CN114456233B

CN114456233B - Cell penetrating peptide-sterol coupling and preparation method thereof

Info

Publication number: CN114456233B
Application number: CN202210245984.4A
Authority: CN
Inventors: 李明媛; 李世勤; 李媛; 贾琳; 苏雯; 户博蕊; 马文林; 孔祥舜; 谢焱博
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2024-02-27
Anticipated expiration: 2042-03-14
Also published as: CN114456233A

Abstract

The invention discloses a cell penetrating peptide-sterol coupling and a preparation method thereof, wherein sterols are cholesterol, ergosterol, beta-sitosterol, stigmasterol and lanosterol, and the amino acid sequence of the cell penetrating peptide is designed autonomously and comprises CGYKK (cysteine-glycine-tyrosine-lysine), CGYRR (cysteine-glycine-tyrosine-arginine) and CGYK (cysteine-glycine-tyrosine-lysine). The Fmoc solid-phase synthesis method is adopted to synthesize the cell penetrating peptide, so that the yield is high and the synthesis speed is high; intermediate compounds are formed by bromoacetic acid or pyridine acid and sterol, and then are connected with polypeptide chains, so that the product has high purity and less impurities. The cell penetrating peptide-sterol coupling is used as a lipid material for a nano drug delivery system, so that on one hand, the transfection efficiency of nano carriers and cells can be improved, and on the other hand, after the transfection into the cells is completed, the cell penetrating peptide-sterol coupling can be degraded into sterols and amino acids by cytolactonase, which are nutrient components available for cell growth, and the cytotoxicity is low. In addition, sterol mother cores with different biological activities can endow corresponding coupling matters with different functions, so that the novel lipid material has wide application prospect in the fields of biological medicines, health care products and cosmetics.

Description

Cell penetrating peptide-sterol coupling and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicine, and relates to a cell penetrating peptide-sterol coupler and a preparation method thereof.

Background

The cell penetrating peptide is a short peptide with positive charges and strong penetrating action on cell membranes, can be used for modifying nano drug delivery systems such as liposome, nanoparticle, micelle and the like, and can promote cell uptake, and is widely focused in the field of drug delivery at present. Taking liposome as an example, the surface of the cell membrane has negative charge, the cell entering capability of the common liposome is limited, the surface of the liposome modified by the cell penetrating peptide has positive charge, and the liposome has electrostatic adsorption effect with the cell membrane, so that the cell entering efficiency of the liposome can be remarkably improved.

A cell penetrating peptide-sterol conjugate refers to sterols modified by cell penetrating peptide, including cholesterol, ergosterol, beta-sitosterol, stigmasterol, lanosterol, etc. Cholesterol is a major component of mammalian cell membranes and interacts with phospholipids to regulate lipid membrane structure, kinetics, etc., and chemical structure is shown in figure 1. Meanwhile, cholesterol has important physiological roles in the human body and is a precursor for synthesis of bile acid and steroid hormone. Cholesterol can be used as pharmaceutic adjuvants such as liposome membrane materials, emulsifying agents, ointment matrixes and the like, and has important effect on improving the stability of the preparation in particular in liposome preparations. When a proper amount of cholesterol is added in the preparation of the liposome, the membrane can keep fluidity and has certain rigidity. Ergosterol is the main sterol component in fungal cell membranes and can be obtained by microbial fermentation synthesis, and has a similar structure to cholesterol and a chemical structure shown in figure 2. Ergosterol has antibacterial, anti-tumor and anti-inflammatory effects, is precursor of progesterone, cortisone, vitamin D2 and other medicines, has important effect on membrane structure, can increase membrane accumulation, and has effect of filling double-layer membrane. Beta-sitosterol (chemical structure shown in figure 3) and stigmasterol (chemical structure shown in figure 4) are plant sterols, are called healthy sterols, and are similar to cholesterol in structure, but can inhibit the absorption of cholesterol in intestinal tracts and influence the metabolism of cholesterol, and have preventive effects on cardiovascular metabolic diseases, cancers and the like, and the dosages of the beta-sitosterol and the stigmasterol have similar thermally induced phase behaviors and liposome oxidation resistance effects; natural plant sterols belong to the non-toxic class of sterols which are allowed to be added in foods, are safe and reliable to use in liposome preparations, have the ability to regulate lipid membranes similar to cholesterol, and the obtained functional liposome can maintain basic characteristics similar to those of original cholesterol liposome; the phytosterol endows the liposome with pharmacological activities such as cholesterol absorption in vivo, anti-tumor and the like; the pharmacological action of the healthy sterols and the absence of cholesterol expand the application of liposome preparations in the field of reducing blood fat. Lanosterol (chemical structure is shown in figure 5) is fungus sterol, is widely used as a safe natural product in the food, medicine and chemical industries, and has the advantages of low production raw material cost, easy industrial production and the like. Lanosterol in an amount of 20% in a certain concentration range has lipid membrane regulating effect. The rich pharmacological activity of the fungus sterols and the slight structural difference endow the liposome with new characteristics.

