Preparation and application of dendritic lipopeptide molecule

Abstract

The invention provides two dendritic lipopeptide molecules, lipopeptide liposomes and a preparation method and application of the dendritic lipopeptide molecules and the lipopeptide liposomes. The invention provides a dendritic lipopeptide molecule head as shown in a formula (a) and a dendritic lipopeptide molecule as shown in a formula (b). The invention also provides a lipopeptide liposome prepared from the dendritic lipopeptide molecule, cationic amino acid is used as a raw material to synthesize a lipopeptide head, the lipopeptide molecule is further synthesized, other auxiliary lipids are introduced to jointly form the cationic lipopeptide liposome, and the cationic lipopeptide liposome is electrostatically bound with siRNA, so that the delivery efficiency of the siRNA is improved, and the siRNA is prevented from being degraded by nucleic acid; the characteristics of good biocompatibility, high in-vivo and in-vitro transfection efficiency and the like are realized. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the preparation and application of a dendritic lipopeptide molecule. Background Art

[0002] When treating human diseases, traditional small molecule drugs and antibodies usually directly interfere with the action of proteins to achieve therapeutic purposes. However, these drugs have their limitations. They cannot regulate a large number of pathogenic proteins, thus limiting their efficacy for certain diseases. Therefore, researchers have begun to focus on a new treatment strategy, which is to precisely control the level of protein production by regulating the expression of pre-protein genes, which is the so-called gene therapy. Thanks to the in-depth research of molecular biology, nucleic acid-derived drugs (such as pDNA, mRNA, siRNA, ASO and miRNA) have become the mainstream of gene therapy. These nucleic acid drugs usually have multiple functions, such as encoding disease-related proteins, inhibiting the expression of specific genes, regulating protein function and promoting gene transcriptional activation. siRNA, also known as short interfering RNA, is a class of double-stranded RNA molecules with a length ranging from 20 to 27 base pairs. After entering the cell, it can block the expression of related proteins and play a role in the cell. The mechanism of action of these molecules is that siRNA binds to RNA-induced RNA silencing complexes and AGO2 proteins. After siRNA binds to AGO2 protein, the siRNA crystal strand is released from the complex and degraded, while the antisense strand binds to the entire mRNA, destroys the pathway and is released. The endonuclease cuts the mRNA into small fragments, preventing the mRNA from being translated into protein. The endonuclease cuts the mRNA into small fragments, preventing the mRNA from being translated into protein. Currently, the design and targeted delivery of siRNA is becoming an important research approach for the treatment of diseases based on abnormal gene expression, and will play an important role in the field of biomedicine. In 2018, with the first therapeutic drug ONPATTRO TM (generic name Patisiran) has been approved by the U.S. Food and Drug Administration (FDA), and DNA therapy will be more widely used in clinical practice. To date, a variety of nucleic acid-based drugs have entered the preclinical and clinical trial stages, including many products that have been approved and successfully used in clinical practice.

[0003] However, the electronegativity, nuclease sensitivity and intracellular effects of nucleic acid drugs limit their clinical application. Therefore, it is necessary to develop suitable drug delivery systems to avoid these shortcomings and improve the efficacy of nucleic acid drugs. Currently, the most commonly used nanocarriers in siRNA delivery systems are liposomes, lipid nanoparticles, inorganic nanoparticles and dendrimer nanoparticles.

[0004] Liposomes are a nano-drug delivery system that can be used as a non-viral transport carrier for nucleic acids. They have a stable nanostructure under physiological conditions, can bind to endosomes, and effectively deliver nucleic acid drugs. Liposomes mainly contain cationic lipids, zwitterions, and cholesterol. Cationic lipids form complexes with the negatively charged phosphate groups of siRNA through electrostatic interactions, thereby inhibiting the absorption of phagocytes and improving the elongation efficiency of siRNA in particles. Therefore, cationic lipids are the key to liposome delivery of RNA, and the development of new cationic lipid molecules is crucial for effective gene therapy in the real world. Summary of the invention

[0005] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a dendritic lipopeptide molecule head, a dendritic lipopeptide molecule and a preparation method thereof, using cationic amino acids as raw materials to synthesize the lipopeptide head, and further synthesizing the lipopeptide molecule, introducing other auxiliary lipids to form cationic lipopeptide liposomes, and electrostatically binding with siRNA, thereby improving the delivery efficiency of siRNA and avoiding degradation by nucleic acids.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides an amphiphilic cationic lipopeptide molecular head, which mainly utilizes compounds such as lysine methyl ester hydrochloride (H-Lys-OMe·2HCl), N'-fluorenylmethoxycarbonyl-L-lysine methyl ester hydrochloride (H-Lys(Fmoc)-OMe·HCl), lysine (Boc-Lys(Boc)-OH) and arginine (Boc-Arg(Pbf)-OH) to synthesize a type of cationic lipopeptide head group through amide condensation reaction and ester hydrolysis reaction, and the structural formula thereof is shown in formula (a).

[0008]

[0009] Among them, R 1 and R 2 are independently selected from lysine and arginine,

[0010] The dendritic lipopeptide molecule head includes M1-OH and N1-OH;

[0011] When R 1 C 5 N 4 H 14 , R 2 C 5 N 2 H 14 When , the head of the dendritic lipopeptide molecule is M1-OH;

[0012] When R 1 C 5 N2 H 14 , R 2 C 5 N 4 H 14 When , the head of the dendritic lipopeptide molecule is N1-OH.

[0013] The invention provides the synthesis of M1-OH of the lipopeptide molecule head, and the reaction is divided into four steps: synthesis of Fmoc-Lys(Boc)-Boc, synthesis of H-Lys(Boc)-Boc, synthesis of M1-OMe and demethylation of M1-OMe.

[0014] As a specific embodiment of the present invention, Fmoc-Lys(Boc)-Boc is prepared by amide condensation reaction, using Fmoc-Lys-OMe·HCl, Boc-Lys(Boc)-OH, HOBT and HBTU as raw materials, dissolving them in a suitable solvent, reacting them at room temperature and under vacuum conditions, and using a spot plate to detect whether the reaction is completed. The resulting crude product is then purified by silica gel column chromatography.

