A cyclic lipopeptide delivery system

Abstract

This invention discloses a cyclic lipopeptide delivery system, comprising a cyclic lipopeptide carrier and nucleic acid; the head of the cyclic lipopeptide carrier is composed of heptapeptides R1R2R2R3R4R2R2 linked together by amide bonds to form a ring, and the hydrophobic tail of the cyclic lipopeptide carrier is composed of a fatty acid with a carbon chain length of 6-44 forming an ester bond with the hydroxyl group on the head amino acid R3, with the following general structural formula: where 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 glutamate (Glu) or glutamine (Gln); n is an integer from 5 to 43; the cyclic lipopeptide carrier encapsulates the nucleic acid drug to form cyclic lipopeptide-nucleic acid nanoparticles.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically to a cyclic lipopeptide delivery system. Background Technology

[0002] Engineered non-viral delivery systems capable of effectively delivering mRNA are diverse, mainly including lipid nanoparticles (LNPs), polymer-based systems, lipid-polymer hybrid nanoparticles, cell-penetrating peptides (CPPs), exosomes, and cationic nanoemulsions (CNEs). Among these, lipid nanoparticles are currently the most widely used mRNA vaccine delivery system in clinical practice. They are multi-component systems composed of ionizable cationic lipids, neutral auxiliary lipids, cholesterol, and polyethylene glycol-modified lipids. However, the four-component LNP delivery system has certain limitations in application, mainly in the following aspects: (1) There are many auxiliary components, and the preparation process mostly adopts microfluidic technology, which makes it difficult to scale up industrially; (2) Significant side effects: Some LNPs containing cationic lipids have been reported to cause adverse events such as high inflammation and cytotoxicity. Moreover, LNPs are prone to accumulate in the liver, and polyethylene glycol-modified lipids, 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 have also disclosed adverse reactions of commercial mRNA vaccines, including local injection pain and local or systemic inflammatory reactions (fever, discomfort, etc.), while the relative contributions of mRNA and LNP to these adverse events are still unclear.

[0003] Research on mRNA vaccines in my country started relatively late. Analysis of patent applications for delivery systems shows that my country's first patent application for an mRNA vaccine LNP delivery system was filed in 2015. The number of related patent applications has increased year by year since then, reaching its peak in 2021. This indicates that China's development of delivery systems lags slightly behind the global trend. Globally, patent barriers for delivery systems pose a high entry barrier for my country's mRNA vaccine and drug development. Breaking through the international patent barriers for delivery systems and developing novel, safe, and efficient nucleic acid delivery systems is a challenge facing Chinese research institutions and enterprises.

[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 surfactantin, fengycin, and iturin, not only possess various biological activities including antibacterial, antitumor, and lipid-lowering effects, but can also self-assemble into nanomicelle structures in aqueous solutions, thus showing broad application prospects in agriculture and medicine.

[0005] In the prior art, for example, invention application No. 202410888049.9 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 6-44, wherein 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); and R3 is any amino acid; 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 cyclic lipopeptide-nucleic acid particles. Although the cyclic lipopeptide prepared by this technology can provide some protection for nucleic acid drugs, its preparation process is relatively complex and the production cost is high. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a cyclic lipopeptide delivery system, comprising a cyclic lipopeptide carrier and encapsulated nucleic acid. This cyclic lipopeptide carrier has high safety, can effectively protect nucleic acid drugs, improve their stability, and has good transfection efficiency for various cell types, thus having broad application prospects in the field of nucleic acid drug delivery.

[0007] The technical solution adopted by this 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 by heptapeptides R1R2R2R3R4R2R2 linked into a ring by amide bonds, and the hydrophobic tail of the cyclic lipopeptide carrier is formed by a fatty acid with a carbon chain length of 6-44 forming an ester bond with the hydroxyl group on the head R3 amino acid; the general structural formula of the cyclic lipopeptide carrier is as follows:

[0008]

[0009] In the formula, R1 is a basic amino acid, including histidine (His), lysine (Lys), or arginine (Arg); preferably lysine (Lys); R2 is a hydrophobic amino acid, including leucine (Leu), isoleucine (Ile), or valine (Val); preferably leucine (Leu); R3 is a hydrophilic amino acid, including tyrosine (Tyr), serine (Ser), or threonine (Thr); preferably tyrosine (Tyr); R4 is glutamate (Glu) or glutamine (Gln); preferably glutamate (Glu); n is an integer from 5 to 43; n is preferably 14;

[0010] The cyclic lipopeptide carrier encapsulates the nucleic acid drug, forming cyclic lipopeptide-nucleic acid nanoparticles.