Through retrieval, catherine, philina, duoton et al (patent publication No. CN 104530190A) adopts Fmoc and Boc solid phase peptide method to synthesize peptide chain, piperidine is used to remove Fmoc protecting group, then condensing agent is diisopropylcarbodiimide, dicyclohexylcarbodiimide and N-ethyl-N ' - (3-dimethyl-aminopropyl) carbodiimide, and unlike the invention, the invention adopts morpholine to remove Fmoc protecting group, and the condensing agent adopts benzotriazole-N, N, N ', N ' -tetramethyl urea hexafluorophosphate and 1-hydroxybenzotriazole; ralff Ai Senhu et al (patent publication No. CN 111670194A) synthesized glucagon peptide by Fmoc solid phase peptide method, and the condensing agent is TBTU, which is obviously different from the present invention, firstly, the target peptide chain is designed independently, and secondly, the condensing agent is HBTU.

The innovation point of the invention is that: first, cell penetrating peptide chains are designed and synthesized autonomously; secondly, forming an intermediate compound by utilizing bromoacetic acid/pyridine acid and sterol, and then connecting the intermediate compound with a polypeptide chain, wherein a synthetic route is designed autonomously; third, other sterols than cholesterol are rarely applied to the construction of nano-delivery vehicles.

Disclosure of Invention

The invention relates to a cell penetrating peptide-sterol coupling and a preparation method thereof, which have the following advantages: firstly, independently designing cell penetrating peptides CGYKK (figure 6), CGYRR (figure 7) and CGYYK (figure 8), and synthesizing by adopting Fmoc solid-phase synthesis method, wherein the yield is high and the synthesis speed is high; secondly, bromoacetic acid/pyridine acid and sterol are utilized to form an intermediate compound, and then the intermediate compound is connected with a polypeptide chain, so that the cell penetrating peptide-sterol coupling is prepared, and after separation and purification, the purity is high, and the impurities are less; thirdly, in the construction of the cell penetrating peptide-sterol coupling obtained by the invention by using nano-carriers, on one hand, the film forming property of sterols is exerted, on the other hand, the electropositivity of the cell penetrating peptide is exerted, and the transfection efficiency of the carriers into cells is improved; fourth, other sterols, except cholesterol, are hardly applied to the construction of nano delivery vehicles, but each has different biological activities, so that the coupling of the corresponding sterols can be selected as lipid materials according to the use of the nano delivery vehicles; fifth, after transfection, the cell penetrating peptide-sterol coupling can be metabolized and decomposed by esterase in cells to become corresponding sterols and amino acids, which are raw materials available for cell growth, and the general quaternary ammonium salt positive charge phospholipid material has a large structural difference from neutral phospholipids in cell membranes in vivo, and lacks efficient metabolic pathways in cells, so that the biological toxicity is high and the cell damage is large.

A method of preparing a cell penetrating peptide-sterol conjugate comprising the steps of:

step 1, coupling reaction is carried out on resin and amino acid, so that linear peptide is coupled on the resin;

step 2, adding cutting fluid into resin for reaction, filtering, and adding Dimethylformamide (DMF) or isopropanol to wash out redundant fluid;

step 3, adding condensing agent and amino acid for connection;

step 4, repeating the operations of the steps 2 and 3, and connecting the last amino acid;

step 5, adding a cutting fluid, and cutting off all protecting groups to obtain target cell penetrating peptide;

step 6, dissolving bromoacetic acid or pyridine acid, EDCI, DMAP and sterol in dichloromethane, stirring in ice bath, then continuously reacting at room temperature, and sequentially using saturated NaHCO ₃ Washing with saturated NaCl aqueous solution, anhydrous MgSO ₄ Or Na (or) ₂ SO ₄ Drying to obtain intermediate compound B.

Step 7, purifying the intermediate compound B through column chromatography to obtain sterol bromoacetate or sterol pyridine acid ester;

and 8, dissolving the cell penetrating peptide, sterol, bromoacetate or pyridine acid ester in DMF, reacting at room temperature for h, and extracting and concentrating to obtain the cell penetrating peptide-sterol coupling.

Specifically, the amino acid addition sequence of CGYKK is lysine, tyrosine, glycine and cysteine; the amino acid addition sequence of CGYRR is arginine, tyrosine, glycine and cysteine; the amino acid addition sequence of CGYYK is lysine, tyrosine, glycine and cysteine; the sterols include cholesterol, ergosterol, beta-sitosterol, stigmasterol, lanosterol.

In particular to a cell penetrating peptide-sterol coupling and a preparation method thereof, wherein sterols comprise cholesterol, ergosterol, beta-sitosterol, stigmasterol and lanosterol,

comprising the following steps:

step 2, adding cutting fluid into resin for reaction, filtering, and adding Dimethylformamide (DMF)/isopropanol to wash out redundant fluid;

step 3, adding condensing agent and amino acid for connection;

step 4, repeating the operations of the steps 2 and 3 until the last amino acid is connected

And 5, adding a cutting fluid, and cutting off all protecting groups to obtain the target cell penetrating peptide.

