ability to bind to the nucleic acid becomes weaker, resulting in the release of the nucleic acid.

Example 4

Hemolytic activity assay of lipopeptides

Take 2mL of fresh blood from SPF-grade, 8-week-old female KM mice and add it to a centrifuge tube containing 10mL of Alder's solution purchased from Solebol and mix well. Centrifuge at 300×g for 8min, discard the supernatant, add 10mL of Alder's solution to the precipitate, mix gently, centrifuge at 300×g for 8min, and gently remove the supernatant with a pipette. Repeat the above process several times until the supernatant is no longer red. The obtained red blood cells are prepared into a 2% red blood cell suspension with PBS (2mL red blood cells plus PBS to 100mL). Mix 100μL of 10mg/mL lipopeptide solution and 100μL of 2% red blood cell suspension in equal volumes, bathe at 37℃ for 1h, and centrifuge at 300×g for 2min to remove intact red blood cells and red blood cell fragments. Use an enzyme marker to measure the absorbance value of the sample at 540nm. Distilled water mixed with blood cells is used as a positive control, and PBS mixed with blood cells is used as a negative control.

Hemolysis rate = (OD test - OD negative) / (OD positive - OD negative) × 100%

Note: OD measurement: absorbance value of sample; OD negative: absorbance value of negative control (PBS mixed with blood cells); OD positive: positive control (distilled water mixed with blood cells).

As shown in FIG5 , the hemolytic activity of the lipopeptide is much less than that of water and the solvent ethanol, and has only a low hemolytic activity.

Example 5

Cytotoxicity assay of lipopeptides

Cytotoxicity is detected by colorimetric detection of lactate dehydrogenase released by dead cells. The principle is that when cells die, the cell membrane structure is destroyed and lactate dehydrogenase is released. Under the action of lactate dehydrogenase, NAD ⁺ is reduced to generate NAD ⁺ and a strong colorant formazan, which has an absorption peak at 490nm, and the absorbance is proportional to the activity of lactate dehydrogenase. Therefore, this method can be used to detect cell mortality in cytotoxicity.

Hela cells were plated in 24-well plates, with 10,000 cells per well. Lipopeptides were added to each well at a total amount of 150 nmol, 75 nmol, 37.5 nmol, 18.75 nmol, and 9.375 nmol, with three replicates per group. Opti-MEM medium (with 1% serum) was added to 500 μL, and cultured at 37°C and 5% CO2 for 6 hours. 100 μL of LDH release reagent was added to the control wells and cultured for another 1 hour. The cell culture plate was then centrifuged at 400×g for 5 minutes using a multi-well plate centrifuge. 120 μL of the supernatant from each well was taken and added to the corresponding wells of a new 96-well plate, and 60 μL of LDH detection working solution was added to each well. Mix well and incubate at room temperature in the dark for 30 minutes. The absorbance was then measured at 490 nm. Dual wavelength measurement was performed using 600 nm as the reference wavelength.

Calculate (the absorbance of each group should be subtracted from the absorbance of the background blank control well) cytotoxicity or mortality (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum cell enzyme activity - absorbance of sample control well) × 100. As shown in Figure 6, the cytotoxicity of lipopeptide at different concentration gradients was lower than that of commercial lipid Lipofectamine 2000.

Example 6

Study on the efficiency of transfection of Hela cells by mixing lipopeptide and pDNA at different volume ratios

Hela cells were seeded into 24-well plates at a density of 105 cells/well and cultured overnight until the cell density reached more than 70%. Lipopeptide and plasmid DNA were mixed with plasmid according to the ratio in Table 2.

Table 2

Rinse the cells 3 times with PBS. Add 450 μL of Opti-MEM medium to each well. Dilute the nucleic acid and lipopeptide combination solution with Opti-MEM medium and add it to the cells, adding 50 μL to each well to ensure that 1 μg of nucleic acid is added to each well. After adding the mixture to the cells, shake it evenly with a microplate oscillator and place it in an incubator at 37°C and 5% CO2 for ₇₂ hours. Observe and take pictures every 24 hours.

As shown in Figure 7, when the volume ratio of lipopeptide to nucleic acid was 1:3, the number of fluorescent cells was relatively the largest, and the nucleic acid transfection efficiency was relatively the highest, which was the optimal mixing volume ratio. The reference group was Lipofectamine 2000.

