Cyclic lipopeptide delivery system

Abstract

The invention discloses a cyclic lipopeptide delivery system. The cyclic lipopeptide delivery system comprises a cyclic lipopeptide carrier and nucleic acid, the head of the cyclic lipopeptide carrier is formed by connecting heptapeptide R1R2R2R3R4R2R2 into a ring through an amido bond, the hydrophobic tail of the cyclic lipopeptide carrier forms an ester bond through fatty acid with the carbon chain length of 6-44 and hydroxyl on head R3 amino acid, and the structural general formula of the cyclic lipopeptide carrier is as follows: # imgabs0 #, R1 is basic amino acid and comprises one of histidine His, lysine Lys or arginine Arg; r2 is hydrophobic amino acid and comprises one of leucine Leu, isoleucine Ile or valine Val; r3 is hydrophilic amino acid and comprises one of tyrosine Tyr, serine Ser and threonine Thr; r4 is one of glutamic acid Glu or glutamine Gln; n is an integer from 5 to 43; the cyclic lipopeptide carrier wraps a nucleic acid drug to form the cyclic lipopeptide-nucleic acid nanoparticles.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to a cyclic lipopeptide delivery system. Background Art

[0002] The engineered non-viral delivery systems capable of achieving effective mRNA delivery are diverse, mainly including lipid nanoparticles (LNP), polymer-based systems, lipid-polymer hybrid nanoparticles, cell-penetrating peptides (CPP), exosomes, cationic nanoemulsions (CNE), etc. Among them, lipid nanoparticles are currently the most widely used mRNA vaccine delivery system in clinical practice. It is a multi-component system composed of ionizable cationic lipids, neutral co-lipids, cholesterol, and polyethylene glycolylated lipids. However, the four-component LNP delivery system has certain application limitations, mainly reflected in: (1) There are many auxiliary components, and the preparation process mostly uses microfluidic technology, making it difficult to scale up industrially; (2) Obvious side effects: Some LNPs containing cationic lipids have been reported in the literature to cause adverse events such as high inflammation and cytotoxicity, and LNPs tend to accumulate in the liver. The polyethylene glycolylated lipid, one of the auxiliary components, can cause complement activation-related pseudoallergy (CARPA). In addition, the clinical research data of the two COVID-19 vaccines BNT162b2 and mRNA-1273 approved by the US FDA also disclosed the adverse reactions of commercial mRNA vaccines, including local injection pain and local or systemic inflammatory reactions (fever, discomfort, etc.), and the relative contributions of mRNA and LNP to these adverse events are still unclear.

[0003] The research on mRNA vaccines in China started relatively late. Analyzing from the patent application situation of the delivery system, the first patent application in China regarding the LNP delivery system for mRNA vaccines was in 2015. Since then, the number of related patent applications has increased year by year and reached the peak in 2021. It can be seen that the development trend of the delivery system in China lags slightly behind the global trend. Looking at the world, the patent barriers of the delivery system have brought a relatively high entry threshold to the research and development of mRNA vaccines and drugs in China. How to break through the iron wall of international delivery system patents and develop new, safe, and efficient nucleic acid delivery systems is a challenge faced by R & D institutions and enterprises in China.

[0004] Lipopeptides are a class of amphiphilic molecules formed by a hydrophilic peptide chain head and a hydrophobic fatty acid chain tail. Cyclic lipopeptides produced by Bacillus subtilis metabolism, such as surfactin, fengycin, and iturin, not only have various biological activities such as antibacterial, anti-tumor, and lipid-lowering effects, but can also self-assemble into nano-micelle structures in aqueous solutions, thus having broad application prospects in the agricultural and pharmaceutical fields.