Step 6, dissolving bromoacetic acid/pyridine acid, EDCI, DMAP, sterol (cholesterol, ergosterol, beta-sitosterol, stigmasterol, lanosterol) in dichloromethane, stirring for 10-60min in ice bath, then continuously reacting at room temperature for 3-24h, sequentially using saturated NaHCO ₃ Washing with saturated NaCl aqueous solution, anhydrous MgSO ₄ /Na ₂ SO ₄ Drying to obtain intermediate compound B.

And 7, purifying the intermediate compound B through column chromatography to obtain sterol bromoacetate/pyridine acid ester.

And 8, reacting the cell penetrating peptide and sterol bromoacetate solution/pyridine acid ester with DMF at room temperature for 0.5-5h, and extracting and concentrating to obtain the cell penetrating peptide-sterol coupling.

Wherein the amino acid addition sequence of CGYKK is lysine, tyrosine, glycine and cysteine; the amino acid addition sequence of CGYRR is arginine, tyrosine, glycine and cysteine; the amino acid addition sequence of CGYYK is lysine, tyrosine, glycine and cysteine.

Specifically, the cell penetrating peptide-sterol coupling is mixed with a phospholipid material, dissolved by an organic solvent, and then a nano-carrier with an entrapment delivery function is prepared by a thin film dispersion method, an anti-solvent volatilization method or a microfluidic method.

The principle of design of cell penetrating peptide-sterol conjugates is as follows: first, only 3-hydroxyl group can be used as reactive group in the structure of sterol, and the carboxyl group at the C-terminal of polypeptide has weak reactivity, and can not directly react with 3-hydroxyl group of sterol, and the amino group at the N-terminal can not directly combine with hydroxyl group, so that it is necessary to react 3-hydroxyl group of sterol with bromoacetic acid or picolinic acid to form ester, and the reaction condition is mild and the yield is high (for example, the reaction with bromoacetic acid is shown in fig. 9). To introduce it into both ends of the peptide chain, 1 cysteine was added to the N-terminus of the peptide chain, and a thiol (-SH) group of its side chain was used to chemically react with it, thereby linking the sterol to the polypeptide (see FIG. 10 for a reaction scheme, exemplified by the reaction of CYGKK with cholesterol bromoacetate). Secondly, in order to maintain the conformational flexibility of sterols when combined with lipid membranes, 1 glycine is added between cysteine and peptide chain, and the flexibility of the molecules is utilized to achieve the aim of maintaining the conformational flexibility of each of peptide chain and sterols, and simultaneously, the steric hindrance is minimum. Third, the C-terminus of the cell penetrating peptide is required to meet the rules of Cend R motifs, i.e., the C-terminus must be arginine or lysine to provide a positive charge. Fourth, tyrosine is an amino acid containing a benzene ring, which can increase the immunogenicity of peptide chains. Fifth, the amino acids of the peptide chain repeats are designed to provide positive charges and increase immunogenicity; the cell penetrating peptide-sterol coupling of the invention has positive charges, and can assist DNA, siRNA, mRNA and the like to enter cells; the cell penetrating peptide-sterol coupling can be used as a phospholipid material to be applied to a nano-carrier prescription, so that the delivery efficiency of the cell penetrating peptide-sterol coupling is improved, and the cell penetrating peptide-sterol coupling can be applied to the fields of biological medicines, health care products, cosmetics and the like.

Description of the drawings:

FIG. 1 is a chemical structure of cholesterol

FIG. 2 is a chemical structure of ergosterol

FIG. 3 is a chemical structure of beta-sitosterol

FIG. 4 is a chemical structure of stigmasterol

FIG. 5 is a chemical structure of lanosterol

FIG. 6 is a chemical formula of CGYKK

FIG. 7 is a chemical formula of CGYRR

FIG. 8 is a chemical formula of CGYYYK

FIG. 9 is a chemical reaction of sterols (A cholesterol, B ergosterol, C.beta. -sitosterol, D stigmasterol, E lanosterol) with bromoacetic acid

FIG. 10 is a chemical reaction formula of cholesterol bromoacetate and peptide chain CGYKK

FIG. 11 is a synthetic reaction scheme for CGYKK of example 1

FIG. 12 shows MALDI-TOF MS spectra (A) and HPLC (B) of CGYKK of example 1

FIG. 13 is a synthetic reaction formula of CGYRR of example 2

FIG. 14 shows MALDI-TOF MS spectra (A) and HPLC (B) of CGYRR of example 2

FIG. 15 is a synthetic reaction formula of example 3 CGYYYK

FIG. 16 shows MALDI-TOF MS spectra (A) and HPLC (B) of example 3CGYYK

FIG. 17 shows the structures of bromoacetic acid of example 5 (A) and picolinic acid of example 6 (B)

