A lipopeptide, a preparation method thereof, a lipopeptide-based lipid nanoparticle delivery system and applications thereof

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a lipopeptide, a preparation method thereof, a lipopeptide-based lipid nanodelivery system and its applications. The lipopeptide is a compound formed by the ring-opening reaction of a 1,2-epoxide with the partially or fully exposed amino groups of a lysine-histidine polypeptide; the lipopeptide-based lipid nanodelivery system comprises: a lipopeptide, one of cholesterol and cholesterol derivatives, a co-lipid, and a polyethylene glycolylated lipid; the molar ratio of the lipopeptide, cholesterol or cholesterol derivative, co-lipid, and polyethylene glycolylated lipid is (5 to 45):(25 to 40):(25 to 65):(0.8 to 2.5); the above lipopeptide-based lipid nanodelivery system is used for preparing lipopeptide-based lipid nanoparticles. The present invention provides a lipopeptide with a simple preparation process and low toxicity, and the lipopeptide-based lipid nanodelivery system can achieve high-efficiency delivery of various RNA molecules such as siRNA and mRNA in vitro and in vivo.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a lipopeptide, a preparation method thereof, a lipopeptide-based lipid nanoparticle delivery system and applications thereof. Background Art

[0002] RNA therapy, which has emerged in recent years, is a treatment method that can manipulate gene expression or produce therapeutic proteins, and has now been widely studied for the treatment of infectious diseases, cancers, immune diseases, neurodegenerative diseases, etc. The common small interfering RNA (siRNA) therapy reduces the expression of pathogenic genes by mediating the degradation of mRNA (Messenger RNA) through the RNA-induced silencing complex (RISC) in cells to achieve the therapeutic effect. The size of mRNA is much larger than that of siRNA. Based on the fact that mRNA therapy achieves the therapeutic purpose by enhancing the expression of therapeutic proteins in cells, it has been well-known in recent years due to its application in COVID-19 vaccines. However, regardless of the therapeutic mechanism, due to the anionic nature of RNA molecules and their sensitivity to RNases present in blood and tissues, therapeutic RNAs are difficult to effectively enter cells and exert their functions. To overcome the obstacles of RNA delivery in vivo and in vitro, scientists have developed various carrier systems to protect RNAs from degradation so that they can be maximally delivered into target cells to exert therapeutic effects. Among them, the delivery system based on lipid nanoparticles (LNP) has attracted much attention. However, the currently developed LNP systems have the disadvantages of high production cost, high biological toxicity, strong immunogenicity, low endosomal escape efficiency, and difficulty in targeting extrahepatic organs. In addition, the currently developed mature LNP systems are mainly used for the delivery of small molecule nucleic acid siRNA, and the effective delivery of large molecule nucleic acids such as mRNA is still a problem. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a lipopeptide; the second object of the present invention is to provide a preparation method of a lipopeptide; the third object of the present invention is to provide a lipopeptide-based lipid nanoparticle delivery system; the fourth object of the present invention is to provide applications of a lipopeptide-based lipid nanoparticle delivery system.

[0004] To achieve the first object, the technical solution adopted by the present invention is as follows:

[0005] A lipopeptide, which is a compound formed by the ring-opening reaction of a 1,2-epoxide with the partially or fully exposed amino groups of a lysine-histidine polypeptide;

[0006] Among them, the structural formula of the lipopeptide is:

[0007] Or .

[0008] Furthermore, the 1,2-epoxide includes 2 to 22 carbon atoms.

[0009] Furthermore, the 1,2-epoxide is any one of 1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, and 1,2-epoxyoctadecane.

[0010] Furthermore, in the structural formula of the lipopeptide, the value range of x is 0 to 10, the value range of y is 1 to 10, the value range of n is 1 to 5, and the value range of m is 0 to 5.

[0011] Furthermore, in the structural formula of the lipopeptide, R is any one of the following structural formulas, or R is hydrogen and at least one R is any one of the following structural formulas:

[0012] , , ,

[0013] And .