Example 7

Study on the efficiency of transfection of Hela cells by mixing lipopeptides and pDNA at different mass ratios

Hela cells were inoculated into 24-well plates at a density of 105 cells/well and cultured overnight until the cell density reached more than 70%. Take 11.55 μL of 10 mg/mL lipopeptide and manually mix with 11.5 μL of 0.13 mg/mL, 0.26 mg/mL, 0.39 mg/mL, 0.78 mg/mL, and 1 mg/mL pGreenpuro plasmids (the mass ratio of lipopeptide to nucleic acid is 76.9, 38.5, 25.6, 12.8, and 10, respectively). Rinse the cells 3 times with PBS. Add 450 μL of Opti-MEM medium to each well. Take 30.8 μL of the combination of nucleic acid and lipopeptide, add 119.2 μL of Opti-MEM medium and add to the cells, add 50 μL to each well, and ensure that 1 μg of nucleic acid is added to each well. After the mixture was added to the cells, it was shaken evenly with a microplate oscillator and placed in an incubator at 37°C _and 5% CO2 for a further 72 h. The cells were observed and photographed every 24 h using a fluorescence microscope.

As shown in Figure 8, the optimal mixing mass ratio of lipopeptide to nucleic acid is 25.6. The reference group is Lipofectamine 2000.

Example 8

Study on the efficiency of transfection of Hela cells by lipopeptide mixed with nucleic acid under different pH solvent conditions

Hela cells were seeded into 24-well plates at a density of 105 cells/well and cultured overnight until the cell density reached more than 70%. Lipopeptide (10 mg/mL) and nucleic acid solution (0.13 mg/mL) in Table 3 were mixed into a mixed solution at a volume ratio of 1:3.

Table 3

Rinse the cells 3 times with PBS. Add 450 μL of Opti-MEM medium to each well. Take 30.8 μL of the nucleic acid and lipopeptide combination solution, add 119.2 μL of Opti-MEM medium and add to the cells, add 50 μL to each well, and ensure that 1 μg of nucleic acid is added to each well. After the mixture is added to the cells, shake it evenly with a microplate oscillator, place it in an incubator at 37°C _and 5% CO2 and continue to culture for 72 hours. Observe with a fluorescence microscope and take pictures every 24 hours.

As shown in Figures 9 to 11, the best pH for each nucleic acid transfection effect is different: (1) pH 5 nucleic acid buffer has the best transfection effect on pDNA; (2) For the same double-stranded DNA, the double-stranded (ds) DNA fragment encoding the GFP gene expression cassette has a better transfection effect in the nucleic acid buffer at pH 4; (3) For the single-stranded (ss) DNA encoding the GPP gene expression cassette, the nucleic acid buffer at pH 6.8 has a better transfection effect. (4) The best transfection effect for the mixed transfection of mRNA and lipopeptide is the nucleic acid buffer at pH 6. The reference group is Lipofectamine 2000.

Example 9

Study on the efficiency of Hela cells transfected with different nucleic acids by different mixing methods

Hela cells were inoculated into 24-well plates at a density of 105 cells/well and cultured overnight until the cell density reached more than 70%. 10 mg/mL lipopeptide was mixed with pDNA, dsDNA, ssDNA, and mRNA (0.13 mg/mL) by microfluidics at a volume ratio of 1:3, with a flow rate of 4.5 mL/min for lipopeptide and 13.5 mL/min for nucleic acid. The cells were rinsed 3 times with PBS. 450 μL of Opti-MEM medium was added to each well. 30.8 μL of the nucleic acid and lipopeptide combination solution was taken, 119.2 μL of Opti-MEM medium was added to the cells, and 50 μL was added to each well to ensure that 1 μg of nucleic acid was added to each well. After the mixture was added to the cells, it was shaken with a microplate oscillator and placed in an incubator at 37°C and 5% CO ₂ for 72 hours. It was observed and photographed every 24 hours under a fluorescence microscope.

As shown in Figure 12, the transfection effect of microfluidic mixed particles is better than that of manual mixing, and most of the particles obtained by microfluidic mixing are smaller than those obtained by manual mixing. Specific operation of manual mixing of small systems: In a 0.2mL centrifuge tube, use a pipette to absorb a certain volume of lipopeptide and nucleic acid, mix them according to a volume ratio of 1:3, and gently blow and suck the pipette several times to mix the system evenly. The reference group is Lipofectamine 2000.