FIG. 18 is a synthetic route for cholesterol esters of example 6

FIG. 19 shows HNMR (A) and HPLC (B) graphs of cholesterol esters of example 6

FIG. 20 shows MALDI-TOF MS and HPLC images (A) and (B) of CGYKK-cholesterol of example 7

FIG. 21 shows MALDI-TOF MS and HPLC patterns (A) and (B) of CGYRR-cholesterol of example 8

FIG. 22 shows MALDI-TOF MS and HPLC images (A) and (B) of CGYYK-cholesterol of example 9

FIG. 23 is a synthetic route for ergosterol ester of example 10

FIG. 24 is a HNMR (A) and HPLC (B) graphs of ergosterol esters of example 10

FIG. 25 shows MALDI-TOF MS and HPLC graphs (A) of CGYKK-ergosterol of example 11

FIG. 26 is a MALDI-TOF MS plot (A) and an HPLC plot (B) of CGYRR-ergosterol of example 12

FIG. 27 is a MALDI-TOF MS plot (A) and an HPLC plot (B) of CGYYK-ergosterol of example 13

FIG. 28 is a particle size potential diagram of example 15

FIG. 29 is a scanning image of a transmission electron microscope of example 15

FIG. 30 is an inverted fluorescence microscope image of cell uptake in example 15 and example 16

FIG. 31 is a graph showing transfection efficiency of example 14 and comparative example 1

FIG. 32 shows the results of the cytotoxicity MTT assay of examples 7, 8, 9, 11, 12 and 13

FIG. 33 shows the results of the cytotoxicity MTT assay of examples 1, 15 and 16

Detailed Description

The technical scheme of the invention is further described by specific examples.

EXAMPLE 1 Synthesis of CGYKK peptide chain

1g of 2-chloro-3 benzyl chloride resin and 2.23g of Fmoc-Lys (Boc) -OH are weighed and dissolved in 10mL of DCM, 1.77mL of DIEA is added dropwise into the mixture for reaction for 3.5h, 0.48mL of MeOH is added dropwise into the mixture for reaction for 0.5h, after the reaction is finished, the mixture is filtered (liquid is removed) by suction, isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added for reaction for 30min, and the mixture is detected by ninhydrin, and the resin is blue to indicate that Fmoc protecting groups are removed, and isopropanol/DMF is added for cleaning; 1.67g Fmoc-Lys (Boc) -OH, 1.35g HBTU and 0.48g HOBt are added and dissolved in 10mL DMF, 1.18mL DIEA is added thereto and reacted for 2.5h, after the reaction is finished, the reaction is detected by ninhydrin, the resin is colorless, indicating that the amino acid is already connected, the reaction is filtered (liquid is removed), isopropanol/DMF is added for cleaning, 10mL30% morpholine (morpholine/DMF) is added and reacted for 30min, the resin is blue, indicating that the Fmoc protecting group has been removed, and isopropanol/DMF is added for cleaning; adding 1.64g of Fmoc-Tyr (tBu) -OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA into the mixture, reacting for 2.5h, detecting the reaction to be finished through ninhydrin, detecting the resin to be colorless, indicating that amino acid is connected, performing suction filtration (removing liquid), adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), reacting for 30min, detecting the resin to be blue through ninhydrin, indicating that Fmoc protecting group is removed, and adding isopropanol/DMF for cleaning; adding 1.06g Fmoc-Gly-OH, 1.35g HBTU and 0.48g HOBt into 10mL DMF, adding 1.18mL DIEA, reacting for 2.5h, detecting by ninhydrin, filtering (removing liquid) after the resin is colorless, adding isopropanol/DMF for cleaning, adding 10mL30% morpholine (morpholine/DMF), reacting for 30min, detecting by ninhydrin, and removing Fmoc protecting group after the reaction is completedWashing with propanol/DMF; 2.09g of Fmoc-Cys (Trt) -OH, 1.35g of HBTU and 0.48g of HOBt are added and dissolved in 10mL of DMF, 1.18mL of DIEA is added thereto for reaction for 2.5h, after the reaction is finished, the reaction is detected by ninhydrin, the resin is colorless, which indicates that the amino acid is attached, suction filtration (liquid removal) is carried out, isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added for reaction for 30min, and the resin is blue after ninhydrin detection, which indicates that the Fmoc protecting group has been removed. At this time, CGYKK-resin is formed, CGYKK is cut off from branches, trt, boc, tBU protecting groups are removed, 10mL of lysate (8.25 mL of TFA, 0.5mL of toluene thioether, 0.25mL of water, 0.5mL of phenol and 0.5mL of ethanedithiol) is added to react for 3.5 hours, after the reaction is finished, pumping filtration is carried out, liquid is dripped into frozen 20-30mL of methyl tertiary ether, namely sediment is generated, the sediment is obtained through centrifugation, and argon is dried to obtain CGYKK (chemical reaction formula is shown in figure 11), white solid, total 456mg is obtained, and the product is detected by MALDI-TOF and HPLC, wherein the MALDI-TOF MS value is [ M-1 ]] ^- (FIG. 12A), HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: awater 50% Bmethanol 50%, flow rate: 0.6ml/min, column temperature: 30 ℃ C.; detection wavelength: 220nm, sample introduction amount: 20. Mu.l, purity of 92% was detected (FIG. 12B).