[0014] To achieve the second objective, the technical solution adopted by the present invention is:

[0015] A method for preparing a lipopeptide, used to prepare the lipopeptide, comprising the following steps:

[0016] S101. Under the protection of an inert gas, add a lysine-histidine polypeptide and a 1,2-epoxide to a reaction solvent, and stir at a temperature of 50 °C to 110 °C for 48 h to 72 h to prepare a mixture;

[0017] S102. Purify the mixture to prepare the lipopeptide.

[0018] To achieve the third objective, the technical solution adopted by the present invention is:

[0019] A lipopeptide-based lipid nanodelivery system, comprising the lipopeptide, and also comprising one of cholesterol and cholesterol derivatives, a co-lipid, and a polyethylene glycolated lipid;

[0020] Among them, the molar ratio of lipopeptide, cholesterol or cholesterol derivative, co-lipid, and polyethylene glycolated lipid is (5-45):(25-40):(25-65):(0.8-2.5).

[0021] Furthermore, the co-lipid is at least one of cationic lipid, anionic lipid, phospholipid, and phospholipid derivative;

[0022] The polyethylene glycolated lipid is at least one of PEG-DMG, PEG-C-DMG, and PEG-DSPE.

[0023] To achieve the fourth objective, the technical solution adopted by the present invention is:

[0024] An application of a lipopeptide-based lipid nanodelivery system for preparing lipopeptide-based lipid nanoparticles, where the lipopeptide-based lipid nanoparticles include the lipopeptide-based lipid nanodelivery system and an RNA molecule.

[0025] Furthermore, the RNA molecule is at least one of messenger RNA, small interfering RNA, microRNA, and self-amplifying RNA; the mass ratio of the lipopeptide-based lipid nanodelivery system to the RNA molecule is 40-10:1.

[0026] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0027] The present invention provides a lipopeptide with low toxicity. The preparation process of the lipopeptide is simple. Based on the lipopeptide, the present invention also provides a lipopeptide-based lipid nanodelivery system and its application. The lipopeptide-based lipid nanodelivery system is used to prepare lipopeptide-based lipid nanoparticles, and the lipopeptide-based lipid nanoparticles can achieve high-efficiency delivery of various RNA molecules such as siRNA and mRNA in vitro and in vivo. The in vitro experimental results show that the lipopeptide-based lipid nanoparticles provided by the present invention can deliver RNA molecules in multiple cell lines with high delivery efficiency; the in vivo experimental results show that the lipopeptide-based lipid nanoparticles provided by the present invention have high selectivity for liver, spleen, or lung organs, and thus can achieve the purpose of precisely delivering RNA molecules to multiple organs in vivo.

[0028] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0029] Figure 1 is the transmission electron microscope image (TEM) (scale bar: 50 nm) of C 10 -KH2 / DOTAP provided in Example 2 of the present invention.

[0030] Figure 2The LP-LNPs prepared from the lipopeptide and the co-lipid DOTAP provided in Experimental Example 1 of the present invention, and Lipofectamine 2000 TM Statistical chart of the 24-hour toxicity of A549 cells.

[0031] Figure 3 The LP-LNPs prepared from the lipopeptide and the co-lipid DOPE provided in Experimental Example 1 of the present invention, and Lipofectamine 2000 TM Statistical chart of the 24-hour toxicity of A549 cells.

[0032] Figure 4 The LP-LNPs prepared from the lipopeptide and the co-lipid DSPC provided in Experimental Example 1 of the present invention, and Lipofectamine 2000 TM Statistical chart of the 24-hour toxicity of A549 cells.

[0033] Figure 5 The LP-LNPs prepared from the lipopeptide and the co-lipid 14PA provided in Experimental Example 1 of the present invention, and Lipofectamine 2000 TM Statistical chart of the 24-hour toxicity of A549 cells.