[0015] As a specific embodiment of the present invention, H-Lys(Boc)-Boc is obtained by reacting Fmoc-Lys(Boc)-Boc with piperidine at room temperature.

[0016] As a specific embodiment of the present invention, M1-OME is obtained by reacting H-Lys(Boc)-Boc, Boc-Arg(Pbf)-OH, HOBT and HBTU as raw materials under vacuum and room temperature conditions, and the crude product is purified by silica gel column chromatography.

[0017] As a specific embodiment of the present invention, the M1-OME product is mixed with a methanol solution of NaOH under ice bath conditions to obtain the M1-OH final product.

[0018] The invention provides the synthesis of N1-OH in the head of a lipopeptide molecule. The reaction is divided into four steps: synthesis of Boc-Lys(Boc)-Fmoc, synthesis of Boc-Lys(Boc)-H, synthesis of N1-OMe and demethylation of N1-OMe.

[0019] As a specific embodiment of the present invention, Boc-Lys(Boc)-Fmoc is prepared by amide condensation reaction, using H-Lys(Fmoc)-OMe·HCl, Boc-Lys(Boc)-OH, HOBT, HBTU as raw materials, dissolving them in a suitable solvent, reacting at room temperature and vacuum conditions, and using a spot plate to detect whether the reaction is complete, and the obtained crude product is then purified by silica gel column chromatography.

[0020] As a specific embodiment of the present invention, Boc-Lys(Boc)-Fmoc is reacted with piperidine at room temperature to obtain Boc-Lys(Boc)-H.

[0021] As a specific embodiment of the present invention, N1-OME is obtained using Boc-Lys(Boc)-H, Boc-Arg(Pbf)-OH, HOBT, and HBTU as raw materials under vacuum and room temperature conditions, and the crude product is further purified by silica gel column chromatography.

[0022] As a specific embodiment of the present invention, N1-OME is further hydrolyzed with a methanol solution of NaOH under ice bath conditions to obtain the final product N1-OH.

[0023] In the specific example of lipopeptide head synthesis provided by the present invention, the first step (synthesis of Fmoc-Lys(Boc)-Boc or Boc-Lys(Boc)-Fmoc) and the third step (synthesis of M1-OMe or N1-OMe) both need to use DMF to dissolve the solid powder of the raw material under ice bath conditions, and then slowly add a certain amount of DIPEA under stirring to react at room temperature. And the reaction is completed by spot plate, and then ethyl acetate is used as a solvent to transfer the product, and the reaction container is washed with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution and saturated sodium chloride solution in turn, and finally a crude product solution of ethyl acetate is obtained. Anhydrous sodium sulfate is added to dry overnight, vacuum evaporation and concentration are obtained to obtain a solid crude product, which is further vacuum dried. Finally, a silica gel chromatography column is used for purification.

[0024] As a specific embodiment of the present invention, when purifying by silica gel chromatography column, the initial purification ratio for the first step is dichloromethane: methanol = 100: 1 (V / V), and a pure product can be obtained when the dichloromethane: methanol ratio is about 60: 1 (V / V); the initial purification ratio for the third step (synthesis of M1-OMe or N1-OMe) is dichloromethane: methanol = 60: 1 (V / V), and a pure product can be obtained when the dichloromethane: methanol ratio is about 30: 1 (V / V).

[0025] In the synthesis of Fmoc-Lys(Boc)-Boc / Boc-Lys(Boc)-Fmoc, the molar ratio of Fmoc-Lys-OMe·HCl or H-Lys(Fmoc)-OMe·HCl, Boc-Lys(Boc)-OH, HOBT, and HBTU is 1:1.1~2:1.1~2:1.1~2.

[0026] In the synthesis of Boc-Lys(Boc)-OH / Boc-Lys(Boc)-OH, the molar ratio of Fmoc-Lys(Boc)-Boc or Boc-Lys(Boc)-Fmoc to piperidine is 1:19-21.

[0027] In the synthesis of M1-OMe or N1-OMe, the molar ratio of H-Lys(Boc)-Boc, Boc-Arg(Pbf)-OH, HOBT, and HBTU is 1:1.1~2:1.1~2:1.1~2; the molar ratio of Boc-Lys(Boc)-H, Boc-Arg(Pbf)-OH, HOBT, and HBTU is 1:1.1~2:1.5~2.5:1.5~2.5.

[0028] In the synthesis of M1-OH or N1-OH, the molar ratio of M1-OMe or N1-OMe to NaOH is 1:11-12.

[0029] The present invention also provides an amphiphilic cationic lipopeptide molecule, the structure of which is shown in formula (b):

[0030]

[0031] Among them, R 1 and R 2 independently selected from lysine and arginine;

[0032] R3 is the structure shown in DSPE or OA below;

[0033] DSPE:

[0034]

[0035] OA:

[0036]

[0037] As a specific embodiment of the present invention, a dendritic lipopeptide molecule includes compounds with structures shown below: M1-DSPE, N1-DSPE, M1-OA, N1-OA;

[0038] M1-DSPE:

[0039]

[0040] N1-DSPE:

[0041]

[0042] M1-OA:

[0043] N1-OA:

[0044]

[0045] In the present invention, the dendritic lipopeptide molecule M 1 -DSPE,M 1 -OA, N 1 -DSPE,N 1 -OA is synthesized by amide condensation reaction of the dendritic lipopeptide molecule head with DSPE compound or OA compound. The dendritic lipopeptide molecule is further used to prepare liposomes, and the obtained liposomes can be applied to non-viral nucleic acid vectors. The molar ratio of the DSPE compound to the dendritic lipopeptide molecule head is 1:2.5-3.0; the molar ratio of the OA compound to the dendritic lipopeptide molecule head is 1:0.2-0.8.