EXAMPLE 2 Synthesis of CGYRR peptide chain

1g of 2-chloro-3 benzyl chloride resin and 3.08g of Fmoc-Arg (Pbf) -OH are weighed and dissolved in 10mL of DCM, 1.77mL of DIEA is added dropwise into the mixture for reaction for 3.5h, 0.48mL of MeOH is added dropwise into the mixture for reaction for 0.5h, the mixture is filtered (liquid is removed) after the reaction is finished, isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added for reaction for 30min, after the reaction is finished, ninhydrin is detected, the resin is blue, the Fmoc protecting group is removed, and isopropanol/DMF is added for cleaning; adding 2.32g of Fmoc-Arg (Pbf) -OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA into the mixture, reacting for 2.5h, detecting the reaction to be finished by ninhydrin, detecting the resin to be colorless, indicating that amino acid is connected, filtering (removing liquid), adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), detecting the resin to be blue, indicating that Fmoc protecting group is removed, and adding the iso-isomerWashing with propanol/DMF; 1.64g of Fmoc-Tyr (tBu) -OH, 1.35g of HBTU and 0.48g of HOBt are added and dissolved in 10mL of DMF, 1.18mL of DIEA is added to the mixture to react for 2.5h, after the reaction is finished, the reaction is detected by ninhydrin, the resin is colorless, which indicates that the amino acid is connected, the reaction is performed by suction filtration (liquid removal), isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added, the resin is detected by ninhydrin, the resin is blue, which indicates that the Fmoc protecting group is removed, and isopropanol/DMF is added for cleaning; adding 1.06g of Fmoc-Gly-OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA into the mixture, reacting for 2.5h, detecting by ninhydrin, filtering (removing liquid) after the resin is colorless to indicate that amino acid is connected, adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), detecting by ninhydrin, and adding isopropanol/DMF for cleaning after the resin is blue to indicate that Fmoc protecting group is removed; 2.09g of Fmoc-Cys (Trt) -OH, 1.35g of HBTU and 0.48g of HOBt are added and dissolved in 10mL of DMF, 1.18mL of DIEA is added thereto and reacted for 2.5h, after the reaction is finished, the reaction is detected by ninhydrin, the resin is colorless, indicating that the amino acid is attached, suction filtered (liquid removed), and washed by adding isopropanol/DMF, and after 10mL of 30% morpholine (morpholine/DMF) is added, the resin is blue, indicating that the Fmoc protecting group has been removed. At this time, CGYRR-resin is formed, CGYRR is cut off from branches, a Trt, boc, tBU protecting group is removed, 10mL of lysate (8.25 mL of TFA, 0.5mL of toluene thioether, 0.25mL of water, 0.5mL of phenol and 0.5mL of ethanedithiol) is added to the mixture to react for 3.5 hours, after the reaction is finished, suction filtration is performed, liquid is dripped into frozen 20-30mL of methyl tertiary ether, precipitation is generated, centrifugation is performed, precipitation is taken out, and argon is used for drying, so that CGYRR (chemical reaction formula shown in figure 13) is obtained, and the white solid is 436mg. MALDI-TOF detection MS value is [ M-1 ]] ^- See fig. 14A. The product was detected by HPLC, method: chromatographic column: ZORBAX SB-C18 (4.6mm. Times.250 mm,5 μm); mobile phase: water 50% b methanol: 50%; flow rate: 0.6ml/min; column temperature: 30 ℃; detection wavelength: 220nm; sample injection amount: 20 μl, with purity of 90% as measured in FIG. 14B.