[0011] Furthermore, the cyclic lipopeptide carrier forms a micromicelle structure in aqueous solution, encapsulating the nucleic acid drug to form cyclic lipopeptide-nucleic acid nanoparticles.

[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 nucleic acid is 15–70:1.

[0014] Furthermore, the preparation method of the cyclic lipopeptide complex solution encapsulating nucleic acid drugs is as follows: First, the cyclic lipopeptide is dissolved in ethanol to prepare a solution of 5-10 mg / mL. The nucleic acid drug is dissolved 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. The cyclic lipopeptide and nucleic acid are mixed according to a mass ratio of 1:1 to 1:3. After mixing, the mixture is allowed to stand for 5-120 min to obtain the cyclic lipopeptide complex solution encapsulating nucleic acid drugs.

[0015] Furthermore, the cyclic lipopeptide carrier is mainly used in 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 this invention are as follows: Compared with the prior art, in the cyclic lipopeptide delivery system provided by this invention, the cyclic lipopeptide carrier and nucleic acid are not bound by electrostatic interaction, but rather the cyclic lipopeptide carrier spontaneously forms micromicelles to encapsulate nucleic acid molecules under certain pH conditions, so that the surface of the formed complex particles carries a negative charge, which greatly reduces the toxicity of the material (existing LNP technology is mostly cationic liposomes, the particle surface carries a positive charge, and has high cytotoxicity). Moreover, the complex particles have uniform particle size, stable structure, and the ability to effectively deliver nucleic acids to cells and animals. Compared with the prior art, the cyclic lipopeptide provided by this invention has amino acids that are cyclically linked by amide bonds. Compared with the cyclic lipopeptide synthesis process disclosed in application No. 202410888049.9 (the amino acids of the cyclic peptide are cyclically linked by β-hydroxy fatty acids through lactone bonds), the difficulty of cyclization is reduced, the process is simplified, and the cost is lower. Attached Figure Description

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

[0018] Figure 2 The image shows the primary mass spectrum of the cyclic lipopeptide prepared in Example 1.

[0019] Figure 3 The transfection effect of cyclic lipopeptides on different forms of nucleic acids in HeLa cells.

[0020] Figure 4 The transfection effect of cyclic lipopeptides and nucleic acids mixed under different pH conditions in Raw264.7 cells.

[0021] Figure 5 The transfection effects of different mass ratios of cyclic lipopeptides and nucleic acids in Raw264.7 cells were investigated.

[0022] Figure 6 The transfection effects of different mixed volume ratios of cyclic lipopeptides and nucleic acids in Raw264.7 cells were investigated.

[0023] Figure 7 Transmission electron microscopy (TEM) images of cyclic lipopeptide@GFP-mRNA complex particles: (a) cyclic lipopeptide empty vector system; (b) cyclic lipopeptide@GFP-mRNA complex particles.

[0024] Figure 8 The transfection effect of the cyclic lipopeptide@GFP-mRNA complex in different cells.

[0025] Figure 9 To evaluate the toxicity of cyclic lipopeptides to Raw264.7 cells.

[0026] Figure 10The results show the in vivo transfection of cyclic lipopeptide@Luc-mRNA in mice. Detailed Implementation

[0027] Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of this invention shall fall within the scope of this invention.