[0005] In the prior art, as disclosed in the invention application with the application number 202410888049.9, a cyclic lipopeptide carrier for encapsulating nucleic acid drugs is disclosed, including a cyclic lactone structure formed by a heptapeptide R 1 R 2 R 2 R 3 R 1 R 2 R 2 and a β-hydroxy fatty acid with a carbon chain length of 6-44, where R 1 is a basic amino acid, including one of histidine (His), lysine (Lys), or arginine (Arg), R 2 is a hydrophobic amino acid, one of leucine (Leu), isoleucine (Ile), or valine (Val), R 3 is one of any amino acids; n is an integer from 2 to 40; the positively charged basic amino acid in the cyclic lipopeptide binds to the negatively charged nucleic acid drug through electrostatic interaction to form a cyclic lipopeptide-nucleic acid particle. Although the cyclic lipopeptide prepared by this technology can play a certain protective role on nucleic acid drugs, its preparation process is relatively complex and the production cost is relatively high. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a cyclic lipopeptide delivery system, including a cyclic lipopeptide carrier and the encapsulated nucleic acid. 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, having broad application prospects in the field of nucleic acid drug delivery.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a cyclic lipopeptide delivery system, characterized in that: the delivery system includes a cyclic lipopeptide carrier and nucleic acid encapsulated by the cyclic lipopeptide carrier; the head of the cyclic lipopeptide carrier is formed into a ring by amide bonds with a heptapeptide R 1 R 2 R 2 R 3 R 4 R 2 R 2 The hydrophobic tail of the cyclic lipopeptide carrier is formed by a fatty acid with a carbon chain length of 6-44 and the head R3 The hydroxyl group on the amino acid forms an ester bond, and the general structural formula of the cyclic lipopeptide carrier is as follows:

[0008]

[0009] In the formula, R 1 is a basic amino acid, including one of histidine (His), lysine (Lys), or arginine (Arg); preferably lysine (Lys); R 2 is a hydrophobic amino acid, including one of leucine (Leu), isoleucine (Ile), or valine (Val); preferably leucine (Leu); R 3 is a hydrophilic amino acid, including one of tyrosine (Tyr), serine (Ser), or threonine (Thr); preferably tyrosine (Tyr); R 4 is one of glutamic acid (Glu) or glutamine (Gln); preferably glutamic acid (Glu); n is an integer from 5 to 43; n is preferably 14;

[0010] The cyclic lipopeptide carrier encapsulates the nucleic acid drug to form a cyclic lipopeptide-nucleic acid nanoparticle.

[0011] Furthermore, the cyclic lipopeptide carrier forms a micelle structure in an aqueous solution, encapsulates the nucleic acid drug, and forms a cyclic lipopeptide-nucleic acid nanoparticle.

[0012] Furthermore, the nucleic acid drug includes RNA, plasmid, double-stranded DNA fragment, or single-stranded DNA fragment.

[0013] Furthermore, the mass ratio of the cyclic lipopeptide carrier to the nucleic acid is 15 - 70:1.

[0014] Furthermore, the preparation method of the cyclic lipopeptide complex solution encapsulating the nucleic acid drug is as follows: First, dissolve the cyclic lipopeptide in ethanol to prepare a solution with a concentration of 5 - 10 mg / mL, dissolve the nucleic acid drug in a 10 - 50 mM sodium acetate solution with a pH of 5.0 - 7.5 to obtain a nucleic acid solution with a concentration of 0.01 - 1.0 mg / mL, mix the cyclic lipopeptide and the nucleic acid according to the mass ratio, the mixing volume ratio is 1:1 - 1:3, and after mixing, let it stand for 5 - 120 min to obtain the cyclic lipopeptide complex solution encapsulating the nucleic acid drug.

[0015] Furthermore, the cyclic lipopeptide carrier is mainly applied to mRNA vaccines, mRNA drugs, DNA vaccines, DNA drugs, or nucleic acid transfection. It can transfect a variety of animal cells, including but not limited to cervical cancer Hela cells, DC2.4 cells, C2C12 cells, and Raw264.7 cells.

[0016] The beneficial effects of the present invention are as follows: Compared with the prior art, in the cyclic lipopeptide delivery system provided by the present invention, the cyclic lipopeptide carrier does not bind to nucleic acids through electrostatic interaction. Instead, the cyclic lipopeptide carrier spontaneously forms micelles to encapsulate nucleic acid molecules under certain pH conditions, making the surface of the formed complex particles carry negative charges, greatly reducing the toxicity of the material (most of the existing LNP technologies are cationic liposomes, and the particle surface carries positive charges, with high cytotoxicity). Moreover, the complex particles have uniform particle sizes, stable structures, and the ability to effectively deliver nucleic acids into cells and animals. Compared with the prior art, for the cyclic lipopeptide provided by the present invention, the amino acids of the cyclic peptide form a ring through amide bonds. Compared with the synthesis process of the cyclic lipopeptide disclosed in the invention application with the application number 202410888049.9 (the amino acids of the cyclic peptide form a ring through lactone bonds with β-hydroxy fatty acids), the ring-forming difficulty is reduced, the process is more simplified, and the cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 HPLC purity analysis chromatogram of the cyclic lipopeptide prepared in Example 1.

