A cyclic lipopeptide carrier for encapsulating nucleic acid drugs

Abstract

The present invention discloses a cyclic lipopeptide carrier for encapsulating nucleic acid drugs, comprising 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: #imgabs0# wherein 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, and R3 is one of any amino acid; n is an integer of 2 to 40; the positively charged basic amino acids in the cyclic lipopeptide bind to the negatively charged nucleic acid drug through electrostatic interaction to form cyclic lipopeptide-nucleic acid particles. The cyclic lipopeptide carrier for encapsulating nucleic acid drugs provided by the present invention has high safety, can effectively protect nucleic acid drugs, improves their stability, and has good transfection efficiency for a variety of cells, and has broad application prospects in the field of nucleic acid drug delivery.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a cyclic lipopeptide carrier for encapsulating nucleic acid drugs. Background Art

[0002] Gene therapy and nucleic acid vaccines involve the introduction of exogenous genetic material into cells. However, the effective delivery of genetic material and its efficient transport to target cells is hindered by numerous extracellular and intracellular barriers. Nucleic acids are negatively charged, hydrophilic substances have difficulty penetrating cell membranes, and nucleic acids are easily degraded by nucleases after entering cells, making gene delivery vehicles particularly important. Currently, the most commonly used nanoliposomes are based on cationic lipids, but the cationic lipids currently available for nucleic acid delivery are almost all patented by foreign pharmaceutical companies.

[0003] Cellular uptake of nonviral vectors primarily occurs through endocytosis, most commonly through the use of nanoliposomes. Nucleic acids are conjugated to cationic lipids to form nanoliposomes (LNPs), which then enter cells. These LNPs are then internalized by the cell surface and release the nucleic acids (Dries et al. 2012). Because cell membranes are typically negatively charged, while the lipids in the nanoparticles used for nucleic acid delivery are positively charged, electrostatic interactions promote the adsorption and fusion of LNPs with cell membranes; this attraction drives membrane fusion and endocytosis. After entering the cell, the nucleic acid is released from the cationic lipid complex. Anionic lipids in the cell may neutralize the charge of the cationic lipid carrier, disrupting the electrostatic interactions between the lipid carrier and the nucleic acid, thereby facilitating the release of nucleic acids from LNPs (Tarahovsky et al. 2000, Tarahovsky et al. 2004).

[0004] Lipopeptides are compounds of lipids and amino acids. They were first isolated in the 1950s and 1960s, with Bacillus being the most important microorganism producing them. As a type of biosurfactant, lipopeptides have applications in biocontrol, drug delivery, and other fields (Raju et al. 2023).

[0005] Lipids (such as nanoliposomes) and peptides (such as polylysine) can be used as carriers for nucleic acid delivery to cells, but both suffer from instability. For example, polylysine forms irregular coils at a pH of 7, a structure that significantly impacts the properties of bioactive substances. Several studies have found that introducing fatty acids into peptides can increase the stability of the complex and enhance membrane degradation activity (He et al. 2020), thereby enhancing the delivery of nucleic acids into cells. Lipopeptides have also been documented as being useful for drug delivery, for example, using diacylated lysine as a delivery vehicle for the local anesthetic lipopivacaine (Eixeira et al. 2014).

[0006] This 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

[0007] In response to the shortcomings of the existing technology, 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 various cells. It has broad application prospects in the field of nucleic acid drug delivery.

[0008] The technical solution adopted by the present invention to solve the technical problem is: a cyclic lipopeptide carrier for encapsulating nucleic acid drugs, wherein the cyclic lipopeptide carrier comprises a cyclic lactone structure formed by the 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:

[0009]

[0010] wherein 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; and n is an integer from 2 to 40.

[0011] The positively charged basic amino acids in the cyclic lipopeptide and the negatively charged nucleic acid drug are combined through electrostatic interaction to form cyclic lipopeptide-nucleic acid particles.

[0012] 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.

[0013] Furthermore, the nucleic acid drug includes mRNA, plasmid DNA, double-stranded DNA fragments or single-stranded DNA fragments.

[0014] 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.