[0028] Example 1

[0029] Synthesis and purification of cyclic lipopeptides

[0030] Cyclic lipopeptides were synthesized using a liquid-phase polypeptide synthesis method. The synthetic route is as follows:

[0031]

[0032] The specific implementation steps are as follows:

[0033] (1) Add 5g Fmoc-Leu-OH, 100mL DCM and 1.79g HOSu to a 250mL three-necked flask, stir under nitrogen and cool to 0-5℃, add 3.21g DCC / 10mL dichloromethane solution dropwise, and stir for 1h after the addition is complete. After the addition is complete, filter, add 2.57g leucine methyl ester hydrochloride to the filtrate, and add 4.57g diisopropyl ethylamine dropwise under stirring and controlled temperature of 15-25℃, and stir for 2h. Wash the reaction solution twice with water, 50mL each time, dry the organic phase with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 6.32g Fmoc-Leu-Leu-Me;

[0034] (2) Dissolve 6.32g of Fmoc-Leu-Leu-Me in 30mL of DMF, add 4.5mL of diethylamine while stirring, and stir at room temperature for 2h. After the reaction is complete, diethylamine and DMF are distilled off under reduced pressure at 30℃. Add 65mL of n-hexane and 6.5mL of ethyl acetate to the residue in the flask, stir for 30min, filter and dry to obtain 3.22g of Leu-Leu-Me;

[0035] (3) Add 5.43 g of fluorenemethoxycarbonyl-L-glutamic acid-5-tert-butyl ester, 110 mL of DCM and 1.62 g of HOSu to a 250 mL three-necked flask, stir under nitrogen and cool to 0-5 °C, then add 2.90 g of DCC / 10 mL of dichloromethane solution dropwise, stirring for 1 h after the addition is complete. After the addition is complete, filter the solution, add 3.22 g of Leu-Leu-Me to the filtrate, and add 2.47 g of diisopropylethylamine dropwise under stirring and controlled temperature of 15-25 °C, stirring for 2 h. Wash the reaction solution twice with water, 50 mL each time, 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 7.86g of Fmoc-Glu(γ-t-Bu)-Leu-Leu-Me in 40mL of DMF, add 6mL of diethylamine while stirring, and stir at room temperature for 2h. After the reaction is complete, diethylamine and DMF are distilled off under reduced pressure at 30℃. Add 80mL of n-hexane and 8mL of ethyl acetate to the residue in the flask, stir for 1h, filter and dry to obtain 4.82g of Glu(γ-t-Bu)-Leu-Leu-Me;

[0037] (5) Add 3g myristic acid, 60mL DCM, and 1.66g HOSu to a 250mL three-necked flask. Purge with nitrogen, stir, and cool to 0-5℃. Add 3.0g DCC / 10mL dichloromethane solution dropwise, stirring for 1h after the addition is complete. After filtration, add 5.30g Fmoc-Tyr-OH to the filtrate. Add 2.12g diisopropylethylamine dropwise at 0-5℃ with stirring, stirring at 10-20℃ for 2h after the addition is complete. Cool the reaction solution to 0-5℃, add 1.66g HOSu, and add 3.0g DCC / 10mL dichloromethane solution dropwise, stirring for 1h after the addition is complete. After filtration, add 4.82g Glu(γ-t-Bu)-Leu-Leu-Me to the filtrate. Add 2.12g diisopropylethylamine dropwise at 15-25℃ with stirring, stirring for 2h after the reaction is complete. The reaction solution was washed twice with 50 mL of water each time. After drying the organic phase with anhydrous sodium sulfate, the solvent was evaporated under reduced pressure to obtain 9.0 g of Fmoc-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me.

[0038] (6) 9.0 g of Fmoc-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me was dissolved in 50 mL of DMF, and 7.5 mL of diethylamine was added with stirring. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the diethylamine and DMF were distilled off under reduced pressure at 30 °C. 90 mL of n-hexane and 13.5 mL of ethyl acetate were added to the residue in the flask, and the mixture was stirred for 1 h. After filtration and drying, 6.5 g of Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me was obtained.