[0046] As a specific embodiment of the present invention, Hp is used to represent the lipopeptide head group, which can be the dendritic lipopeptide head M1 or N1 provided by the present invention, so that four lipopeptide molecules, M1-DSPE, M1-OA, N1-DSPE, and N1-OA, can be obtained.

[0047] In the present invention, the synthesis of the dendritic lipopeptide molecule Hp-DSPE is divided into two steps: the synthesis of Pbf(Boc)-Hp-DSPE and the deprotection of Pbf(Boc)-Hp-DSPE.

[0048] Among them, the synthesis of Pbf(Boc)-Hp-DSPE is to use DSPE compound, dendritic lipopeptide head, HOBT, HBTU under vacuum conditions, dissolve them in a suitable solvent, react for a period of time under ice bath conditions, and then heat to 37°C to continue the reaction until the reaction is completed under spot plate monitoring. After drying, the crude product is obtained, and then further purified using a silica gel chromatography column.

[0049] As a specific embodiment of the present invention, DMF, CHCl 3 Dissolve, add DIPEA after 30 minutes of ice bath, react again for 30 minutes under ice bath condition, and then raise the temperature to 37°C to continue the reaction.

[0050] As a specific embodiment of the present invention, the initial ratio of purification using silica gel chromatography is dichloromethane:methanol=40:1 (V / V), and the pure product of Pbf(Boc)-Hp-DSPE is obtained when the ratio of dichloromethane:methanol is about 15:1 (V / V).

[0051] Among them, Pbf(Boc)-Hp-DSPE was deprotected in dichloromethane and trifluoroacetic acid to obtain the corresponding dendritic lipopeptide molecule.

[0052] As a specific embodiment of the present invention, Pbf(Boc)-Hp-DSPE is dissolved in dichloromethane as a solvent under ice bath conditions, trifluoroacetic acid is added and the reaction is carried out at room temperature. After the reaction is completed, the solvent is evaporated and extracted with ice anhydrous ether, and then further dried to obtain a pure product.

[0053] In the present invention, the synthesis of the dendritic lipopeptide molecule Hp-OA is divided into two steps: the synthesis of Pbf(Boc)-Hp-OA and the deprotection of Pbf(Boc)-Hp-OA.

[0054] Among them, the synthesis of Pbf(Boc)-Hp-OA uses Hp-OH, HOBT, EDCI, and OA compounds as raw materials, which are fully dissolved under suitable solvent conditions, and then reacted at room temperature. The reaction is completed by spot plate detection. The obtained crude product is dried and further purified by silica gel column chromatography.

[0055] As a specific embodiment of the present invention, the powdered raw material is dissolved in dichloromethane under ice bath conditions, and the OA compound and DIPEA are added to complete the reaction at room temperature.

[0056] As a specific embodiment of the present invention, the initial ratio of dichloromethane:methanol = 50:1 (V / V) for purification by silica gel column chromatography, and the pure product Pbf(Boc)-M1-OA is obtained when the ratio of dichloromethane:methanol is about 20:1 (V / V).

[0057] As a specific embodiment of the present invention, dichloromethane is used as a solvent under ice bath conditions, trifluoroacetic acid is added and the reaction is carried out at room temperature. After the reaction is completed, the solvent is evaporated and extracted with ice anhydrous ether, and then further dried to obtain a pure product.

[0058] Among them, Pbf(Boc)-Hp-OA is deprotected in dichloromethane and trifluoroacetic acid to obtain the corresponding lipopeptide molecule.

[0059] The present invention also provides a liposome that can be used for drug delivery, which is mainly formed by self-assembly of cationic lipopeptides, zwitterions and cholesterol through a thin film dispersion method. This type of nano delivery system can be used as a non-viral nucleic acid carrier, which can improve the stability of the drug and improve the in vivo and in vitro transfection efficiency of the drug.

[0060] As a specific embodiment of the present invention, the liposome provided by the present invention is a cationic lipopeptide and DOPE, DSPE-PEG 2000 and cholesterol as raw materials, and the corresponding cationic lipopeptide liposomes were prepared by thin film dispersion method in a certain proportion.

[0061] As a specific embodiment of the present invention, the cationic lipopeptide can be selected from the four dendritic lipopeptide molecules M1-DSPE, M1-OA, N1-DSPE, and N1-OA provided above.

[0062] As a specific embodiment of the present invention, the molar ratio of the components of the liposome is cationic lipopeptide: DOPE: DSPE-PEG 2000 : Cholesterol = 30:20:3:1.5.

[0063] As a specific embodiment of the present invention, the above-mentioned non-viral nucleic acid vector adopts siRNA, which is loaded onto liposomes through electrostatic interaction, and is further characterized by particle size and potential. The electrostatic interaction between lipopeptide liposomes and siRNA is observed by agarose gel electrophoresis, and the in vitro transfection efficiency of cationic lipopeptide liposomes is verified by in vitro transfection experiments.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The present invention provides a type of dendritic lipopeptide molecular head shown in formula (a), and a type of dendritic lipopeptide molecule shown in formula (b). The present invention also provides lipopeptide liposomes prepared from such dendritic lipopeptide molecules, wherein the cationic head group in the lipopeptide molecular structure can bind to siRNA through electrostatic interaction, thereby avoiding degradation of siRNA by nucleases in vivo, promoting siRNA uptake into cells, and improving the endosomal escape efficiency of siRNA in cells, while other auxiliary lipid components in the lipopeptide liposomes maintain the stability of the liposomes, and jointly improve the transfection efficiency of siRNA, effectively solving the application limitations of nucleic acid drugs currently on the market due to electronegativity, nuclease sensitivity, and intracellular effects, and is a type of gene delivery carrier with excellent performance, having the characteristics of good biocompatibility and efficient in vivo and in vitro transfection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a particle size characterization diagram of different cationic lipopeptide liposomes shown in Example 8;

[0067] Figure 2 It is a particle size characterization diagram of different siRNA / cationic lipopeptide liposome complexes shown in Example 8;

[0068] Figure 3 This is a potential characterization diagram of different cationic lipopeptide liposomes shown in Example 9;