Example 10

Investigation of the efficiency of lipopeptide transfection into various cells

Hela, DC2.4, C2C12, Raw264.7, and 293T cells were inoculated into 24-well plates at a density of 105 cells/well and cultured overnight until the cell density reached more than 70%. 10 mg/mL lipopeptide was mixed with pDNA and mRNA (0.13 mg/mL) in a volume ratio of 1:3 using microfluidics, with a flow rate of 4.5 mL/min for lipopeptide and 13.5 mL/min for nucleic acid. The cells were rinsed 3 times with PBS. 450 μL of Opti-MEM medium was added to each well. 30.8 μL of the nucleic acid and lipopeptide combination solution was taken, 119.2 μL of Opti-MEM medium was added to the cells, and 50 μL was added to each well to ensure that 1 μg of nucleic acid was added to each well. After the mixture was added to the cells, it was shaken with a microplate oscillator and placed in an incubator at 37°C and 5% CO ₂ for 72 hours. It was observed and photographed every 24 hours using a fluorescence microscope.

As shown in Figures 13 to 16, the reporter gene can be successfully expressed in all cells, and the best effect is achieved in 293T cells. The reference group is Lipofectamine 2000.

In summary, the lipopeptide vector provided by the present invention can bind to nucleic acid and protect DNA from being degraded by DNase I; it has basically no hemolytic activity and its cytotoxicity is lower than that of Lipofectamine 2000; it can efficiently deliver nucleic acids in a variety of cells, and can deliver a variety of nucleic acids including mRNA, plasmid DNA, double-stranded DNA fragments, single-stranded DNA fragments, etc., and has the potential to become a non-viral vector in nucleic acid vaccines and gene therapy.

The above implementation modes are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention should be defined by the claims.

Claims (8)

1. A cyclic lipopeptide vector for encapsulating a nucleic acid drug, comprising: the cyclic lipopeptide carrier comprises a cyclic lactone structure formed by heptapeptide R ₁R₂R₂R₃R₁R₂R₂ and beta-hydroxy fatty acid with a carbon chain length of 6-44, and the structural general formula is as follows:

Wherein R ₁ is a basic amino acid comprising one of histidine His, lysine Lys or arginine Arg, R ₂ is one of hydrophobic amino acids leucine Leu, isoleucine Ile or valine Val, and R ₃ is one of any amino acid; n is an integer of 2 to 40;

the positively charged basic amino acid of the cyclic lipopeptide is combined with the negatively charged nucleic acid drug by electrostatic interaction to form a cyclic lipopeptide-nucleic acid particle.

2. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 1, wherein: in the cyclic lipopeptid, the carbon chain length of the beta-hydroxy fatty acid is 13-19, and n is an integer of 9-15.

3. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 1, wherein: the nucleic acid drug comprises mRNA, plasmid DNA, double-stranded DNA fragments or single-stranded DNA fragments.

4. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 3, wherein: the molar ratio of the cyclic lipopeptide to the mRNA is 5-20: 1, a step of; the mass ratio of the cyclic lipopeptide to the plasmid DNA is 20-30: 1, a step of; the mass ratio of the cyclic lipopeptide to the double-stranded DNA is 20-30: 1, a step of; the mass ratio of the cyclic lipopeptide to the single-stranded DNA is 20-30: 1.

5. A cyclic lipopeptide carrier for encapsulating a nucleic acid drug according to any one of claims 1 to 4, wherein the preparation method of the cyclic lipopeptide complex solution encapsulating a nucleic acid drug is as follows: dissolving cyclic lipopeptide in ethanol to prepare a solution with the concentration of 5-10 mg/mL, and dissolving nucleic acid medicine in a 50mM sodium acetate solution with the pH of 4-7 to obtain a nucleic acid solution with the concentration of 0.13-1.0 mg/mL; mixing the cyclic lipopeptide and the nucleic acid according to a certain molar ratio or mass ratio, wherein the mixing volume ratio of the cyclic lipopeptide to the nucleic acid is 1:1 to 1:3, the mixing flow rate ratio of the cyclic lipopeptide to the nucleic acid is 1:3, standing for 5-30 min after the mixing is completed, and obtaining the cyclic lipopeptide composite solution for coating the nucleic acid medicine.

6. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 5, wherein: the mixing mode of the cyclic lipopeptide and the nucleic acid is manual mixing or microfluidic mixing.

7. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 5, wherein: the pH response range of the cyclic lipopeptide composite solution for coating the nucleic acid medicine is 6-9, and the coated nucleic acid is released when the pH is 6-9.

8. A cyclic lipopeptide vector for encapsulating a nucleic acid agent according to claim 1, wherein: the cyclic lipopeptide vector is mainly applied to mRNA vaccines, mRNA medicaments, DNA vaccines, DNA medicaments or nucleic acid transfection.