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

[0040] (8) Dissolve 5.92g of Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me in 60mL of DMF, add 3.2g of Fmoc-Leu-OSu, cool to 0-10℃ and add 1.24g of diisopropylethylamine dropwise, and react at room temperature for 2h after the addition is complete. After the reaction is complete, add 9mL of diethylamine to the reaction solution and stir at room temperature for 2h. After the reaction is complete, diethylamine and DMF are distilled off under reduced pressure at 30℃, and 90mL of n-hexane and 15mL of ethyl acetate are added to the residue in the flask, stirred for 1h, filtered and dried to obtain 5.64g of Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me;

[0041] (9) Add 3.2g Fmoc-Lys(Boc)-OH, 50mL DCM, and 0.87g HOSu to a three-necked flask. Purge with nitrogen, stir, and cool to 0-5℃. Add 1.55g DCC / 10mL dichloromethane solution dropwise, stirring for 1h after the addition is complete. After the addition is complete, filter the solution, concentrate the filtrate under reduced pressure to remove the solvent, dissolve it in 80mL DMF, add 5.64g Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me, and add 1.32g diisopropylethylamine dropwise while stirring and controlling the temperature at 15-25℃. Stir for 2h. After the reaction is complete, add 1mL of the reaction solution, stir for 15min, add 12mL diethylamine, and stir for 1-2h. After the solvent was removed by vacuum distillation, 80 mL of n-hexane and 20 mL of ethyl acetate were added to the residue in the flask, stirred for 1 h and filtered. After drying the solid, 5.83 g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me was obtained.

[0042] (10) 5.83g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-Me was added to 30mL of acetonitrile. 0.2g of lithium hydroxide / 10mL aqueous solution was added dropwise while stirring at room temperature. After the addition was complete, the reaction was stirred for 3h. The acetonitrile was concentrated by vacuum distillation at room temperature. 30mL of water was added, and 5N acetic acid solution was added dropwise while stirring to adjust the pH to 5.5. A large amount of solid precipitated. The solid was filtered and dried to obtain 5.21g of Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu-COOH.

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

[0044] (12) 4g of Cyclo(Lys(Boc)-Leu-Leu-Tyr(Myristyl)-Glu(γ-t-Bu)-Leu-Leu) was added to 30mL of trifluoroacetic acid and stirred at room temperature for 2h. After the reaction was completed, the reaction solution was added dropwise to 80mL of methyl tert-butyl ether to crystallize. The crystals were filtered to obtain a white solid, which was dried under vacuum to obtain crude Cyclo(Lys-Leu-Leu-Tyr(Myristyl)-Glu-Leu-Leu).

[0045] (13) Crude Cyclo (Lys-Leu-Leu-Tyr(Myristyl)-Glu-Leu-Leu) was prepared and purified, and lyophilized to obtain 1.5g of sample with a purity of 93.26%. The HPLC purity analysis chromatogram is shown below. Figure 1 As shown, the sample was analyzed using high-resolution mass spectrometry, and the molecular weight was 1082.7342 Da. The first-order mass spectrum is shown below. Figure 2 As shown.

[0046] Example 2

[0047] Validating the efficacy of cyclic lipopeptides in delivering mRNA, plasmid DNA, and single-stranded DNA at the cellular level.

[0048] Circular lipopeptides were dissolved in anhydrous ethanol to prepare a 10 mg / mL solution. Green fluorescent protein (GFP) mRNA, GFP plasmid DNA, and GFP single-stranded DNA were diluted with sodium acetate buffer (pH 6.0) to prepare 0.13 mg / mL solutions. The circular lipopeptide solution was added to each of the different nucleic acid solutions (volume ratio 1:3), and the mixture was thoroughly mixed by pipetting several times. The mixture was then incubated at room temperature for 30 min. HeLa cells were cultured at a concentration of 1.0 × 10⁶ cells / mL. 5Cells / well were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The cell culture medium in the 24-well plates was then replaced with Opti-MEM medium. A cyclic lipopeptide@nucleic acid complex containing 1 μg GFP-mRNA, GFP-plasmid DNA, and GFP-single-stranded DNA was added to 450 μL of Opti-MEM medium, and GFP expression was observed after 48 h. The cell transfection results of the cyclic lipopeptide@nucleic acid complex are shown below. Figure 3 As shown, cyclic lipopeptides enable the efficient delivery of different forms of nucleic acids in cells.