[0034] Figure 6 Statistical chart of the gene silencing efficiency of the LP-LNPs prepared from the lipopeptide and the co-lipid DOTAP provided in Experimental Example 1 of the present invention for delivering siCCNB1 to A549 cells.

[0035] Figure 7 Statistical chart of the gene silencing efficiency of the LP-LNPs prepared from the lipopeptide and the co-lipid DOPE provided in Experimental Example 1 of the present invention for delivering siCCNB1 to A549 cells.

[0036] Figure 8 Statistical chart of the gene silencing efficiency of the LP-LNPs prepared from the lipopeptide and the co-lipid DSPC provided in Experimental Example 1 of the present invention for delivering siCCNB1 to A549 cells.

[0037] Figure 9 Statistical chart of the gene silencing efficiency of the LP-LNPs prepared from the lipopeptide and the co-lipid 14PA provided in Experimental Example 1 of the present invention for delivering siCCNB1 to A549 cells.

[0038] Figure 10 It is C provided in Experimental Example 1 of the present invention 10 Statistical chart of the gene silencing efficiency of C-KH2 / DOTAP for delivering siRNA to A549, Hela, 4T1, and MCF-7 cells.

[0039] Figure 11 is C provided in Experimental Example 1 of the present invention 10 - Inverted fluorescence microscopy images of GFP gene expression after KH2 / DOTAP delivered GFP-mRNA to A549, MCF-7, CHO, Vero, HEK 293T, PC 12, Hela, RAW264.7, and 4T1 cells.

[0040] Figure 12 is C provided in Experimental Example 1 of the present invention 10 - Histogram of cell positive rate after KH2 / DOTAP delivered GFP-mRNA to A549, MCF-7, CHO, Vero, HEK 293T, PC 12, Hela, RAW264.7, and 4T1 cells.

[0041] Figure 13 is the histogram of the relative expression levels of the PTEN gene in the heart, liver, spleen, lung, and kidney tissues of mice 48 h after LP-LNPs delivered siPTEN into the mice provided in Experimental Example 2 of the present invention.

[0042] Figure 14 is the bioluminescence image of the heart, liver, spleen, lung, and kidney tissues of mice 6 h after LP-LNPs delivered Luciferase-mRNA into the mice provided in Experimental Example 2 of the present invention.

[0043] Figure 15 is the histogram of the average luminescence intensity of the heart, liver, spleen, lung, and kidney tissues of mice 6 h after LP-LNP delivered Luciferase-mRNA into the mice provided in Experimental Example 2 of the present invention. Detailed implementation manners

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0045] A lipopeptide, which is a compound formed by the ring-opening reaction of a 1,2-epoxide with the partially or fully exposed amino groups of a lysine-histidine polypeptide;

[0046] Among them, the structural formula of the lipopeptide is:

[0047] Or .

[0048] Preferably, the 1,2-epoxide contains 2 to 22 carbon atoms.

[0049] Preferably, the 1,2-epoxide is any one of 1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, and 1,2-epoxyoctadecane.

[0050] Preferably, in the lipopeptide structural formula, the value range of x is 0 to 10, the value range of y is 1 to 10, the value range of n is 1 to 5, and the value range of m is 0 to 5.

[0051] Preferably, R is any one of the following structural formulas, or R is hydrogen and at least one R is any one of the following structural formulas:

[0052] , , ,

[0053] and .

[0054] A method for preparing a lipopeptide for preparing the lipopeptide, comprising the following steps:

[0055] S101. Under the protection of an inert gas, lysine-histidine polypeptide and 1,2-epoxide are added to a reaction solvent, and the mixture is stirred at a temperature of 50 °C to 110 °C for 48 h to 72 h to prepare a mixture;

[0056] S102. Purify the mixture to prepare the lipopeptide.