[0069] Figure 4 It is a potential characterization diagram of different siRNA / cationic lipopeptide liposome complexes shown in Example 9;

[0070] Figures 5 to 8The complex effects of different cationic lipopeptide liposomes and siRNA shown in Example 10;

[0071] Fig. 9 The cytotoxicity results of different siRNA / cationic lipopeptide liposome complexes shown in Example 11;

[0072] Fig.10 Example 12 shows the in vitro silencing effects of different siRNA / cationic lipopeptide liposome complexes. DETAILED DESCRIPTION

[0073] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] Example 1

[0075] Synthesis of Dendritic Lipopeptide Head M1-OH

[0076] S1-1: Accurately weigh Fmoc-Lys-OMe·HCl 5.00g, Boc-Lys(Boc)-OH 4.96g, HOBT2.43g, and HBTU 4.08g and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add DMF40mL to dissolve the above solid powder in an ice bath, and slowly add DIPEA20.80 mL under stirring, and react at room temperature for 8h. After the reaction is completed, transfer the reacted liquid to a separatory funnel, wash the three-necked flask with ethyl acetate in small amounts and multiple times, and transfer it to a separatory funnel. Wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution, and saturated sodium chloride solution in turn to obtain a crude product solution of ethyl acetate, and add anhydrous sodium sulfate to dry overnight. Vacuum evaporation and concentration to obtain a solid crude product, and then place it in a vacuum drying oven to dry for 2h. The crude product was purified by silica gel column chromatography. The starting ratio was dichloromethane:methanol = 100:1 (V / V). The pure product Fmoc-Lys(Boc)-Boc was obtained when the ratio of dichloromethane:methanol was about 60:1 (V / V).

[0077] S1-2: Accurately weigh 2.00g of Fmoc-Lys(Boc)-Boc and place it in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 10mL of DMF to dissolve it in an ice bath, add 1.5mL of piperidine under stirring, and react at room temperature for 6h. After the reaction is completed, evaporate the solvent under reduced pressure, repeatedly add ethyl acetate in small amounts and pressurize and rotary evaporate to remove piperidine, add 100mL of a solution of petroleum ether: ethyl acetate = 10:1, and the precipitate is the pure product of H-Lys(Boc)-Boc.

[0078] S1-3: Accurately weigh H-Lys(Boc)-Boc1.4g, Boc-Arg(Pbf)-OH 1.82g, HOBT 0.58g, and HBTU1.63g and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 20mL of DMF to dissolve the above solid powder in an ice bath, and slowly add 2.00mL of DIPEA under stirring, and react at room temperature for 24h. After the reaction is completed, transfer the reacted liquid to a separatory funnel, wash the three-necked flask with a small amount of ethyl acetate several times and transfer it to a separatory funnel, wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution, and saturated sodium chloride solution in turn to obtain a crude product solution of ethyl acetate, and add anhydrous sodium sulfate to dry overnight. Vacuum evaporation and concentration to obtain a solid crude product, and then place it in a vacuum drying oven to dry for 2h. The crude product was purified by silica gel column chromatography. The starting ratio was dichloromethane:methanol = 60:1 (V / V). The pure product M1-OMe was obtained when the ratio of dichloromethane:methanol was about 30:1 (V / V).

[0079] S1-4: 2.00 g of M1-OMe was accurately weighed and placed in a round-bottom flask. 50 mL of methanol solution of NaOH (2 g of NaOH was dissolved in 50 mL of methanol solution) was added under ice bath, and the reaction was carried out under ice bath for 6 h. After the reaction, 1 M dilute hydrochloric acid was added to adjust the pH to 2-3 to neutralize the excess NaOH. The methanol solution was removed by evaporation and concentration under reduced pressure, and ethyl acetate was added to dissolve it. The solution was then washed repeatedly three times with saturated sodium chloride solution. Anhydrous sodium sulfate was added to the organic reagent layer and dried for 2 h. The ethyl acetate was removed by evaporation under reduced pressure, and the product was dried in a vacuum drying oven for 24 h to obtain the final product of M1-OH.

[0080] 1 H NMR (300 MHz, CDCl 3)δ7.87-7.03(m,3H),6.46(s,2H),5.99(s,1H),5.79(s,1H),4.94(s,1H),4.54-3.98(m,3H),3.12(d ,J=40.8Hz,6H),2.93(d,J=8.8Hz,2H),2.51(d,J=18.5Hz,6H),2.08(s,3H),1.32(d,J=43.7Hz,49H).

[0081] Example 2

[0082] Synthesis of N1-OH Head of Dendritic Lipopeptide Molecules

[0083] S1-1: Accurately weigh H-Lys(Fmoc)-OMe·HCl 5.00g, Boc-Lys(Boc)-OH 4.96g, HOBT2.43g, and HBTU 9.04g and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add DMF40mL to dissolve the above solid powder in an ice bath, and slowly add DIPEA 16.60mL under stirring, and react at room temperature for 8h. After the reaction is completed, transfer the reacted liquid to a separatory funnel, wash the three-necked flask with ethyl acetate in small amounts and multiple times, and transfer it to a separatory funnel. Wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution, and saturated sodium chloride solution in turn to obtain a crude product solution of ethyl acetate, and add anhydrous sodium sulfate to dry overnight. Vacuum evaporation and concentration to obtain a solid crude product, and then place it in a vacuum drying oven to dry for 2h. The crude product was purified by silica gel column chromatography. The starting ratio was dichloromethane:methanol = 100:1 (V / V). The pure Boc-Lys(Boc)-Fmoc product was obtained when the ratio of dichloromethane:methanol was about 60:1 (V / V).

[0084] S1-2: Accurately weigh 2.00g of Boc-Lys(Boc)-Fmoc and place it in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 10mL of DMF to dissolve it in an ice bath, add 1.5mL of piperidine under stirring, and react at room temperature for 6h. After the reaction is completed, evaporate the solvent under reduced pressure, repeatedly add ethyl acetate in small amounts and pressurize and rotary evaporate to remove piperidine, add 100mL of a solution of petroleum ether: ethyl acetate = 10:1, and the precipitate is the pure product of Boc-Lys(Boc)-H.