[0049] Example 3

[0050] The effect of mixed pH of cyclic lipopeptides and nucleic acids on nucleic acid delivery efficiency

[0051] The cyclic lipopeptide was dissolved in anhydrous ethanol to prepare a 10 mg / mL solution. Green fluorescent protein (GFP) mRNA was diluted with sodium acetate buffer at pH 5.0, 6.0, 6.5, 7.0, and 7.5 to prepare 0.13 mg / mL solutions. The cyclic lipopeptide solution was added to nucleic acid solutions at different pH values ​​(volume ratio 1:3), and mixed thoroughly by pipetting several times. The mixture was then incubated at room temperature for 30 min. Raw264.7 cells were then cultured at a density of 1.0 × 10⁻⁶ cells / mL. 5 Cells / well were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The cell culture medium in the 24-well plates was then replaced with Opti-MEM medium. Cyclic lipopeptide@GFP-mRNA complexes containing 1 μg of GFP-mRNA at different pH values ​​were added to 450 μL of Opti-MEM medium. GFP expression was observed after 24 h. Cell transfection results are shown below. Figure 4 As shown, cyclic lipopeptides and mRNA were mixed under different pH conditions, and both achieved efficient mRNA delivery in cells.

[0052] Example 4

[0053] The effect of the mass ratio of cyclic lipopeptides to nucleic acids on nucleic acid delivery efficiency

[0054] Cyclic lipopeptides were dissolved in anhydrous ethanol to prepare solutions with concentrations of 6.5 mg / mL, 10 mg / mL, 13 mg / mL, 19.5 mg / mL, and 26 mg / mL, respectively. Green fluorescent protein (GFP) mRNA was diluted with sodium acetate buffer at pH 6.5 to a concentration of 0.13 mg / mL. Cyclic lipopeptide solutions were added to nucleic acid solutions at mass ratios of 16.6:1, 25.6:1, 33.3:1, 50:1, and 66.6:1 (volume ratio 1:3). The solutions were mixed thoroughly by pipetting several times and incubated at room temperature for 30 min. Raw264.7 cells were then cultured at a concentration of 1.0 × 10⁶ cells / mL. 5 Cells / well were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The cell culture medium in the 24-well plates was then replaced with Opti-MEM medium. Circular lipopeptide@GFP-mRNA complexes containing 1 μg GFP-mRNA at different mass ratios were added to 450 μL of Opti-MEM medium. GFP expression was observed after 24 h. Cell transfection results are shown below. Figure 5 As shown, cyclic lipopeptides and mRNA were mixed under different mass ratios, and both achieved efficient mRNA delivery in cells.

[0055] Example 5

[0056] The effect of the mixed volume ratio of cyclic lipopeptides and nucleic acids on nucleic acid delivery efficiency

[0057] The cyclic lipopeptide was dissolved in anhydrous ethanol to prepare a 10 mg / mL solution. Green fluorescent protein (GFP) mRNA was diluted with sodium acetate buffer (pH 6.5) to prepare concentrations of 0.39 mg / mL and 0.13 mg / mL, respectively. The cyclic lipopeptide solutions were mixed with the nucleic acid solutions at volume ratios of 1:1 and 1:3, respectively. The mixtures were thoroughly mixed by pipetting several times and incubated at room temperature for 30 min. Raw264.7 cells were then cultured at a density of 1.0 × 10⁶ cells / mL. 5 Cells / well were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The cell culture medium in the 24-well plates was then replaced with Opti-MEM medium. Circular lipopeptide@GFP-mRNA complexes containing 1 μg GFP-mRNA at different mass ratios were added to 450 μL of Opti-MEM medium. GFP expression was observed after 24 h. Cell transfection results are shown below. Figure 6 As shown, cyclic lipopeptides and mRNA were mixed under different volume ratios, and both achieved efficient mRNA delivery in cells.

[0058] Example 6

[0059] Preparation and characterization of cyclic lipopeptide@GFP-mRNA complex

[0060] The cyclic lipopeptide was dissolved in anhydrous ethanol to prepare a 10 mg / mL solution. The green fluorescent protein (GFP) mRNA was diluted with sodium acetate buffer at pH 6.5 to prepare a 0.13 mg / mL solution. The cyclic lipopeptide solution was added to the nucleic acid solution at a mass ratio of 25.6:1 (volume ratio 1:3). The mixture was pipetted several times to mix thoroughly. The mixture was then incubated at room temperature for 30 min. The ethanol molecules were removed by dialyzing with sodium acetate buffer (pH 6.5). The molecular weight cutoff of the dialysis bag was 1 kDa, thus obtaining the cyclic lipopeptide@GFP-mRNA complex.