[0015] The preparation method of the cyclic lipopeptide complex solution encapsulating a nucleic acid drug is as follows: first, the cyclic lipopeptide is dissolved in ethanol to prepare a solution of 5-10 mg / mL, and the nucleic acid drug is dissolved 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; the cyclic lipopeptide and the nucleic acid are mixed according to a certain molar ratio or mass ratio, the mixing volume ratio of the cyclic lipopeptide to the nucleic acid is 1:1-1:3, and the mixing flow rate ratio of the cyclic lipopeptide to the nucleic acid is 1:3; after the mixing is completed, the solution is allowed to stand for 5-30 minutes to obtain the cyclic lipopeptide complex solution encapsulating the nucleic acid drug.

[0016] Furthermore, the cyclic lipopeptide and the nucleic acid are mixed by manual mixing or microfluidic mixing.

[0017] Furthermore, the pH response range of the cyclic lipopeptide complex solution encapsulating the nucleic acid drug is 6 to 9, and the encapsulated nucleic acid is released when the pH is 6 to 9.

[0018] 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.

[0019] The present invention has the beneficial effects of: Compared with the prior art, the cyclic lipopeptide carrier for encapsulating nucleic acid drugs provided by the present invention can tightly bind to the nucleic acid drug, protecting the nucleic acid drug from degradation by DNase I, enter cells through endocytosis, and release the encapsulated nucleic acid drug in response to changes in pH, achieving intracellular release. The cyclic lipopeptide of the present invention has low hemolytic activity and, as shown in cytotoxicity experiments, has low cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the electrophoresis diagram of the mixture of lipopeptides and nucleic acids at different concentrations;

[0021] Figure 2 This is the electrophoresis diagram of the ethanol and pDNA mixture group to investigate the DNA protection ability of lipopeptides;

[0022] Figure 3 This is the electrophoresis diagram of the lipopeptide and pDNA mixed group to investigate the DNA protection ability of the lipopeptide;

[0023] Figure 4 This is the electrophoresis diagram of the lipopeptide nucleic acid complex in response to pH changes;

[0024] Figure 5 is a bar graph of the relative hemolytic activity of lipopeptides;

[0025] Figure 6is a histogram of cytotoxicity of lipopeptides;

[0026] Figure 7 is the transfection efficiency of Hela cells mixed with different volume ratios of lipopeptide and pDNA;

[0027] Figure 8 is the transfection efficiency of lipopeptide and pDNA mixed at different mass ratios in Hela cells;

[0028] Figure 9-11 It is the transfection efficiency of lipopeptides mixed with different nucleic acids under different pH solvent conditions in Hela cells;

[0029] Figure 12 It is the transfection efficiency of different nucleic acids in Hela cells under different mixing methods;

[0030] Figure 13 is the transfection efficiency of lipopeptide in C2C12 cells;

[0031] Figure 14 is the transfection efficiency of lipopeptides in 293T cells;

[0032] Figure 15 is the transfection efficiency of lipopeptide in DC2.4 cells;

[0033] Figure 16 is the transfection efficiency of lipopeptides in Raw264.7 cells. DETAILED DESCRIPTION

[0034] The present invention will be further described below by way of specific examples, which are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0035] The meanings of the English abbreviations involved in the following examples are shown in Table 1.

[0036] Table 1

[0037] English abbreviation Chinese meaning English abbreviation Chinese meaning DNA DNA mRNA messenger RNA DNase I Deoxyribonuclease 1 pDNA Plasmid DNA PBS Phosphate buffered saline KM Kunming LDH Lactate dehydrogenase <![CDATA[NAD + ]]> Nicotinamide adenine dinucleotide ssDNA single-stranded DNA fragments dsDNA double-stranded DNA fragments

[0038] 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, and the mixture was stirred. 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU were added and stirred for 30 min. 3.98 g (0.01 mol) of Trt-histidine was added and the mixture was stirred for 2 h.

[0039] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

[0040] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

[0041] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.17 g (0.01 mol) of valine, stir, and react for 2 h.

[0042] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.17 g (0.01 mol) of valine, stir, and react for 2 h.

[0043] Add 2.06 g (0.01 mol) of DCC and 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.

[0044] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

[0045] Add 2.06 g (0.01 mol) of DCC and 1.15 g of HOSU, stir for 30 min, add 1.31 g (0.01 mol) of leucine, stir, and react for 2 h.