[0057] A lipopeptide-based lipid nanoparticle delivery system comprising the lipopeptide, and further comprising one of cholesterol and cholesterol derivatives, co-lipid, and polyethylene glycolated lipid;

[0058] Wherein, the molar ratio of the lipopeptide, cholesterol or cholesterol derivative, co-lipid, and polyethylene glycolated lipid is (5 to 45):(25 to 40):(25 to 65):(0.8 to 2.5).

[0059] Preferably, the co-lipid is at least one of cationic lipid, anionic lipid, phospholipid, and phospholipid derivative;

[0060] The polyethylene glycolated lipid is at least one of PEG-DMG, PEG-C-DMG, and PEG-DSPE.

[0061] An application of a lipopeptide-based lipid nanoparticle delivery system for preparing lipopeptide-based lipid nanoparticles, wherein the lipopeptide-based lipid nanoparticles comprise the lipopeptide-based lipid nanoparticle delivery system and an RNA molecule.

[0062] Preferably, the RNA molecule is at least one of messenger RNA, small interfering RNA, microRNA, and self-amplifying RNA; the mass ratio of the lipopeptide-based lipid nanoparticle delivery system to the RNA molecule is 40 to 10:1.

[0063] The present invention will be further described below in conjunction with specific examples and experimental examples.

[0064] In the following examples, the experimental methods used are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can all be obtained from commercial sources unless otherwise specified.

[0065] Example 1 Preparation of lipopeptide.

[0066] Under N2 protection, lysine-histidine polypeptide and 1,2-epoxide were added to a 10 mL pressure-resistant bottle. The composition and amount of different lysine-histidine polypeptides and 1,2-epoxides are shown in Table 1. The reaction solvent was methanol, and the added amount was preferably 2 ml. After stirring at 90 °C for 96 h, the reaction was stopped. 25 different lipopeptides were prepared as shown in Table 2. The reaction mixture obtained was purified by dialysis (for in vitro application) or silica gel column chromatography (the eluent was dichloromethane:methanol = 20:1 2:1) (for in vivo application) to obtain the lipopeptide. The yield of 25 different lipopeptides is shown in Table 3.

[0067] Among them, the amino acid sequences of different lysine-histidine polypeptides are respectively represented as follows:

[0068] SEQ ID NO.1 is: KH-NH2;

[0069] SEQ ID NO.2 is: KKHH-NH2;

[0070] SEQ ID NO.3 is: KKKHHH-NH2;

[0071] SEQ ID NO.4 is: KKKKHHHH-NH2;

[0072] SEQ ID NO.5 is: KHHKHHKHHKHH-NH2.

[0073] Table 1 Composition of 25 different lipopeptides

[0074]

[0075] Table 2 25 different lipopeptides obtained by preparation

[0076]

[0077] Table 3 Yield of 25 different lipopeptides after purification

[0078]

[0079] Example 2 Preparation of lipopeptide-based lipid nanoparticles

[0080] Mix the RNA molecule with the lipopeptide-based lipid nanoparticle delivery system to obtain nanoparticles encapsulating the RNA, which are lipopeptide-based lipid nanoparticles. The preparation process is as follows:

[0081] Dissolve 25 lipopeptides, co-lipids, cholesterol, and DMG-PEG 2000 prepared in Example 1 in absolute ethanol respectively. Among them, in in vitro applications, the mass ratio of lipopeptide, co-lipid, cholesterol, and DMG-PEG 2000 is 9.5:8.5:3:1; in in vivo applications, the molar ratio of lipopeptide, co-lipid, cholesterol, and DMG-PEG 2000 is 25:45:28.5:1.5. The co-lipids added are cationic liposome 1,2-dioleoyl-3-trimethylammonium propane (abbreviated as DOTAP), zwitterionic liposome 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (abbreviated as DOPE), zwitterionic liposome 1,2-distearoyl-sn-glycero-3-phosphocholine (abbreviated as DSPC), or anionic liposome 1,2-dimyristoyl-sn-glycero-3-phosphate (abbreviated as 14PA).