[0085] S1-3: Accurately weigh Boc-Lys(Boc)-H 1.37g, Boc-Arg(Pbf)-OH 2.22g, HOBT 0.76g, and HBTU 2.13g and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 30mL of DMF to dissolve the above solid powder in an ice bath, and slowly add 1.96mL of DIPEA under stirring, and react at room temperature for 24h. After the reaction is completed, transfer the reacted liquid to a separatory funnel, wash the three-necked flask with a small amount of ethyl acetate several times and transfer it to a separatory funnel, wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution, and saturated sodium chloride solution in turn to obtain a crude product solution of ethyl acetate, and add anhydrous sodium sulfate to dry overnight. Vacuum evaporation and concentration to obtain a solid crude product is placed in a vacuum drying oven and dried for 2h. The crude product was purified by silica gel column chromatography. The starting ratio was dichloromethane:methanol = 60:1 (V / V). The pure product N1-OMe was obtained when the ratio of dichloromethane:methanol was about 30:1 (V / V).

[0086] S1-4: 2.00 g of N1-OMe was accurately weighed and placed in a round-bottom flask. 50 mL of methanol solution of NaOH (2 g of NaOH was dissolved in 50 mL of methanol solution) was added under ice bath, and the reaction was carried out under ice bath for 6 h. After the reaction, 1 M dilute hydrochloric acid was added to adjust the pH to 2-3 to neutralize the excess NaOH. The methanol solution was removed by evaporation and concentration under reduced pressure, and ethyl acetate was added to dissolve it. The solution was then washed repeatedly three times with saturated sodium chloride solution. Anhydrous sodium sulfate was added to the organic reagent layer and dried for 2 h. The ethyl acetate was removed by evaporation under reduced pressure, and the product was dried in a vacuum drying oven for 24 h to obtain the final product of N1-OH.

[0087] 1 H NMR (300 MHz, CDCl 3 )δ7.99-7.22(m,2H),6.17(d,J=136.1Hz,5H),4.82(s,1H),4.60–4.08(m,3H),3.51-2.98(m ,6H),2.93(d,J=9.0Hz,2H),2.50(dd,J=17.7,7.1Hz,6H),2.07(s,3H),1.73-1.25(m,49H).

[0088] Example 3

[0089] Synthesis of Lipopeptide Molecule M1-DSPE

[0090] Step 1: Accurately weigh 1.00 g of DSPE compound, 1.57 g of M1-OH, 0.54 g of HOBT, and 1.52 g of HBTU and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 5.00 mL of DMF and CHCl in an ice bath.3 Dissolve the solid powder in 45 mL of water, stir in an ice bath for 30 min, then slowly add DIPEA in small amounts for a total of 2.30 mL. React the mixed solution in an ice bath for 30 min and then at 37°C for 24 h. After the reaction is completed by spot plate monitoring, add CHCl 3 300mL was transferred to a separatory funnel and washed three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution and saturated sodium chloride solution in sequence. The organic layer was collected and dried for 0.5h by adding anhydrous sodium sulfate. The solid crude product was concentrated by vacuum evaporation and then dried in a vacuum drying oven for 2h. The crude product was purified by silica gel column chromatography, with a starting ratio of dichloromethane: methanol = 40: 1 (V / V). The pure product of Pbf(Boc)-M1-DSPE was obtained when the ratio of dichloromethane: methanol was about 15: 1 (V / V).

[0091] Step 2: Accurately weigh 0.50 g of Pbf(Boc)-M1-DSPE in a three-necked flask and repeatedly evacuate the flask three times under nitrogen protection. Add 2 mL of dichloromethane to make a solution under ice bath, add 2 mL of trifluoroacetic acid under stirring, and react at room temperature for 24 hours. After the reaction is completed, concentrate and evaporate to remove trifluoroacetic acid, add ice anhydrous ether, and a white solid precipitates. Discard the supernatant, repeat washing several times, and vacuum dry for 3 hours to obtain pure M1-DSPE product.

[0092] 1 H NMR (300MHz, DMSO) δ5.11 (s, 1H), 4.35-4.07 (m, 3H), 3.79 (s, 6H), 2.92 (d, J = 113.5Hz, 8H), 2.26 (s, 4H), 1.22 (s, 76H), 0.84 (t, J = 6.2Hz, 6H).

[0093] Example 4

[0094] Synthesis of Lipopeptide Molecule M1-OA

[0095] Step 1: Accurately weigh 0.50g of M1-OH, 0.21g of HOBT, and 0.29g of EDCI and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 10mL of dichloromethane to dissolve the above solid powder under an ice bath, and add 0.25mL of OA compound and 0.72mL of DIPEA. Remove the ice bath and react at room temperature for 24h. After the reaction is completed by spot plate monitoring, add an appropriate amount of dichloromethane to the reaction solution and transfer it to a separatory funnel. Wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution and saturated sodium chloride solution in turn, collect the organic layer, and add anhydrous sodium sulfate to dry for 0.5h. Vacuum evaporation and concentration to obtain a solid crude product is placed in a vacuum drying oven and dried for 2h. The crude product was purified by silica gel column chromatography. The starting ratio was dichloromethane:methanol = 50:1 (V / V). The pure product Pbf(Boc)-M1-OA was obtained when the ratio of dichloromethane:methanol was about 20:1 (V / V).

[0096] Step 2: Accurately weigh 0.50 g of Pbf(Boc)-M1-OA in a three-necked flask and repeatedly evacuate the flask three times under nitrogen protection. Add 2 mL of dichloromethane to make a solution under ice bath, add 2 mL of trifluoroacetic acid under stirring, and react at room temperature for 24 hours. After the reaction is completed, concentrate and evaporate to remove trifluoroacetic acid, add ice anhydrous ether, and a white solid precipitates. Discard the supernatant, repeat washing several times, and vacuum dry for 3 hours to obtain pure M1-OA product.