[0046] Sodium hydroxide solution was added to adjust the pH to 10-10.5, stirred for 30 min, and the pH was adjusted to neutral to precipitate a solid.

[0047] The solid was added to 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.

[0048] Hydrochloric acid solution was added to adjust the pH to 2 and stirred for 1 h. The precipitated solid was slurried with methyl tert-butyl ether and dried to obtain 7.47 g of the desired product in a yield of 71%.

[0049] The specific reaction equation is:

[0050]

[0051]

[0052] Example 1

[0053] Study the binding ability of different concentrations of lipopeptides to plasmid DNA

[0054] Lipopeptide at different concentrations of 0, 2, 4, 6, 8, and 10 mg / mL was manually mixed with an equal volume of 0.13 mg / mL DNA and allowed to stand at room temperature for 3 hours. Agarose gel electrophoresis was then performed to observe changes in the brightness of the nucleic acid bands at different ratios to determine the effectiveness of cationic liposome encapsulation of nucleic acids and to select the optimal ratio of nucleic acid-lipopeptide complexes. The principle of the gel retardation assay is that plasmid DNA alone, due to its negative charge, readily migrates through the gel. When the plasmid DNA and lipopeptide are not bound or the binding is incomplete, it readily migrates through the gel, displaying a strong fluorescence intensity. When the plasmid DNA-lipopeptide complex is positively charged and larger in size, its migration through the gel is hindered, resulting in a decrease in the amount of free plasmid DNA detected in the agarose gel and a weakened fluorescence intensity. When the plasmid DNA-lipopeptide complex is fully bound, no free plasmid DNA is detected in the agarose gel, and no fluorescence is displayed.

[0055] like Figure 1 As shown, lipopeptides can bind to DNA. After DNA binds to the lipopeptide, its molecular weight increases, preventing it from entering the gel and instead being retained in the sample wells. The wells display a green color, indicating the binding of nucleic acids to the dye. As the lipopeptide concentration increases, its binding to DNA becomes tighter, and its molecular weight increases. At a lipopeptide concentration of 8 mg / mL, the lipopeptide completely binds to DNA.

[0056] Example 2

[0057] Investigation of the DNA protection ability of lipopeptides

[0058] Taking advantage of the property of DNase I that can degrade DNA, we explored whether the binding of lipopeptides to DNA can resist degradation by DNase I. Each lipopeptide at the highest concentration (10 mg / mL) was mixed with 10 μL of 0.13 mg / mL pDNA by manual pipetting until uniform. After standing at room temperature, one tube was added with DNase I and incubated in a 37°C water bath for 30 minutes. Then, an equal volume of DNA extraction solution was added. The components of the DNA extraction solution are phenol, chloroform, and isoamyl alcohol. It can inactivate DNase I and disrupt the binding of lipopeptides to nucleic acids, so that the nucleic acids no longer remain in the gel pores in the agarose gel and run out of the swimming lane.

[0059] like Figure 2 As shown, lane a shows the lipopeptide or its solvent, anhydrous ethanol, bound to pDNA; lane b shows the lipopeptide-nucleic acid binding system directly disrupted with a DNA extraction solution, releasing the nucleic acid; and lane c shows the nucleic acid extracted from the lipopeptide-nucleic acid binding solution after DNase I treatment. Naked DNA is degraded by DNase I, resulting in diffuse bands in the agarose gel. For example, the control ethanol mixed with pDNA is degraded by DNase I, resulting in diffuse bands.

[0060] like Figure 3 As shown, lane c has an intact plasmid band, indicating that the lipopeptide can protect DNA from degradation by DNase I.

[0061] Example 3

[0062] Investigation of the ability of nucleic acid-lipopeptide complexes to release nucleic acids in response to pH changes

[0063] Manually mix 40 μL of a 10 mg / mL lipopeptide with 40 μL of a 0.13 mg / mL DNA fragment dissolved in 50 mM sodium acetate solution at pH 4 by pipetting repeatedly. Mix thoroughly until the mixture is homogeneous and free of stratification. Incubate on ice for 2 hours. Divide the mixture evenly into five tubes, 10 μL per tube. Leave one tube unadjusted, and adjust the pH of the remaining tubes to 3-4, 6-7, 7-8, and 8-9, respectively, using hydrochloric acid and sodium hydroxide. Because the system contains ethanol, mix 6× DNA loading buffer and glycerol in a 1:2 volume ratio. 10 μL of this mixture, along with 10 μL of the lipopeptide-nucleic acid mixture, is added to the wells and examined by agarose gel electrophoresis.