EXAMPLE 3 Synthesis of CGYYYK peptide chain

Weighing and weighing1g of 2-chloro-3 benzyl chloride resin and 2.23g of Fmoc-Lys (Boc) -OH are dissolved in 10mL of DCM, 1.77mL of DIEA is added dropwise into the mixture for 3.5h, 0.48mL of MeOH is added dropwise into the mixture for 0.5h, after the reaction is finished, the mixture is filtered (liquid is removed), isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added, and the mixture is detected by ninhydrin, and the resin is blue, which indicates that Fmoc protecting groups are removed, and isopropanol/DMF is added for cleaning; adding 1.64g of Fmoc-Tyr (tBu) -OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA into the mixture, reacting for 2.5h, detecting the reaction to be finished through ninhydrin, detecting the resin to be colorless, indicating that amino acid is connected, carrying out suction filtration (removing liquid), adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), detecting through ninhydrin, detecting the resin to be blue, indicating that Fmoc protecting groups are removed, and adding isopropanol/DMF for cleaning; 1.64g of Fmoc-Tyr (tBu) -OH, 1.35g of HBTU and 0.48g of HOBt are added and dissolved in 10mL of DMF, 1.18mL of DIEA is added to the mixture to react for 2.5h, after the reaction is finished, the reaction is detected by ninhydrin, the resin is colorless, which indicates that the amino acid is connected, the reaction is performed by suction filtration (liquid removal), isopropanol/DMF is added for cleaning, 10mL of 30% morpholine (morpholine/DMF) is added, the resin is detected by ninhydrin, the resin is blue, which indicates that the Fmoc protecting group is removed, and isopropanol/DMF is added for cleaning; adding 1.06g of Fmoc-Gly-OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA into the mixture, reacting for 2.5h, detecting by ninhydrin, filtering (removing liquid) after the resin is colorless to indicate that amino acid is connected, adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), detecting by ninhydrin, and adding isopropanol/DMF for cleaning after the resin is blue to indicate that Fmoc protecting group is removed; adding 2.09g of Fmoc-Cys (Trt) -OH, 1.35g of HBTU and 0.48g of HOBt into 10mL of DMF, adding 1.18mL of DIEA thereto, reacting for 2.5h, detecting the end of the reaction, detecting the presence of ninhydrin, filtering (removing liquid) the resin until the amino acid is connected, adding isopropanol/DMF for cleaning, adding 10mL of 30% morpholine (morpholine/DMF), detecting the presence of ninhydrin, detecting the resin until the resin is blue, indicating that the Fmoc protecting group is removed, forming CGYYK-resin, cutting CGYRR from the branches, and removing Trt, boc, tBU A protecting group, adding 10mL of lysate (8.25 mL of TFA, 0.5mL of toluene thioether, 0.25mL of water, 0.5mL of phenol, 0.25mL of ethanedithiol) to react for 3.5 hours, after the reaction is finished, filtering, dripping the liquid into frozen 20-30mL of methyl tertiary ether, namely generating precipitate, centrifuging, taking the precipitate, drying the precipitate by argon to obtain CGYYK (chemical reaction formula shown in figure 15), adding 532mg of white solid, and detecting MALDI-TOF to obtain MS value of [ M-1 ]] ^- See fig. 16A. The product was detected by HPLC, method: chromatographic column: ZORBAX SB-C18 (4.6mm. Times.250 mm,5 μm); mobile phase: 0-60min, from 5% water, 95% acetonitrile to 95% water, 5% acetonitrile; flow rate: 0.6ml/min; column temperature: 30 ℃; detection wavelength: 220nm; sample injection amount: 20 μl, with a purity of 83% as measured in FIG. 16B.

EXAMPLE 4 Synthesis of cholesterol bromoacetate

4.5g bromoacetic acid, 5.0g cholesterol, 8.7g EDCI and 79mg DMAP were dissolved in 35mL Dichloromethane (DCM), stirred in an ice bath for 30min, then continued to react at room temperature for 12h, followed by sequential use of saturated NaHCO ₃ Washing with saturated NaCl aqueous solution, anhydrous MgSO ₄ And (5) drying. Purifying by column chromatography, eluting with eluent: v (Petroleum ether): V (ethyl acetate) =10:1, yielding 3.6g of a white solid with MS (m/z) of 524[ M+18 ]] ⁺ The nuclear magnetism is as follows: HNMR (CDCl 3, 250 MHz), δ:5.4 (d, 1h, j=3.9hz, 6-CH-cholestenol); 4.6 (m, 1h, 3-CH-cholestenol); 3.8 (s, 2H, CH2 Br); 0.6-2.25 (m, 43H,cholesterol protons) to obtain the cholesterol bromoacetate.

EXAMPLE 5 Synthesis of ergosterol bromoacetate

4.4g bromoacetic acid (structure shown in FIG. 17B), 5.0g ergosterol, 8.5g EDCI and 77mg DMAP were dissolved in 35mL Dichloromethane (DCM), stirred in an ice bath for 30min, then reacted for 12h at room temperature, followed by sequential use of saturated NaHCO ₃ Washing with saturated NaCl aqueous solution, anhydrous MgSO ₄ And (5) drying. Purifying by column chromatography, eluting with eluent: v (Petroleum ether): V (ethyl acetate) =10:1, yielding 3.2g of a white solid with MS (m/z) 534[ M+18 ]] ⁺ The nuclear magnetism is as follows: 1H NMR (CDCl 3, 400 MHz), δ:5.58-5.56 (m, 1H); 5.39-5.38 (m, 1H); 5.22-5.18 (m, 2H); 3.8 (s, 2H, CH2 Br); 2.49-2.44 (m, 1H); 2.28 (t, 1h, j=12 Hz); 1.91-1.90 (m, 3H); 1.88-1.87 (m, 3H); 1.55-1.25 (m, 16H); 1.04-1.01 (m, 2H); 0.95-0.91 (m, 5H); 0.85-0.80 (m, 5H); 0.63 (s, 2H) to obtain ergosterol bromoacetate.