[0082] The RNA molecule (siRNA or mRNA) is dissolved in a citric acid-sodium citrate enzyme-inactivated buffer with pH = 3 to prepare an aqueous phase. The aqueous phase and the organic phase are mixed manually or by microfluidics at a ratio of 3:1 (v / v). The mass ratio of the lipopeptide-based lipid nanoparticle delivery system to the RNA molecule is preferably 20:1, and lipopeptide-based lipid nanoparticles (LP-LNPs) can be prepared, denoted as lipopeptide / co-lipid, such as C 10 -KH2 / DOTAP, C 10 -KH4 / DOPE, C 10 -KH4 / DOPE, C 18 -KH4 / DOPE, which are not listed one by one here. Figure 1 For C 10 -KH2 / DOTAP, it is the transmission electron microscopy image.

[0083] The obtained LP-LNPs can be directly used for characterization experiments and in vitro applications. For in vivo applications, the above-prepared LP-LNPs are dialyzed with 1×PBS in a dialysis cassette with a molecular weight cut-off of 3500 Da at 4°C for 2 hours before use.

[0084] Example 3 Characterization of lipopeptide-based lipid nanoparticles

[0085] The LP-LNPs samples prepared by manual mixing in Example 2 were detected for particle size, Polymer Dispersity Index (PDI), and Zeta potential using a Malvern laser particle size analyzer. The measurement results are shown in Tables 4 and 5.

[0086] As can be seen from Tables 4 and 5, the particle size of the LP-LNPs samples obtained by manual mixing was between 59 nm and 361 nm, and the Zeta potential was between -5.6 mV and 3.8 mV.

[0087]

[0088]

[0089] Experimental Example 1 In vitro cell experiment.

[0090] I. Cell culture:

[0091] The cells were cultured in a complete medium containing fetal bovine serum; the volume fraction of fetal bovine serum in the complete medium was preferably 10%; the culture conditions were preferably continuous culture in an incubator at 37°C with 5% carbon dioxide by volume.

[0092] II. Cytotoxicity test:

[0093] The CCK-8 kit was used to evaluate the cytotoxicity of LP-LNPs. The specific procedure was as follows: Lung cancer cells A549 were seeded in a 96-well culture plate at a density of 1x10 4 cells per well and cultured in an incubator at 37°C with 5% carbon dioxide by volume for another 24 h until the cell confluence reached 80% - 90%. Then, LP-LNPs at a concentration of 10 μg / mL and the commercial transfection reagent Lipofectamine 2000 at a concentration of 0.75 μg / mL TM were co-cultured with the cells for 24 h. Then, 10 μL of CCK-8 reagent was added to each well. The mixture was allowed to act at 37°C for another 2 h. Then, the absorbance of each well at 450 nm was measured using a microplate reader. The cell survival rate was calculated according to the following formula:

[0094] Cell survival rate (%) = [(As - Ab) / (Ac - Ab)] x 100;

[0095] where As, Ac, and Ab represent the absorbance of the sample, control well, and blank well, respectively. Each experiment was repeated three times. LP-LNPs prepared by compounding lipopeptides with different co-lipids and Lipofectamine 2000 TMThe 24-hour toxicity results for A549 cells are as follows Figures 2 to 5 shown

[0096] III. Determination of the gene silencing efficiency of LP-LNPs for in vitro delivery of siRNA (siRNA@LP-LNPs).

[0097] A549, MCF-7, Hela, RAW264.7, and 4T1 cells were seeded in 24-well culture plates at a density of 2 x 10 5 cells per well and incubated in an incubator at 37°C with 5% carbon dioxide in the volume fraction for another 24 h until the cell confluence reached 60% - 80%. The complete medium was replaced with minimum essential medium (MEM medium, abbreviated) containing siRNA@LP-LNPs (concentration of 10 μg / mL) (0.5 μg siRNA per well). After transfection for 4 h, the medium was replaced with complete medium and the cells were cultured for another 48 h. Then, RNA was extracted for qPCR to detect the relative expression level of the target gene compared to the housekeeping gene to evaluate the efficiency of LP-LNPs in delivering siRNA. (The control group siRNA transfection reagent was Lipofectamine 2000 TM , and Lipofectamine 2000 TM was used according to the instructions, with a dosage of 1.5 μL per well.)