[0097] 1 H NMR (300MHz, DMSO) δ4.18 (d, J = 7.9 Hz, 1H), 3.79 (d, J = 35.4 Hz, 2H), 2.93 (d, J = 104.5 Hz, 8H), 2.05-1.03 (m, 44H), 0.88-0.80 (m, 3H).

[0098] Example 5

[0099] Synthesis of Lipopeptide Molecule N1-DSPE

[0100] Step 1: Accurately weigh 1.00 g of DSPE compound, 1.57 g of N1-OH, 0.72 g of HOBT, and 2.02 g of HBTU and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 5.00 mL of DMF and CHCl in an ice bath. 3 Dissolve the solid powder in 45 mL of water, stir in an ice bath for 30 min, then slowly add DIPEA in small amounts for a total of 1.16 mL. React the mixed solution in an ice bath for 30 min and then at 37°C for 24 h. After the reaction is completed by spot plate monitoring, add CHCl 3300 mL was transferred to a separatory funnel and washed three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution and saturated sodium chloride solution in sequence. The organic layer was collected and dried over anhydrous sodium sulfate for 0.5 h. The solid crude product was concentrated by vacuum evaporation and then dried in a vacuum oven for 2 h. The crude product was purified by silica gel column chromatography with a starting ratio of dichloromethane: methanol = 40: 1 (V / V). The pure product of Pbf(Boc)-N1-DSPE was obtained when the ratio of dichloromethane: methanol was about 15: 1 (V / V).

[0101] Step 2: Accurately weigh 0.50 g of Pbf(Boc)-N1-DSPE in a three-necked flask and repeatedly evacuate the flask three times under nitrogen protection. Add 2 mL of dichloromethane to make a solution under ice bath, add 2 mL of trifluoroacetic acid under stirring, and react at room temperature for 24 hours. After the reaction is completed, evaporate the trifluoroacetic acid under reduced pressure, add ice anhydrous ether, and a white solid precipitates. Discard the supernatant, repeat washing several times, and vacuum dry for 3 hours to obtain pure N1-DSPE product.

[0102] 1 H NMR (300MHz, DMSO) δ5.10 (d, J=6.9Hz, 1H), 4.33-4.00 (m, 3H), 3.87-3.65 (m, 6H), 3.25-2.71(m,8H),2.25(dt,J=7.3,3.8Hz,4H),1.22(s,76H),0.90-0.81(m,6H).

[0103] Example 6

[0104] Synthesis of Lipopeptide Molecule N1-OA

[0105] Step 1: Accurately weigh 0.50g of N1-OH, 0.21g of HOBT, and 0.29g of EDCI and place them in a three-necked flask. Repeat the vacuum three times under nitrogen protection. Add 0.25mL of OA compound and 0.72mL of DIPEA to dissolve the above solid powder in an ice bath. Remove the ice bath and react at room temperature for 24h. After the reaction is completed by spot plate monitoring, add an appropriate amount of dichloromethane to the reaction solution and transfer it to a separatory funnel. Wash it three times with saturated sodium bicarbonate solution, 0.1M dilute hydrochloric acid solution and saturated sodium chloride solution in turn, collect the organic layer, and add anhydrous sodium sulfate to dry for 0.5h. Vacuum evaporation and concentration to obtain a solid crude product, which is then placed in a vacuum drying oven to dry for 2h. The crude product is purified by silica gel column chromatography, with the starting ratio of dichloromethane: methanol = 50:1 (V / V), and the pure product of Pbf(Boc)-N1-OA is obtained when the ratio of dichloromethane: methanol is about 20:1 (V / V).

[0106] Step 2: Accurately weigh 0.50 g of Pbf(Boc)-N1-OA in a three-necked flask and repeatedly evacuate the flask three times under nitrogen protection. Add 2 mL of dichloromethane to make a solution under ice bath, add 2 mL of trifluoroacetic acid under stirring, and react at room temperature for 24 hours. After the reaction is completed, concentrate and evaporate to remove trifluoroacetic acid, add ice anhydrous ether, and a white solid precipitates. Discard the supernatant, repeat washing several times, and vacuum dry for 3 hours to obtain pure N1-OA product.

[0107] 1 H NMR (300MHz, DMSO) δ4.22 (d, J = 7.6 Hz, 1H), 3.76 (d, J = 45.0 Hz, 2H), 3.19-2.63 (m, 8H), 1.23 (s, 44H), 0.93-0.78 (m, 3H).

[0108] Example 7

[0109] Preparation of lipopeptide liposomes

[0110] The ratio of each raw material of the liposome is cationic lipopeptide molecule (M1-DSPE or M1-OA or N1-DSPE or N1-OA): DOPE: DSPE-PEG 2000 : cholesterol = 30:20:3:1.5 (mol / mol). When preparing liposomes, the concentration of the mother solution of each liposome raw material was 1 mg / mL.

[0111] The preparation of the mother solution of each liposome raw material includes the following steps: weighing cationic lipopeptide, DOPE, DSPE-PEG 2000 , and cholesterol (about 10 mg), which were placed in four bacterial culture bottles respectively. An appropriate amount of a mixed solution of chloroform:methanol = 3:1 was added and dissolved by ultrasonication in a water bath to prepare mother solutions of liposome raw materials with a concentration of 1 mg / mL for later use.

[0112] Take 1 mL of cationic lipopeptide molecule mother liquor, and add the mother liquors of the other three raw materials into a 50 mL eggplant-shaped bottle according to the above proportions. Evaporate and concentrate to remove the solvent under reduced pressure. A uniform film is formed on the bottom of the bottle. Add 1 mL of UP water to hydrate it. Shake it in a water bath under ultrasound until the film completely falls off. Transfer it to a 4 mL centrifuge tube and place it under an ultrasonic cell disruptor for probe ultrasound. Set the power to 50% and ultrasound for 3 minutes to obtain cationic lipopeptide liposomes; mark them as L-M1-D, L-M1-O, L-N1-D, and L-N1-O.