[0018] Figure 2 First-level mass spectrum of the cyclic lipopeptide prepared in Example 1.

[0019] Figure 3 Transfection effects of different forms of nucleic acids by the cyclic lipopeptide in Hela cells.

[0020] Figure 4 Transfection effects of the mixture of cyclic lipopeptide and nucleic acid in Raw264.7 cells under different pH conditions.

[0021] Figure 5 Transfection effects of different mass ratios of cyclic lipopeptide and nucleic acid in Raw264.7 cells.

[0022] Figure 6 Transfection effects of different mixing volume ratios of cyclic lipopeptide and nucleic acid in Raw264.7 cells.

[0023] Figure 7 Transmission electron microscopy morphological map of the cyclic lipopeptide@GFP-mRNA complex particles, (a) cyclic lipopeptide empty system; (b) cyclic lipopeptide@GFP-mRNA complex particles.

[0024] Figure 8 Transfection effects of the cyclic lipopeptide@GFP-mRNA complex in different cells.

[0025] Figure 9 Toxicity evaluation of the cyclic lipopeptide on Raw264.7 cells.

[0026] Figure 10In vivo transfection results of cyclic lipopeptide @Luc-mRNA in mice. Detailed implementation manners

[0027] Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0028] Example 1

[0029] Synthesis and purification of cyclic lipopeptide

[0030] The cyclic lipopeptide was synthesized by solid-phase peptide synthesis. The synthetic technical route is as follows:

[0031]

[0032] The specific implementation steps are as follows:

[0033] (1) Add 5 g of Fmoc-Leu-OH, 100 mL of DCM and 1.79 g of HOSu to a 250 mL three-necked flask. While stirring under a nitrogen atmosphere, cool the temperature to 0 - 5 °C, and slowly add a solution of 3.21 g of DCC in 10 mL of dichloromethane dropwise. After the addition, stir for 1 h. After completion, filter by suction. Add 2.57 g of leucine methyl ester hydrochloride to the filtrate, and while stirring, control the temperature at 15 - 25 °C and slowly add 4.57 g of diisopropylethylamine dropwise. Stir and react for 2 h. Wash the reaction solution twice with 50 mL of water each time. After separating the layers, dry the organic phase with anhydrous sodium sulfate, and then evaporate the solvent under reduced pressure to obtain 6.32 g of Fmoc-Leu-Leu-Me;

[0034] (2) Dissolve 6.32 g of Fmoc-Leu-Leu-Me in 30 mL of DMF, add 4.5 mL of diethylamine while stirring, and stir and react at room temperature for 2 h. After completion, distill off diethylamine and DMF under reduced pressure at 30 °C. Add 65 mL of n-hexane and 6.5 mL of ethyl acetate to the residue in the flask, stir for 30 min, filter by suction and then dry to obtain 3.22 g of Leu-Leu-Me;

[0035] (3) Add 5.43 g of fluorenylmethoxycarbonyl-L-glutamic acid 5-tert-butyl ester, 110 mL of DCM and 1.62 g of HOSu to a 250 mL three-necked flask. While stirring under a nitrogen atmosphere, cool the temperature to 0 - 5 °C, and slowly add a solution of 2.90 g of DCC in 10 mL of dichloromethane dropwise. After the addition, stir for 1 h. After completion, filter by suction. Add 3.22 g of Leu-Leu-Me to the filtrate, and while stirring, control the temperature at 15 - 25 °C and slowly add 2.47 g of diisopropylethylamine dropwise. Stir and react for 2 h. Wash the reaction solution twice with 50 mL of water each time. After separating the layers, dry the organic phase with anhydrous sodium sulfate, and then evaporate the solvent under reduced pressure to obtain 7.86 g of Fmoc-Glu(γ-t-Bu)-Leu-Leu-Me;