[0064] like Figure 4 As shown, without adjusting the pH of the system, the lipopeptide nucleic acid particles were retained in the gel pores, and the gel pores were red. When the pH of the system was adjusted to 3-4 and 5-6, no nucleic acid was released. When the pH of the system was adjusted to 6-7, 7-8, and 8-9, the nucleic acids were gradually released. When the pH of the system was adjusted to 8-9, most of the nucleic acids were released. The theoretical isoelectric point of the polypeptide portion of the lipopeptide is 6.92. When the solution pH is below the isoelectric point, the lipopeptide is positively charged and can bind to the negatively charged nucleic acids. When the solution pH is above the isoelectric point, the positive charge of the lipopeptide decreases, and the binding ability with nucleic acids weakens, resulting in the release of nucleic acids.

[0065] Example 4

[0066] Hemolytic activity assay of lipopeptides

[0067] 2 mL of fresh blood from 8-week-old SPF female KM mice was added to a centrifuge tube containing 10 mL of Aldrich's solution (Solarbio). The mixture was centrifuged at 300 × g for 8 minutes. The supernatant was discarded, and the precipitate was added to 10 mL of Aldrich's solution and gently mixed. The mixture was centrifuged again at 300 × g for 8 minutes, and the supernatant was gently removed with a pipette. This process was repeated several times until the supernatant no longer appeared red. The resulting red blood cells were prepared into a 2% red blood cell suspension in PBS (2 mL of red blood cells were added to 100 mL of PBS). Equal volumes of 100 μL of a 10 mg / mL lipopeptide solution and 100 μL of the 2% red blood cell suspension were mixed, incubated in a 37°C water bath for 1 hour, and centrifuged at 300 × g for 2 minutes to remove intact red blood cells and red blood cell debris. The absorbance of the samples at 540 nm was measured using a microplate reader. A mixture of distilled water and blood cells served as a positive control, and a mixture of PBS and blood cells served as a negative control.

[0068] Hemolysis rate = (OD positive - OD negative) / (OD positive - OD negative) × 100%

[0069] Note: OD measurement: absorbance value of the sample; OD negative: absorbance value of the negative control (PBS mixed with blood cells); OD positive: positive control (distilled water mixed with blood cells).

[0070] like Figure 5 As shown, the hemolytic activity of lipopeptides is much smaller than that of water and solvent ethanol, and only has a low hemolytic activity.

[0071] Example 5

[0072] Cytotoxicity assay of lipopeptides

[0073] 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 + Generates NAD through reduction + The strong colorant formazan 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 death rate in cytotoxicity.

[0074] HeLa cells were plated in a 24-well plate, with 10,000 cells per well. Lipopeptide was added at total concentrations of 150 nmol, 75 nmol, 37.5 nmol, 18.75 nmol, and 9.375 nmol per well, with triplicate wells per group. The volume was made up to 500 μL with Opti-MEM medium (supplemented with 1% serum) and incubated at 37°C, 5% CO₂ for 6 h. 100 μL of LDH-releasing reagent was added to the control wells and incubated for an additional 1 h. The cell culture plate was then centrifuged at 400 × g for 5 min in a multiwell plate centrifuge. 120 μL of supernatant from each well was added to the corresponding well of a new 96-well plate, and 60 μL of LDH assay working solution was added to each well. The mixture was mixed and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 490 nm. Dual-wavelength measurements were performed using 600 nm as a reference wavelength.

[0075] 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. The results are as follows Figure 6 As shown, the cytotoxicity of lipopeptides at different concentration gradients was lower than that of the commercial lipid Lipofectamine 2000.

[0076] Example 6

[0077] Investigation of the transfection efficiency of Hela cells with different volume ratios of lipopeptides and pDNA

[0078] HeLa cells were seeded into 24-well plates at a density of 10 5 cells / well and cultured overnight until the cell density reached 70% or higher. Lipopeptide and plasmid DNA were mixed with the plasmid according to the ratios listed in Table 2.