[0098] The measurement results are as follows Figures 6 to 10 shown, and the results indicate that LP-LNPs can effectively deliver siRNA to multiple cell lines and achieve high gene silencing efficiency. C 10 -KH2 / DOTAP achieved target gene silencing efficiencies of 96%, 93.5%, 92.2%, 95.4%, and 72.3% in delivering siRNA to A549, RAW264.7, Hela, 4T1, and MCF-7 cells, respectively, which were all significantly higher than the gene silencing efficiencies achieved by the commercial transfection reagent Lipofectamine 2000 TM in the above cells.

[0099] IV. Determination of the gene expression efficiency of LP-LNPs for in vitro delivery of green fluorescent protein mRNA (GFP-mRNA) (GFP-mRNA@LP-LNPs).

[0100] A549, MCF-7, CHO, HEK 293T, PC 12, Hela, RAW264.7, and 4T1 cells were seeded in 24-well culture plates at a density of 2 x 10 5Cells were seeded in a 24-well culture plate at a density and incubated in an incubator at 37 °C with 5% carbon dioxide by volume for another 24 h until the cell confluence reached 60% - 80%. The complete medium was replaced with MEM medium containing GFP-mRNA@LP-LNPs (at a concentration of 10 μg / mL) (1 μg siRNA per well). After 4 h of transfection, the medium was replaced with the complete medium and the cells were cultured for another 48 h. Then, inverted fluorescence microscopy and flow cytometry were used for characterization to evaluate the efficiency of LP-LNPs in delivering mRNA. (For the control group, the siRNA transfection reagent was Lipofectamine 2000 TM , Lipofectamine 2000 TM was used according to the instructions at a dosage of 1.5 μL.)

[0101] The measurement results are shown in Figure 11 and Figure 12 . The results show that LP-LNPs can effectively deliver mRNA to various cell lines and achieve high gene expression efficiency. C 10 -KH2 / DOTAP in delivering GFP-mRNA to Hela, CHO, A549, HEK 293T, PC12, 4T1, MCF-7, and RAW264.7 cells achieved positive GFP expression rates of 95.3%, 98.7%, 96.8%, 92.2%, 85.0%, 83.9%, 72.5%, and 78.6% respectively, which were all significantly higher than the positive GFP expression rates achieved by the commercial transfection reagent Lipofectamine 2000 TM in the above cells.

[0102] Experimental Example 2 In vivo animal experiment.

[0103] I. The situation of LP-LNPs delivering siPTEN into mice.

[0104] C 10 -KH2 / DOTAP, C 10 -KH4 / DOPE and the assembly complex of C 18 -KH4 / DOPE with siPTEN (10 μg) were injected into C57BL / 6 mice (20 g) via the tail vein. After 48 h, the mice were sacrificed and dissected, and the main organs of the mice, including the heart, liver, spleen, lungs, and kidneys, were isolated, and RNA was extracted from each tissue. The expression levels of the target gene PTEN relative to the housekeeping gene GAPDH in each tissue were detected by qPCR to evaluate the efficiency of LP-LNPs in delivering siRNA in vivo.

[0105] The detection results are shown in Figure 13 . The results show that C 10 -KH2 / DOTAP, C10 -KH4 / DOPE and C 18 -KH4 / DOPE selectively delivered siPTEN to the lung, liver, and spleen target tissues with high selectivity and achieved target gene silencing efficiencies of 76.9%, 79.2%, and 76.3% in the corresponding target tissues, respectively.