[0036] (4) Dissolve 7.86 g of Fmoc-Glu(γ-t-Bu)-Leu-Leu-Me in 40 mL of DMF. While stirring, add 6 mL of diethylamine, and stir the reaction at room temperature for 2 h. After completion, distill off diethylamine and DMF under reduced pressure at 30 °C. Add 80 mL of n-hexane and 8 mL of ethyl acetate to the residue in the flask, stir for 1 h, filter by suction and dry to obtain 4.82 g of Glu(γ-t-Bu)-Leu-Leu-Me;

[0037] (5) Add 3 g of myristic acid, 60 mL of DCM and 1.66 g of HOSu to a 250 mL three-necked flask. While stirring and purging with nitrogen, cool the temperature to 0 - 5 °C, and dropwise add 3.0 g of DCC / 10 mL dichloromethane solution. After addition, stir for 1 h. After completion, filter by suction. Add 5.30 g of Fmoc-Tyr-OH to the filtrate, and while stirring, control the temperature at 0 - 5 °C and dropwise add 2.12 g of diisopropylethylamine. After addition, stir the reaction at 10 - 20 °C for 2 h. Cool the reaction solution to 0 - 5 °C, add 1.66 g of HOSu, dropwise add 3.0 g of DCC / 10 mL dichloromethane solution. After addition, stir for 1 h. After completion, filter by suction. Add 4.82 g of Glu(γ-t-Bu)-Leu-Leu-Me to the filtrate, and while stirring, control the temperature at 15 - 25 °C and dropwise add 2.12 g of diisopropylethylamine, and stir the reaction for 2 h. Wash the reaction solution twice with 50 mL of water each time. After layering, dry the organic phase with anhydrous sodium sulfate, and distill off the solvent under reduced pressure to obtain 9.0 g of Fmoc-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0038] (6) Dissolve 9.0 g of Fmoc-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me in 50 mL of DMF. While stirring, add 7.5 mL of diethylamine, and stir the reaction at room temperature for 2 h. After completion, distill off diethylamine and DMF under reduced pressure at 30 °C. Add 90 mL of n-hexane and 13.5 mL of ethyl acetate to the residue in the flask, stir for 1 h, filter by suction and dry to obtain 6.5 g of Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0039] (7) Dissolve 6.5 g of Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me in 65 mL of DMF, add 4 g of Fmoc-Leu-OSu, cool to 0 - 10 °C and dropwise add 1.54 g of diisopropylethylamine. After addition, react at room temperature for 2 h. After completion, add 10 mL of diethylamine to the reaction solution, and stir the reaction at room temperature for 2 h. After completion, distill off diethylamine and DMF under reduced pressure at 30 °C. Add 80 mL of n-hexane and 12 mL of ethyl acetate to the residue in the flask, stir for 1 h, filter by suction and dry to obtain 5.92 g of Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0040] (8) Dissolve 5.92 g of Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me in 60 mL of DMF, add 3.2 g of Fmoc-Leu-OSu, cool down to 0 - 10 °C and dropwise add 1.24 g of diisopropylethylamine. After addition, react at room temperature for 2 h. After completion, add 9 mL of diethylamine to the reaction solution and stir at room temperature for 2 h. After completion, distill off diethylamine and DMF under reduced pressure at 30 °C. Add 90 mL of n-hexane and 15 mL of ethyl acetate to the residue in the flask, stir for 1 h, filter by suction and dry to obtain 5.64 g of Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0041] (9) Add 3.2 g of Fmoc-Lys(Boc)-OH, 50 mL of DCM and 0.87 g of HOSu to a three-necked flask, cool down to 0 - 5 °C while stirring under a nitrogen atmosphere, and dropwise add 1.55 g of DCC / 10 mL dichloromethane solution. After addition, stir for 1 h. After completion, filter by suction, concentrate the filtrate under reduced pressure to remove the solvent and dissolve it in 80 mL of DMF. Add 5.64 g of Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me, control the temperature at 15 - 25 °C and dropwise add 1.32 g of diisopropylethylamine while stirring, and stir for 2 h. After completion, add 1 mL to the reaction solution, stir for 15 min, add 12 mL of diethylamine, and stir for 1 - 2 h. After completion, distill off the solvent under reduced pressure. Add 80 mL of n-hexane and 20 mL of ethyl acetate to the residue in the flask, stir for 1 h, filter by suction, and dry the solid to obtain 5.83 g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0042] (10) Add 5.83 g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me to 30 mL of acetonitrile, dropwise add 0.2 g of lithium hydroxide / 10 mL aqueous solution while stirring at room temperature. After addition, stir for 3 h. Concentrate acetonitrile by distillation under reduced pressure at room temperature, add 30 mL of water, adjust the pH to 5.5 by dropwise adding 5N acetic acid solution while stirring, a large amount of solid precipitates, filter by suction, and dry the solid to obtain 5.21 g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-COOH;