[0079] Table 2

[0080]

[0081] Rinse the cells three times with PBS. Add 450 μL of Opti-MEM medium to each well. Dilute the nucleic acid-lipopeptide conjugate with Opti-MEM medium and add 50 μL to each well, ensuring 1 μg of nucleic acid is added to each well. After adding the mixture to the cells, shake well using a microplate shaker and continue incubating in an incubator at 37°C, 5% CO2 for 72 hours. Observe and photograph the cells every 24 hours using a fluorescence microscope.

[0082] like Figure 7 As shown in the figure, when the volume ratio of lipopeptide to nucleic acid was 1:3, the number of fluorescent cells was relatively the highest and the nucleic acid transfection efficiency was relatively the highest, which is the optimal mixing volume ratio. The reference group was Lipofectamine 2000.

[0083] Example 7

[0084] Investigation of the transfection efficiency of Hela cells with different mass ratios of lipopeptides and pDNA

[0085] 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%. 11.55 μL of 10 mg / mL lipopeptide was manually mixed 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 ratios of lipopeptide to nucleic acid were 76.9, 38.5, 25.6, 12.8, and 10, respectively). The cells were rinsed 3 times with PBS. 450 μL of Opti-MEM medium was added to each well. 30.8 μL of the combined solution of nucleic acid and lipopeptide 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 evenly with a microplate shaker and placed in an incubator at 37°C and 5% CO2 for a further 72 hours. The cells were observed and photographed every 24 hours using a fluorescence microscope.

[0086] like Figure 8 As shown in FIG, the optimal mixing mass ratio of lipopeptide to nucleic acid is 25.6. The reference group is Lipofectamine 2000.

[0087] Example 8

[0088] Study on the efficiency of Hela cell transfection by mixing lipopeptides with nucleic acids under different pH solvent conditions

[0089] HeLa cells were seeded into 24-well plates at a density of 105 cells / well and cultured overnight until the cell density reached 70% or higher. Lipopeptide (10 mg / mL) and the nucleic acid solution (0.13 mg / mL) listed in Table 3 were mixed at a volume ratio of 1:3 to form a mixture.

[0090] Table 3

[0091]

[0092]

[0093]

[0094] Rinse the cells three times with PBS. Add 450 μL of Opti-MEM medium to each well. Take 30.8 μL of the nucleic acid-lipopeptide conjugate solution, add 119.2 μL of Opti-MEM medium, and then add 50 μL to the cells, ensuring 1 μg of nucleic acid is added to each well. After adding the mixture to the cells, shake well using a microplate shaker and continue incubating in an incubator at 37°C and 5% CO2 for 72 hours. Observe and photograph the cells every 24 hours using a fluorescence microscope.

[0095] like Figures 9-11 As shown in the figure, the best pH for each nucleic acid transfection effect is different: (1) pH 5 nucleic acid buffer is the best for pDNA transfection; (2) For double-stranded DNA, the double-stranded (ds) DNA fragment encoding the GFP gene expression cassette is better transfected in a nucleic acid buffer at pH 4; (3) For single-stranded (ss) DNA encoding the GPP gene expression cassette, the nucleic acid buffer at pH 6.8 is better transfected. (4) The best transfection effect for the mixed mRNA and lipopeptide is the nucleic acid buffer at pH 6. The reference group is Lipofectamine 2000.

[0096] Example 9

[0097] Investigation of the efficiency of Hela cells transfected with different nucleic acids by different mixing methods

[0098] 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%. 10 mg / mL lipopeptide was mixed with pDNA, dsDNA, ssDNA, and mRNA (0.13 mg / mL) in a 1:3 volume ratio 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 three 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 added to 119.2 μL of Opti-MEM medium and then added to the cells, with 50 μL added to each well, ensuring 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 shaker and placed in an incubator at 37°C and 5% CO2 for another 72 hours. The cells were observed and photographed every 24 hours using a fluorescence microscope.

[0099] like Figure 12 As shown, microfluidic mixing of particles resulted in better cell transfection than manual mixing, and the majority of particles obtained using microfluidic mixing were smaller in size than those obtained using manual mixing. To perform manual mixing in a small system, pipette a specific volume of lipopeptide and nucleic acid into a 0.2 mL centrifuge tube and mix them in a 1:3 volume ratio. Gently pipette several times to mix the mixture evenly. The reference group was Lipofectamine 2000.