EXAMPLE 6 Synthesis of Cholesterol picolinate

184mg of picolinic acid (structure shown in FIG. 17A), 300mg of cholesterol, 4.7mg of DMAP (4-lutidine) were precisely weighed out and dissolved in 3mL of methylene chloride, 178mg of EDCI was added thereto, and the reaction was continued at 0℃for 30 minutes and at 25℃for 6 hours, and the route was shown in FIG. 18. Monitoring (filipin for cholesterol development, other substances developed in ultraviolet (254 nm) PE: ea=5: 1, R _f =0.35. Adding 2mL of water for quenching reaction, adding 5mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 5mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Combining the organic phases, adding saturated saline, shaking thoroughly, separating the organic phases, and adding anhydrous Na ₂ SO ₄ Drying and finally the solvent was removed under reduced pressure to give 417mg of yellow oil. Mixing 417mg crude product with 1.5 times 200-300 mesh silica gel, loading into column by dry method, and filling the column with the proportion of 30:1, the eluent is PE: ea=100: 1-20:1, 100mg of a colourless solid are finally obtained. HNMR demonstrated structural correctness by HPLC and HNMR detection, see fig. 19A. And HPLC (method: chromatographic column: ZORBAX SB-C18 (4.6mm. Times.250 mm,5 μm), mobile phase: methanol 100%, flow rate: 1ml/min, column temperature: 30 ℃ C., detection wavelength: 220nm, sample injection amount: 20. Mu.l) to confirm that the purity is about 85%, to obtain cholesterol picolinate, see FIG. 19B.

EXAMPLE 7 Synthesis of CGYKK-Cholesterol conjugate

The peptide chain CGYKK (53.8 mg) was dissolved in 0.5mL of DMF, and cholesterol ester (50.0 mg) was dissolved in 0.5mL of DMF and added dropwise to the solution of the peptide chain CGYKK, and reacted at 25℃for 2 hours. PE: ea=5:1, rf=0.02. Adding 2mL of water for quenching reaction, adding 1mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Will beMixing the organic phases, adding saturated saline, shaking thoroughly, separating the organic phase, adding anhydrous Na ₂ SO ₄ Drying and finally, the solvent was removed under reduced pressure from the organic phase to obtain 45mg of a yellow oily substance. Drying in a vacuum oven for 24 hours gave 30mg of pale yellow solid. The product was subjected to HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: A water 25%, B methanol 75%, flow rate: 1ml/min, column temperature: 30 ℃ C.; detection wavelength: 220nm; sample injection amount: 20. Mu.l), purity about 95%, see FIG. 20B, and MALDI-TOF detection, MS value of [ M+18 ]] ⁺ See FIG. 20A, to obtain CGYKK-cholesterol conjugate.

EXAMPLE 8 Synthesis of CGYRR-Cholesterol conjugate

Peptide chain CGYRR (109.1 mg) was dissolved in 0.5mL of DMF, and cholesterol ester (50.0 mg) was dissolved in 0.5mL of DMF and added dropwise to the solution of peptide chain CGYRR, and reacted at 25℃for 2 hours. PE: ea=5: 1, rf=0.02. Adding 2mL of water for quenching reaction, adding 1mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Combining the organic phases, adding saturated saline, shaking thoroughly, separating the organic phases, and adding anhydrous Na ₂ SO ₄ Drying and finally, the solvent was removed under reduced pressure from the organic phase to obtain 67mg of a yellow oily substance. Drying in a vacuum oven for 24 hours gave 53mg of pale yellow solid. The product was subjected to HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: A water 25%, B methanol 75%, flow rate: 1ml/min, column temperature: 30 ℃ C.; detection wavelength: 220nm; sample injection amount: 20. Mu.l), purity about 81%, see FIG. 21B, and MALDI-TOF detection, MS value of [ M+18 ]] ⁺ See FIG. 21A, to obtain CGYRR-cholesterol conjugate.

EXAMPLE 9 Synthesis of CGYYK-cholesterol conjugate

Peptide chain CGYYK (105.6 mg) was dissolved in 0.5mL of DMF, and cholesterol ester (50.0 mg) was dissolved in 0.5mL of DMF and added dropwise to a solution of peptide chain CGYK, and reacted at 25℃for 2 hours. PE: ea=5: 1, rf=0.02. 2mL of water is added for quenching reaction, 1mL of ethyl acetate is added for extraction, and the mixture is fully subjected toAfter mixing, standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Combining the organic phases, adding saturated saline, shaking thoroughly, separating the organic phases, and adding anhydrous Na ₂ SO ₄ Drying and finally, the solvent was removed under reduced pressure from the organic phase to obtain 56mg of a yellow oily substance. Drying in a vacuum oven for 24 hours gave 38mg of a pale yellow solid. The product was subjected to HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: A water 25%, B methanol 75%, flow rate: 1ml/min, column temperature: 30 ℃ C.; detection wavelength: 220nm; sample injection amount: 20. Mu.l), purity about 90%, see FIG. 22B, and MALDI-TOF detection, MS value of [ M+18 ]] ⁺ See FIG. 22A, to obtain CGYKK-cholesterol conjugate.