[0043] (11) 5.21 g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-COOH was added to 52 mL of DMF, 1.2 g of DCC was added, and the mixture was stirred at 0 - 10 °C for 2 h. After completion, it was filtered, and the filtrate was concentrated under reduced pressure at room temperature to remove about 25 mL of DMF. 25 mL of water was added dropwise, and the mixture was stirred for 1 h to crystallize. It was filtered by suction and dried to obtain 4 g of Cyclo(Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu);

[0044] (12) 4 g of Cyclo(Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu) was added to 30 mL of trifluoroacetic acid, and the mixture was stirred at room temperature for 2 h. After completion, the reaction solution was added dropwise to 80 mL of methyl tert-butyl ether to crystallize, and the white solid was obtained by suction filtration and dried in vacuo to obtain the crude product of Cyclo(Lys-Leu-Leu-Tyr(Myristyl)-Glu-Leu-Leu);

[0045] (13) The crude product of Cyclo(Lys-Leu-Leu-Tyr(Myristyl)-Glu-Leu-Leu) was purified by preparative purification and freeze-dried to obtain 1.5 g of a sample with a purity of 93.26%. The HPLC purity analysis chromatogram is as Figure 1 shown. The sample was analyzed using a high-resolution mass spectrometer, and the molecular weight was 1082.7342 Da. The first-order mass spectrum is as Figure 2 shown.

[0046] Example 2

[0047] Verify the effects of cyclic lipopeptides on delivering mRNA, plasmid DNA, and single-stranded DNA at the cellular level

[0048] The cyclic lipopeptide was dissolved in absolute ethanol to prepare a 10 mg / mL solution; green fluorescent protein GFP-mRNA, GFP-plasmid DNA, and GFP-single-stranded DNA were respectively diluted with sodium acetate buffer at pH 6.0 to 0.13 mg / mL. The cyclic lipopeptide solution was added to different forms of nucleic acid solutions (volume ratio 1:3), and the mixture was pipetted several times and mixed evenly, and then incubated at room temperature for 30 min. Hela cells were seeded into 24-well plates at 1.0×10 5 cells / well and placed in an incubator at 37 °C and 5% CO 2After incubation for 24 h, replace the cell culture medium in the 24-well plate with Opti-MEM medium. Add the cyclic lipopeptide@nucleic acid complexes containing 1 μg of GFP-mRNA, GFP-plasmid DNA, and GFP-single-stranded DNA into 450 μL of Opti-MEM medium respectively, and observe GFP expression after 48 h. The results of cell transfection with the cyclic lipopeptide@nucleic acid complexes are as shown in Figure 3 shown that the cyclic lipopeptide achieved efficient delivery of different forms of nucleic acids in cells.

[0049] Example 3

[0050] Effect of the mixing pH of cyclic lipopeptide and nucleic acid on the nucleic acid delivery effect

[0051] Dissolve the cyclic lipopeptide in absolute ethanol to prepare a 10 mg / mL solution; dilute green fluorescent protein GFP-mRNA with sodium acetate buffer solutions at pH 5.0, pH 6.0, pH 6.5, pH 7.0, and pH 7.5 to 0.13 mg / mL respectively. Add the cyclic lipopeptide solution to the nucleic acid solutions at different pH values (volume ratio 1:3), pipette several times to mix evenly, and let it stand at room temperature for incubation for 30 min. Seed Raw264.7 cells into a 24-well plate at 1.0×10 5 cells / well, and place it in an incubator at 37 °C and 5% CO 2 After incubation for 24 h, replace the cell culture medium in the 24-well plate with Opti-MEM medium. Add the cyclic lipopeptide@GFP-mRNA complexes with different pH values containing 1 μg of GFP-mRNA into 450 μL of Opti-MEM medium respectively, and observe GFP expression after 24 h. The results of cell transfection are as shown in Figure 4 shown that the cyclic lipopeptide and mRNA were mixed under different pH conditions, and efficient delivery of mRNA was achieved in cells.