[0113] Example 8

[0114] Particle size determination of lipopeptide liposomes and siRNA / lipopeptide liposome complexes

[0115] Take 200 μL of each lipopeptide liposome solution, dilute it to 1.5 mL with UP water, and place it in a quartz dish with four sides transparent to light, and measure the particle size distribution of each liposome under a laser particle size analyzer; dilute each siRNA / lipopeptide liposome complex solution to 1.5 mL with UP water, and place it in a quartz dish with four sides transparent to light, and measure the particle size distribution of each siRNA / lipopeptide liposome complex under a laser particle size analyzer. The above experiments were measured three times in parallel.

[0116] like Figure 1 As shown, the particle sizes of the above four lipopeptide liposomes are all less than 100 nm, and the particle sizes of the lipopeptide liposomes are uniform and meet the standards of nano-preparations.

[0117] like Figure 2 As shown, the particle sizes of the above four lipopeptide liposomes after complexing with siRNA are all less than 200 nm, indicating that the particle sizes of the above four siRNA / lipopeptide liposome complexes are uniform.

[0118] Example 9

[0119] Potentiometric determination of lipopeptide liposomes and siRNA / lipopeptide liposome complexes

[0120] Take 200 μL of each lipopeptide liposome solution, dilute it to 1 mL with UP water and place it in a potential dish, and measure the potential distribution of each liposome under a laser particle size analyzer; dilute each siRNA / lipopeptide liposome complex solution to 1 mL with UP water and place it in a potential dish, and measure the potential distribution of each liposome under a laser particle size analyzer. The above experiments were measured three times in parallel.

[0121] like Figure 3 As shown, the potential of each lipopeptide liposome is between 20-30mV. This is because the head group of the lipopeptide liposome is a basic amino acid such as lysine or arginine, the pH of the lipopeptide liposome in UP water is lower than the isoelectric point of the amino acid, and the amine or guanidine group in the head group of the lipopeptide molecule is positively charged, so the potential of the lipopeptide liposome is positive. Because the ionization ability of the amine group is stronger than that of the guanidine group, the charge value of each lipopeptide liposome is different.

[0122] like Figure 4 As shown in the figure, the potential of each siRNA / lipopeptide liposome complex is lower than that of lipopeptide liposome, but still positive. Since the phosphate group of siRNA is negatively charged and the lipopeptide liposome is positively charged, the potential of the two is reduced after the electrostatic interaction. Since the N / P of the complex is 10, the number of positively charged groups of the lipopeptide liposome is greater than the phosphate group of the siRNA, so the potential of the above complex remains positive.

[0123] Example 10

[0124] The complex effect of liposome and siRNA

[0125] Step 1: Accurately weigh 1.00 g of agarose powder and place it in 100 mL of UP water. Boil and let it stand. When the temperature of the agarose solution drops to about 50°C, add 10 μL of GelRed staining solution. Shake it evenly and pour it into a mold to remove bubbles and let it stand for 30 minutes. After it solidifies, transfer it to an electrophoresis tank filled with TAE buffer.

[0126] Step 2: Set the N / P of siRNA / lipopeptide liposome complex to 0, 1, 1.5, 2, 5, 10, 15, 20. After the above complex is prepared, add different volumes of DEPC water to each sample to make up the sample volume to 20 μL, and then add 4 μL of 6× glycerol gel loading buffer to all samples. After the above samples are mixed evenly, load 20 μL per well, and set the electrophoresis parameters to 100V, 30min. After the electrophoresis is completed, place the agarose gel under the BioRad imager for photography and observation.

[0127] like Figure 5-Figure 8 As shown, siRNA can be completely complexed in the siRNA / L-M1-D complex and the siRNA / L-N1-D complex when N / P is greater than 5, and siRNA can be completely complexed in the siRNA / L-M1-O complex and the siRNA / L-N1-O complex when N / P is greater than 2.

[0128] Embodiment 11

[0129] Cytotoxicity assay

[0130] Step 1: HaCaT cells in the logarithmic growth phase were digested and counted, and the cell suspension after counting was transferred to a 96-well plate (100 μL / well) and cultured for 24 h.

[0131] Step 2: Take the negative control nucleic acid siNC and place it in a refrigerated centrifuge at 5000rpm and centrifuge at 4℃ for 3min. Carefully remove the nucleic acid and add 125uL of DEPC water to the tube to dissolve it to obtain siNC solution. Prepare siNC / LKD complex and siNC / LRD complex. After the above complexes are prepared, dilute them with DMEM basal medium to 20μg / mL, 15μg / mL, 10μg / mL, 5μg / mL, and 2.5μg / mL.

[0132] Step 3: Take out the cell culture plate, discard the supernatant, and wash twice with PBS buffer. Add the dilution of the above complex to each group and place it in the incubator for 6 hours. After 6 hours, take out the cell culture plate, discard the supernatant, wash the cells twice with PBS buffer, and add complete culture medium to continue culturing for 18 hours.

[0133] Step 4: Take out the cell culture plate, discard the supernatant and wash twice with PBS buffer, add 100 μL / well of 1 mg / mL MTT solution, place in an incubator for 4 hours, discard the supernatant in a light-proof condition, add 150 μL / well of DMSO solution, shake on a 37°C shaking plate for 30 minutes, place the 96-well plate under a multi-function microplate reader to detect the absorbance at 490 nm, and calculate the cell survival rate.

[0134] like Fig. 9 As shown, the cell survival rate of each type of liposome is above 90% within the concentration range of 2.5ug / ml to 20ug / ml, indicating that each liposome is non-toxic to cells and the cells are in good condition.

[0135] Example 12

[0136] In vitro silencing efficiency screening

[0137] Step 1: Digest and count 4T1-Luc cells in the logarithmic growth phase, transfer the counted cell suspension to a 24-well plate (500 μL / well) and culture for 24 hours.