[0061] The particle size, polydispersity index (PDI), and zeta potential of the above complex were characterized by dynamic light scattering (DLS) using a particle size potentiometer (Zetasizer Lab) at 25 °C. As shown in Table 1, the particle size of the complex is in the nanometer range, the PDI is less than 0.3, the particle surface carries a negative charge, and the system is stable, making it suitable for in vivo and in vitro nucleic acid delivery.

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

[0063] Complex particles Particle size (nm) Polydispersion Index (PDI) Zeta potential (mV) Cyclic lipopeptide@GFP-mRNA 630.4 0.1268 -36.02

[0064] The morphology of the composite particles was observed using an 80.0 kV transmission electron microscope (TEM), such as... Figure 7 As shown, cyclic lipopeptides can encapsulate GFP-mRNA to form a nearly regular spherical vesicle-like structure.

[0065] Example 7

[0066] Validating the efficacy of cyclic lipopeptides in delivering mRNA at the cellular level

[0067] HeLa cells, Raw264.7 cells, C2C12 cells, and DC2.4 cells were divided into groups of 1.0 × 10⁻⁶ cells. 5 Cells / well were seeded into 24-well plates and incubated at 37°C with 5% CO2 for 24 h. The cell culture medium in the 24-well plates was then replaced with Opti-MEM medium. A cyclic lipopeptide@GFP-mRNA complex containing 1 μg of GFP-mRNA was added to 450 μL of Opti-MEM medium, and GFP expression was observed after 24 h. The cell transfection results of the cyclic lipopeptide@GFP-mRNA complex are shown below. Figure 8 As shown, the cyclic lipopeptide@GFP-mRNA complex achieved efficient mRNA delivery in different cells without significant cytotoxicity.

[0068] Example 8

[0069] Cytotoxicity assay of cyclic lipopeptides

[0070] The disruption of cell membrane structure caused by apoptosis or necrosis leads to the release of enzymes from the cytoplasm into the culture medium, including lactate dehydrogenase (LDH), which has relatively stable enzyme activity. By detecting the activity of LDH released from ruptured cells into the culture medium, quantitative analysis of cytotoxicity can be achieved.

[0071] Specifically, Raw264.7 cells were divided into 1.0 × 10⁻⁶ cells. 5 Cells were seeded per well into 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The cells were then cultured in serum-containing complete medium for 24 hours, washed with PBS, and the medium was replaced with fresh medium. Cyclic lipopeptides were dissolved in anhydrous ethanol to prepare solutions of specific concentrations, which were then diluted with anhydrous ethanol to concentrations of 25, 50, 100, and 200 μg / mL, respectively, to serve as lipopeptide dissolution systems. Separately, the ethanol solution containing the dissolved lipopeptides was mixed with NaAc buffer at a 1:3 volume ratio to prepare an empty lipopeptide carrier system, which was then diluted with Opti-MEM medium to concentrations of 25, 50, 100, and 200 μg / mL, respectively. The lipopeptide dissolution systems and empty carrier systems at different concentrations were added to cells as experimental groups, with each well containing 12.5, 25, 50, and 100 μg. Cells without lipopeptide stimulation served as the maximum enzyme activity control group, Opti-MEM medium as the blank control, and a solution of anhydrous ethanol mixed with NaAc buffer at a 1:3 volume ratio as the sample control. The cells were incubated at 37°C and 5%... Continue incubation under CO2 for 24 h, centrifuge and transfer 120 μL of the supernatant to a new 96-well cell plate. Add 60 μL of lactate dehydrogenase (LDH) working solution and incubate at room temperature in the dark for 30 min. Measure the absorbance at 490 nm. Subtract the absorbance of the blank control group from the absorbance values ​​of each group and calculate the cell death rate using the following formula:

[0072]

[0073] Test results as follows Figure 9 As shown, the cell mortality rate in the cyclic lipopeptide empty vector system was less than 15% within the dosage range of 12.5–100 μg per well, while the cell mortality rate in the lysate system was slightly higher, ranging from 15% to 20% within the dosage range of 12.5–50 μg per well. Overall, from a cellular safety perspective, cyclic lipopeptides can be considered a safe nucleic acid delivery vector within a certain dosage range.