[0052] Example 4

[0053] Effect of the mass ratio of cyclic lipopeptide and nucleic acid on the nucleic acid delivery effect

[0054] Dissolve the cyclic lipopeptide in absolute ethanol to prepare solutions at 6.5 mg / mL, 10 mg / mL, 13 mg / mL, 19.5 mg / mL, and 26 mg / mL respectively; dilute green fluorescent protein GFP-mRNA with sodium acetate buffer solution at pH 6.5 to 0.13 mg / mL. According to the mass ratio of the cyclic lipopeptide solution to the nucleic acid solution of 16.6:1, 25.6:1, 33.3:1, 50:1, 66.6:1, add the cyclic lipopeptide solution to the nucleic acid solution (volume ratio 1:3), pipette several times to mix evenly, and let it stand at room temperature for incubation for 30 min. Seed Raw264.7 cells into a 24-well plate at 1.0×105 Cells were seeded into 24-well plates at a density of 2 1 cell / well and incubated in an incubator at 37 °C with 5% CO Figure 5 for 24 h. The cell culture medium in the 24-well plates was replaced with Opti-MEM medium. Circular lipopeptide@GFP-mRNA complexes with different mass ratios containing 1 μg of GFP-mRNA were added to 450 μL of Opti-MEM medium respectively, and GFP expression was observed after 24 h. The results of cell transfection are shown as

[0055] Example 5

[0056] Effect of the mixing volume ratio of circular lipopeptide and nucleic acid on nucleic acid delivery efficiency

[0057] The circular lipopeptide was dissolved in absolute ethanol to prepare a 10 mg / mL solution; green fluorescent protein GFP-mRNA was diluted with sodium acetate buffer at pH 6.5 to 0.39 mg / mL and 0.13 mg / mL respectively. The circular lipopeptide solution was mixed with the nucleic acid solution at volume ratios of 1:1 and 1:3 respectively, and pipetted several times to mix evenly, and then incubated at room temperature for 30 min. Raw264.7 cells were seeded into 24-well plates at a density of 1.0×10 5 cells / well and incubated in an incubator at 37 °C with 5% CO 2 for 24 h. The cell culture medium in the 24-well plates was replaced with Opti-MEM medium. Circular lipopeptide@GFP-mRNA complexes with different mass ratios containing 1 μg of GFP-mRNA were added to 450 μL of Opti-MEM medium respectively, and GFP expression was observed after 24 h. The results of cell transfection are shown as Figure 6 shown. The circular lipopeptide and mRNA were mixed at different volume ratios, and effective delivery of mRNA was achieved in cells.

[0058] Example 6

[0059] Preparation and characterization of circular lipopeptide@GFP-mRNA complexes

[0060] Dissolve the cyclic lipopeptide in absolute ethanol to prepare a 10 mg / mL solution; dilute the green fluorescent protein GFP-mRNA with sodium acetate buffer at pH 6.5 to make 0.13 mg / mL. According to the mass ratio of the cyclic lipopeptide solution to the nucleic acid solution of 25.6:1, take the cyclic lipopeptide solution and add it to the nucleic acid solution (volume ratio 1:3), pipette several times to mix evenly, then let it stand at room temperature for incubation for 30 min, and use sodium acetate buffer (pH 6.5) for dialysis to remove ethanol molecules. The molecular weight cut-off of the dialysis bag is 1 kDa, and the cyclic lipopeptide@GFP-mRNA complex is obtained.