[0138] Step 2: Take the negative control nucleic acid siLuc and place it in a refrigerated centrifuge at 5000rpm, centrifuge at 4℃ for 3min, carefully remove the nucleic acid, add 125uL of DEPC water to the tube to dissolve it, and obtain the siLuc solution. According to the amount of siLuc in each complex of 750ng and N / P of 10, prepare siLuc / LKD complex, siLuc / LRD complex, siLuc / L-M1-D complex, siLuc / L-M1-O complex, siLuc / L-N1-D complex, and siLuc / L-N1-O complex. Select Lipo2000 as the positive control, take Lipo20001μL to DMEM basal medium without dual antibodies, and siLuc 750ng to DMEM basal medium without dual antibodies. Mix the two evenly and let stand for 5min to obtain siLuc / Lipo2000 complex. After the preparation of the above complex is completed, dilute it to 500μL with DMEM basal medium without dual antibodies for use.

[0139] Step 3: Remove the cell culture plate, discard the supernatant, and wash twice with PBS buffer. Add the above-mentioned dilution of siLuc / liposome complex to each group and place it in the incubator for 6 hours. After 6 hours, remove the cell culture plate, discard the supernatant, wash the cells twice with PBS buffer, and add complete medium to continue culturing for 18 hours.

[0140] Step 4: Remove the cell culture plate, discard the supernatant and wash twice with PBS buffer, add 200 μL / well of reporter gene cell lysate, and let stand at room temperature for 40 minutes. Take 100 μL of the above lysate to a black 96-well plate and add 100 μL of firefly luciferase detection reagent, place it in a multifunctional microplate reader to detect the chemiluminescence value. Take 20 μL of the above lysate and calculate the protein concentration in each group of lysates according to the instructions of the BCA kit and calculate the gene silencing efficiency

[0141] like Fig.10 As shown, there was no significant difference in the gene silencing efficiency of each group compared with Lipo2000, indicating that each siRNA / cationic lipopeptide liposome complex had good transfection performance.

[0142] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A dendritic lipopeptide molecular head, characterized in that: Its structure is shown in formula (a). Wherein, R1 and R2 are independently selected from lysine and arginine.

2. A dendritic lipopeptide molecular head according to claim 1, characterized in that: The dendritic lipopeptide molecule head includes M1-OH and N1-OH; When R1 is C5N4H 13 、R2 is C5N2H 13 When , the head of the dendritic lipopeptide molecule is M1-OH; When R1 is C5N2H 13 、R2 is C5N4H 13 When , the head of the dendritic lipopeptide molecule is N1-OH.

3. A method for preparing a dendritic lipopeptide molecular head, characterized in that: The following steps are involved: S1-1, using Fmoc-Lys-OMe·HCl or H-Lys(Fmoc)-OMe·HCl, Boc-Lys(Boc)-OH, HOBT, HBTU as raw materials, undergoing amide condensation reaction to obtain Fmoc-Lys(Boc)-Boc or Boc-Lys(Boc)-Fmoc; S1-2, piperidine is added to Fmoc-Lys(Boc)-Boc or Boc-Lys(Boc)-Fmoc to react and obtain H-Lys(Boc)-Boc or Boc-Lys(Boc)-H; S1-3. Using H-Lys(Boc)-Boc or Boc-Lys(Boc)-H, Boc-Arg(Pbf)-OH, HOBT, HBTU as raw materials, react under vacuum conditions to obtain M1-OMe or N1-OMe. S1-4, adding M1-OMe or N1-OMe to a methanol solution of NaOH to react and obtain the dendritic lipopeptide molecular head M1-OH or N1-OH as claimed in claim 2.

4. The method for preparing a dendritic lipopeptide molecular head according to claim 3, characterized in that: In step S1-1, the molar ratio of Fmoc-Lys-OMe·HCl or H-Lys(Fmoc)-OMe·HCl, Boc-Lys(Boc)-OH, HOBT, and HBTU is 1:1.1-2:1.1-2:1.1-2; in step S1-2, the molar ratio of Fmoc-Lys(Boc)-Boc or Boc-Lys(Boc)-Fmoc to piperidine is 1:19-21; in step S1-3, H-Lys(Fmoc)-OMe·HCl, Boc-Lys(Boc)-OH, HOBT, and HBTU is 1:1.1-2:1.1-2:1.1-2; The molar ratio of (Boc)-Boc, Boc-Arg(Pbf)-OH, HOBT and HBTU is 1:1.1~2:1.1~2:1.1~2; the molar ratio of Boc-Lys(Boc)-H, Boc-Arg(Pbf)-OH, HOBT and HBTU is 1:1.1~2:1.5~2.5:1.5~2.5; in S1-4, the molar ratio of M1-OMe or N1-OMe and NaOH is 1:11~12.

5. A dendritic lipopeptide molecule, characterized in that: Its structure is shown in formula (b): wherein R1 and R2 are independently selected from lysine and arginine; R3 is the structure shown in DSPE or OA below; DSPE: OA:

6. A dendritic lipopeptide molecule according to claim 5, characterized in that: Including the following M1-DSPE, N1-DSPE, Compounds with structures shown by M1-OA and N1-OA; M1-DSPE: N1-DSPE: M1-OA: N1-OA:

7. A method for preparing a dendritic lipopeptide molecule as claimed in claim 6, characterized in that: The following steps are involved: The dendritic lipopeptide molecule is obtained by amide condensation reaction using DSPE compound or OA compound and the head of the dendritic lipopeptide molecule as raw materials; the head of the dendritic lipopeptide molecule is M1-OH or N1-OH as shown in claim 2; Among them, DSPE compound: OA compounds:

8. The method for preparing a dendritic lipopeptide molecule according to claim 7, characterized in that: The molar ratio of the DSPE compound to the dendritic lipopeptide molecular head is 1:2.5-3.0; the molar ratio of the OA compound to the dendritic lipopeptide molecular head is 1:0.2-0.

8.

9. A liposome that can be used for drug delivery, characterized in that: It is mainly formed by self-assembly of cationic lipopeptide, zwitterion and cholesterol through a thin film dispersion method, wherein the cationic lipopeptide is the dendritic lipopeptide molecule according to any one of claims 5-6.

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