[0106] II. The in vivo delivery of firefly luciferase mRNA (Luciferase-mRNA) to mice by LP-LNPs.

[0107] C 10 -KH2 / DOTAP, C 10 -KH4 / DOPE and C 18 The assembled complex of -KH4 / DSPC and Luc-mRNA (15 μg) was injected into C57BL / 6 mice (20 g) via the tail vein. After 6 h, 200 μL of the luciferase (Luciferase) substrate d-luciferin at a concentration of 20 mg / mL was injected into the peritoneal cavity of the mice. After 15 minutes, the mice were sacrificed and dissected, and the main organs of the mice, including the heart, liver, spleen, lungs, and kidneys, were isolated. The organs were imaged using a small animal in vivo imager (animal three-dimensional imager, PerkinElmer, USA), and the imaging results are as Figure 14 、 Figure 15 shown.

[0108] The results showed that C 10 -KH2 / DOTAP, C 10 -KH4 / DOPE and C 18 -KH4 / DSPCE-delivered Luciferase-mRNA was selectively and highly expressed in the lung, liver, and spleen target tissues, and the average luminescence intensity (p / sec / cm 2 / sr) in the corresponding target tissues was 2.8x10 6 、7.9 x10 5 and 4.8 x10 4 respectively. Therefore, the purpose of precise delivery of RNA molecules to multiple organs in vivo can be achieved. C 10 -KH2 / DOTAP has more excellent selectivity and high transfection efficiency for lung tissue.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipopeptide, characterized in that The lipopeptide is a compound formed by a ring-opening reaction between a 1,2-epoxy compound and part or all of the exposed amino groups of a lysine-histidine polypeptide; The 1,2-epoxy compound comprises 10 to 18 carbon atoms; Wherein, the structural formula of the lipopeptide is: or ; The value range of x is 0 to 3, the value range of y is 1 to 4; the value range of n is 1 to 2, and the value range of m is 0 to 3; R represents a group on the amino group, R is independently selected from the group consisting of the ring-opened 1,2-epoxy compound group and H, and at least one R in the structural formula of the lipopeptide is selected from the ring-opened 1,2-epoxy compound group.

2. The lipopeptide according to claim 1, characterized in that The 1,2-epoxy compound is any one of 1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane and 1,2-epoxyoctadecane.

3. The lipopeptide according to claim 1, characterized in that The ring-opened 1,2-epoxide group is any one of the following structural formulas: 、 、 、 and .

4. A method for preparing a lipopeptide, characterized in that: For preparing the lipopeptide according to any one of claims 1 to 3, comprising the following steps: S101. Under the protection of an inert gas, add a lysine-histidine polypeptide and 1,2-epoxide to a reaction solvent, and stir at a temperature of 50° C. to 110° C. for 48 h to 72 h to prepare a mixture; S102, purifying the mixture to prepare the lipopeptide.

5. A lipopeptide-based lipid nano-delivery system, characterized in that: A lipopeptide according to any one of claims 1 to 3, further comprising cholesterol, a helper lipid and a pegylated lipid; The molar ratio of lipopeptide, cholesterol, auxiliary lipid and PEGylated lipid is (5-45): (25-40): (25-65): (0.8-2.5); The helper lipid is selected from at least one of 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphate; The PEGylated lipid is selected from at least one of PEG-DMG and PEG-C-DMG.

6. An application of a lipopeptide-based lipid nano-delivery system, characterized in that: Used for preparing lipopeptide-based lipid nanoparticles, the lipopeptide-based lipid nanoparticles include the lipopeptide-based lipid nano delivery system as described in claim 5, and also include RNA molecules.

7. Use of the lipopeptide-based lipid nano-delivery system as claimed in claim 6, characterized in that: The RNA molecule is at least one of messenger RNA, small interfering RNA, micro RNA and self-amplifying RNA; the mass ratio of the lipopeptide-based lipid nano-delivery system to the RNA molecule is 40 to 10:1.

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