[0061] At 25 °C, use a particle size potentiometer (Zetasizer Lab) to characterize the particle size, polydispersity index (PDI), and Zeta potential of the above complex by dynamic light scattering (DLS) method. As shown in Table 1, the particle size of the complex particles is in the nanometer range, the polydispersity index PDI is less than 0.3, the particle surface carries a negative charge and the system is stable, which can be used for nucleic acid delivery in vivo and in vitro.

[0062] Table 1 Particle size, PDI, and Zeta potential of the cyclic lipopeptide@GFP-mRNA complex

[0063] Composite particle Particle size (nm) Polydispersity index PDI Zeta potential (mV) Cyclic lipopeptide@GFP-mRNA 630.4 0.1268 -36.02

[0064] The morphology of the complex particles was observed by a transmission electron microscope (Transmission election microscope, TEM) at 80.0 kV, as Figure 7 shown, the cyclic lipopeptide can encapsulate GFP-mRNA to form a nearly regular spherical vesicle-like structure.

[0065] Example 7

[0066] Verify the effect of cyclic lipopeptide in delivering mRNA at the cellular level

[0067] Seed Hela cells, Raw264.7 cells, C2C12 cells, and DC2.4 cells into 24-well plates at 1.0×10 5 cells / well respectively, place them in an incubator at 37 °C and 5% CO 2 for incubation for 24 h. Replace the cell culture medium in the 24-well plates with Opti-MEM medium. Add the cyclic lipopeptide@GFP-mRNA complex containing 1 μg GFP-mRNA to 450 μL of Opti-MEM medium respectively, and observe GFP expression after 24 h. The cell transfection results of the cyclic lipopeptide@GFP-mRNA complex are as Figure 8 shown, the cyclic lipopeptide@GFP-mRNA complex achieved effective delivery of mRNA in different cells and had no obvious cytotoxicity.

[0068] Example 8

[0069] Cytotoxicity Detection of Cyclic Lipopeptides

[0070] The destruction of the cell membrane structure caused by apoptosis or necrosis of cells will lead to the release of enzymes in the cytoplasm into the culture medium, including lactate dehydrogenase (LDH) with relatively stable enzyme activity. By detecting the activity of LDH released from ruptured cells into the culture medium, quantitative analysis of cytotoxicity is achieved.

[0071] Specifically, Raw264.7 cells were seeded into 24-well plates at a density of 1.0×10 5 cells / well, placed in an incubator at 37°C and 5% CO 2 and incubated for 24 h, then cultured in complete medium containing serum for 24 h. The cells were rinsed with PBS and fresh medium was replaced. The cyclic lipopeptide was dissolved in absolute ethanol to prepare a solution with a certain concentration, and then diluted to 25, 50, 100, and 200 μg / mL with absolute ethanol respectively to form a lipopeptide dissolution system; another ethanol solution for dissolving the lipopeptide was mixed with NaAc buffer at a volume ratio of 1:3 to form a lipopeptide empty vector system, and diluted to 25, 50, 100, and 200 μg / mL with Opti-MEM medium respectively; the lipopeptide dissolution system and empty vector system with different lipopeptide concentrations were added to the cells as experimental groups, with a dose of 12.5, 25, 50, and 100 μg per well. The cell group without lipopeptide system stimulation was used as the maximum enzyme activity control group, Opti-MEM medium was used as the blank control, and the solution of absolute ethanol mixed with NaAc buffer at a volume ratio of 1:3 was used as the sample control. The cells were further incubated at 37°C and 5% CO 2 for 24 h. Then, 120 μL of the supernatant was centrifuged and transferred to a new 96-well cell plate, 60 μL of lactate dehydrogenase (LDH) working solution was added, and the mixture was incubated at room temperature in the dark for 30 min. The absorbance was measured at 490 nm. The absorbance values of each group were subtracted by the absorbance value of the blank control group, and the cell death rate was calculated according to the following formula:

[0072]

[0073] The detection results are as Figure 9 shown. The cell death rate of the lipopeptide empty vector system experimental group was less than 15% within the dose range of 12.5 - 100 μg per well, and the cell death rate of the dissolution system experimental group was slightly higher, ranging from 15% to 20% within the dose range of 12.5 - 50 μg per well. Generally, evaluated from the safety aspect at the cellular level, cyclic lipopeptides can be used as safe nucleic acid delivery vectors within a certain dose range.