[0074] Example 9

[0075] Validating the efficacy of cyclic lipopeptides in delivering mRNA at the animal level

[0076] A complex was prepared by encapsulating luciferase mRNA (Luc-mRNA) with a cyclic lipopeptide and dialyzing with NaAc buffer. The complex was administered to 6-8 week old female BABL / c mice via intramuscular and tail vein injection at a dose of 10 μg mRNA / mouse. At 6 h and 24 h post-injection, mice were intraperitoneally injected with 15 mg / mL D-luciferin sodium solution (200 μL per mouse, 10 μL / g body weight). Fluorescence in vivo was detected using a small animal fluorescence microscopy system to investigate whether the cyclic lipopeptide can effectively deliver mRNA in animals. The results of the fluorescence microscopy system are shown below. Figure 10 As shown, the cyclic lipopeptide@Luc-mRNA complex exhibited good fluorescence after 6 hours of injection, whether administered via tail vein or intramuscular injection, indicating that the cyclic lipopeptide has the ability to deliver mRNA in animals. Furthermore, the mice remained healthy throughout the experiment, with no deaths, further demonstrating the good safety of the cyclic lipopeptide in animals at certain injection doses.

[0077] In summary, this invention provides a cyclic lipopeptide molecule that can bind to nucleic acids to form nanoparticles for delivering nucleic acid molecules into cells or animals. Experimental verification shows that the complex particles formed by the cyclic lipopeptide and nucleic acid have uniform particle size and stable structure, exhibiting transfection capabilities both in vivo and in vitro, and without significant cytotoxicity.

[0078] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those skilled in the art can 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, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. 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 composed of heptapeptides R1R2R2R3R4R2R2 linked into a ring by amide bonds, and the hydrophobic tail of the cyclic lipopeptide carrier is composed of a fatty acid with a carbon chain length of 14 forming an ester bond with the hydroxyl group on the head amino acid R3. The general structural formula of the cyclic lipopeptide carrier is as follows: ； In the formula, R1 is the basic amino acid lysine (Lys); R2 is the hydrophobic amino acid leucine (Leu); R3 is the hydrophilic amino acid tyrosine (Tyr); R4 is glutamic acid (Glu); and n is 12. The cyclic lipopeptide carrier encapsulates the nucleic acid drug to form cyclic lipopeptide-nucleic acid nanoparticles; the mass ratio of the cyclic lipopeptide carrier to the nucleic acid is 15~70:1; the cyclic lipopeptide carrier and the nucleic acid are not bound by electrostatic interaction, but the cyclic lipopeptide carrier spontaneously forms a micromicelle structure in an aqueous solution under certain pH conditions, encapsulating the nucleic acid drug, so that the surface of the formed cyclic lipopeptide-nucleic acid nanoparticles carries a negative charge.

2. The cyclic lipopeptide delivery system as described in claim 1, characterized in that: The nucleic acid drugs mentioned include RNA, plasmids, double-stranded DNA fragments, or single-stranded DNA fragments.

3. A cyclic lipopeptide delivery system as described in any one of claims 1 to 2, characterized in that, The preparation method of the cyclic lipopeptide complex solution encapsulating nucleic acid drugs is as follows: First, dissolve the cyclic lipopeptide in ethanol to prepare a solution 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 nucleic acid according to the mass ratio of 1:1 to 1:

3. After mixing, let it stand for 5-120 min to obtain the cyclic lipopeptide complex solution encapsulating nucleic acid drugs.

4. The cyclic lipopeptide delivery system as described in claim 1, characterized in that: The cyclic lipopeptide carrier is mainly used in mRNA vaccines, mRNA drugs, DNA vaccines, DNA drugs, or nucleic acid transfection.

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