[0100] Example 10

[0101] Investigation of the efficiency of lipopeptide transfection into various cells

[0102] HeLa, DC2.4, C2C12, Raw264.7, and 293T cells were seeded into 24-well plates at a density of 105 cells / well and cultured overnight until the cell density reached 70% or higher. A 10 mg / mL lipopeptide was mixed with pDNA and mRNA (0.13 mg / mL) at a 1:3 ratio using a microfluidic system at a flow rate of 4.5 mL / min for the lipopeptide and 13.5 mL / min for the nucleic acid. The cells were rinsed three times with PBS. 450 μL of Opti-MEM medium was added to each well. 30.8 μL of the nucleic acid-lipopeptide complex was added to 119.2 μL of Opti-MEM medium, and then added to the cells, with 50 μL added to each well, ensuring 1 μg of nucleic acid was added to each well. After the mixture was added to the cells, it was shaken using a microplate shaker and incubated in a 37°C, 5% CO2 incubator for 72 hours. The cells were observed and photographed every 24 hours using a fluorescence microscope.

[0103] like Figures 13-16 As shown, the reporter gene was successfully expressed in all cells, with the best effect in 293T cells. The reference group was Lipofectamine 2000.

[0104] In summary, the lipopeptide vector provided by the present invention can bind to nucleic acids and protect DNA from degradation by DNase I; it has essentially 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, and has the potential to become a non-viral vector for nucleic acid vaccines and gene therapy.

[0105] The above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Ordinary technicians in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention. The scope of patent protection of the present invention should be defined by the claims.

Claims

1. A cyclic lipopeptide carrier for encapsulating a nucleic acid drug, characterized by: The cyclic lipopeptide carrier includes a cyclic lactone structure formed by the heptapeptide R1R2R2R3R1R2R2 and β-hydroxy fatty acid, and its structural formula is as follows: ； In the formula, R1 is histidine His, R2 is leucine Leu, R3 is valine Val; n is 10; The positively charged basic amino acids in the cyclic lipopeptide and the negatively charged nucleic acid drug are combined through electrostatic interaction to form cyclic lipopeptide-nucleic acid particles.

2. A cyclic lipopeptide carrier for encapsulating nucleic acid drugs according to claim 1, characterized in that: The nucleic acid drug includes mRNA, plasmid DNA, double-stranded DNA fragments or single-stranded DNA fragments.

3. A cyclic lipopeptide carrier for encapsulating nucleic acid drugs according to claim 2, characterized in that: 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.

4. A cyclic lipopeptide carrier for encapsulating a nucleic acid drug according to any one of claims 1 to 3, characterized in that: The preparation method of the cyclic lipopeptide complex solution encapsulating a nucleic acid drug is as follows: first, the cyclic lipopeptide is dissolved in ethanol to prepare a solution of 5-10 mg / mL, and the nucleic acid drug is dissolved 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; the cyclic lipopeptide and the nucleic acid are mixed according to a certain molar ratio or mass ratio, the mixing volume ratio of the cyclic lipopeptide to the nucleic acid is 1:1-1:3, and the mixing flow rate ratio of the cyclic lipopeptide to the nucleic acid is 1:

3. After the mixing is completed, the solution is allowed to stand for 5-30 minutes to obtain the cyclic lipopeptide complex solution encapsulating the nucleic acid drug.

5. A cyclic lipopeptide carrier for encapsulating nucleic acid drugs according to claim 4, characterized in that: The cyclic lipopeptide and the nucleic acid are mixed by manual mixing or microfluidic mixing.

6. The cyclic lipopeptide carrier for encapsulating nucleic acid drugs according to claim 4, characterized in that: The pH response range of the cyclic lipopeptide complex solution encapsulating the nucleic acid drug is 6-9, and the encapsulated nucleic acid is released when the pH is 6-9.

7. The cyclic lipopeptide carrier for encapsulating nucleic acid drugs according to claim 1, characterized in that: The cyclic lipopeptide vector is mainly used in mRNA vaccines, mRNA drugs, DNA vaccines, DNA drugs or nucleic acid transfection.

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