[0074] Example 9

[0075] Verification of the effect of cyclic lipopeptide on delivering mRNA at the animal level

[0076] A complex was prepared by wrapping luciferase mRNA (Luc-mRNA) with cyclic lipopeptide and dialyzed with NaAc buffer. Female BABL / c mice at 6 - 8 weeks old were immunized with the complex by intramuscular injection and tail vein injection at a dose of 10 μg mRNA / mouse. At 6 h and 24 h after injection, 15 mg / mL D-luciferin sodium salt solution (200 μL per mouse at a dose of 10 μL / g body weight) was intraperitoneally injected into the mice. The fluorescence in the mice was detected using a small animal fluorescence microscopy imaging system to explore whether cyclic lipopeptide could effectively deliver mRNA in animals. The detection results of the fluorescence microscopy imaging system are as Figure 10 shown. Whether the cyclic lipopeptide@Luc-mRNA complex was injected via the tail vein or intramuscularly, the fluorescence effect was good 6 h after injection, indicating that cyclic lipopeptide has the ability to deliver mRNA in animals. In addition, during the whole experiment, the mice were in good health and no deaths occurred, further proving that cyclic lipopeptide has good safety in animals at a certain injection dose.

[0077] In summary, the present invention provides a cyclic lipopeptide molecule that can bind to nucleic acids to form nanoparticles for delivering nucleic acid molecules into cells or animals. Through experimental verification, the complex particles formed by the binding of the cyclic lipopeptide and nucleic acids have uniform particle sizes and stable structures, have certain transfection abilities both in vivo and in vitro, and have no obvious cytotoxicity.

[0078] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can also 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 shall be defined by the claims.

Claims

1. A cyclic lipopeptide delivery system, characterized in that: The delivery system comprises a cyclic lipopeptide vector and a nucleic acid wrapped by the cyclic lipopeptide vector; the head of the cyclic lipopeptide vector is formed by connecting the seven peptides R1R2R2R3R4R2R2 to form a ring through an amide bond, and the hydrophobic tail of the cyclic lipopeptide vector is formed by an ester bond between a fatty acid with a carbon chain length of 6 to 44 and a hydroxyl group on the amino acid R3 of the head. The general structural formula of the cyclic lipopeptide vector is as follows: In the formula, R1 is a basic amino acid, including one of histidine His, lysine Lys or arginine Arg; R2 is a hydrophobic amino acid, including one of leucine Leu, isoleucine Ile or valine Val; R3 is a hydrophilic amino acid, including one of tyrosine Tyr, serine Ser or threonine Thr; R4 is one of glutamic acid Glu or glutamine Gln; n is an integer of 5 to 43; The cyclic lipopeptide carrier encapsulates the nucleic acid drug to form cyclic lipopeptide-nucleic acid nanoparticles.

2. A cyclic lipopeptide delivery system according to claim 1, characterized in that: The cyclic lipopeptide carrier forms a micro-micelle structure in an aqueous solution, encapsulates the nucleic acid drug, and forms cyclic lipopeptide-nucleic acid nanoparticles.

3. A cyclic lipopeptide delivery system according to claim 1, characterized in that: The nucleic acid drug includes RNA, plasmid, double-stranded DNA fragment or single-stranded DNA fragment.

4. A cyclic lipopeptide delivery system according to claim 1, characterized in that: The mass ratio of the cyclic lipopeptide carrier to the nucleic acid is 15-70:

1.

5. A cyclic lipopeptide delivery system according to any one of claims 1 to 4, characterized in that: The preparation method of the cyclic lipopeptide complex solution encapsulating nucleic acid drugs is as follows: firstly dissolve the cyclic lipopeptide in ethanol to prepare a 5-10 mg / mL solution, dissolve the nucleic acid drug in a 10-50 mM sodium acetate solution with a pH of 5.0-7.5 to obtain a nucleic acid solution with a concentration of 0.01-1.0 mg / mL, mix the cyclic lipopeptide and the nucleic acid according to a mass ratio with a mixing volume ratio of 1:1-1:3, let stand for 5-120 minutes after mixing, and obtain the cyclic lipopeptide complex solution encapsulating the nucleic acid drug.

6. A cyclic lipopeptide delivery system 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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