EXAMPLE 10 Synthesis of ergosterol picolinate

89mg of picolinic acid, 150mg of ergosterol and 2.3mg of DMAP were precisely weighed out and dissolved in 3mL of methylene chloride, and 87mg of EDCI was added thereto, and the reaction was continued at 0℃for 30 minutes, and continued at 25℃for 8 hours, as shown in FIG. 23.PE: ea=5: 1, product ergosterol ester rf=0.35. Adding 2mL of water for quenching reaction, adding 5mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 5mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Combining the organic phases, adding saturated saline, shaking thoroughly, separating the organic phases, and adding anhydrous Na ₂ SO ₄ Drying and finally the organic phase was freed of solvent under reduced pressure to give 150mg of yellow solid. Mixing 150mg crude product with 1.5 times 200-300 mesh silica gel, loading into column by dry method, and filling the column with the proportion of 30:1, the eluent is PE: ea=100: 1-20:1, 30mg of a white solid was finally obtained. The product was tested by HNMR and HPLC, which demonstrated the structural correctness of the product, see fig. 24A. HPLC (method: chromatographic column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: methanol 100%, flow rate: 1ml/min, column temperature: 30 ℃ C., detection wavelength: 220nm, sample injection amount: 20. Mu.l) detects that the purity is about 85%, see FIG. 24B, to obtain ergosterol pyridine acid ester.

EXAMPLE 11 Synthesis of CGYKK-ergosterol conjugate

The peptide chain CGYKK (21.1 mg,1.05eq,0.035 mmol) was dissolved in 0.5mL DMF, ergosterol ester (20.0 mg,1.0eq,0.033 mmol) was dissolved in 0.5mL DMF and added dropwise to the solution of peptide chain CGYKK and reacted at 25℃for 2 hours. PE ea=5:1, rf=0.02. Adding 2mL of water for quenching reaction, adding 1mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. The organic phases were combined, added with saturated brine, shaken well, the organic phase was separated, dried with anhydrous Na2SO4, and finally the solvent was removed under reduced pressure to give 40mg of a yellow oily substance, which was dried in a vacuum oven for 24h to give 20mg of a pale yellow solid. The product was tested by MALDI-TOF and HPLC, and its MS value was [ M+1 ] by MALDI-TOF] ⁺ See fig. 25A. HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: water 25%, methanol 75% B, flow rate: 1ml/min, column temperature: 30 ℃ C., detection wavelength: 220nm, sample injection amount: 20. Mu.l) shows that the purity was about 95%, see FIG. 25B. Thus obtaining the CGYKK-ergosterol coupling product.

EXAMPLE 12 Synthesis of CGYRR-ergosterol conjugates

Peptide chain CGYRR (42.7 mg) was dissolved in 0.5mL of DMF, ergosterol ester (20.0 mg) was dissolved in 0.5mL of DMF, added dropwise to the solution of peptide chain CGYRR, and reacted at 25℃for 2 hours. PE ea=5:1, rf=0.02. Adding 2mL of water for quenching reaction, adding 1mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. The organic phases were combined, added with saturated brine, shaken well, the organic phase was separated, dried with anhydrous Na2SO4, and finally the solvent was removed under reduced pressure to give 36mg of a yellow oily substance, which was dried in a vacuum oven for 24h to give 22mg of a pale yellow solid. The product was tested by MALDI-TOF and HPLC, and its MS value was [ M+18 ] by MALDI-TOF] ⁺ See fig. 26A. The product was tested by HPLC, HPLC (method: chromatography column: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: 25% of A water, 75% of B methanol, flow rate: 1ml/min, column temperature: 30 ℃ C., test wavelength: 220nm, sample injection amount: 20. Mu.l)The purity was about 84%, see fig. 26B. Thus obtaining the CGYRR-ergosterol coupling product.

EXAMPLE 13 CGYYK-ergosterol coupling

Peptide chain CGYYK (41.3 mg) was dissolved in 0.5mL of DMF, ergosterol ester (20.0 mg) was dissolved in 0.5mL of DMF, and added dropwise to a solution of peptide chain CGYYK, and reacted at 25℃for 2 hours. PE: ea=5: 1, rf=0.02. Adding 2mL of water for quenching reaction, adding 1mL of ethyl acetate for extraction, fully and uniformly mixing, and standing to separate out upper liquid (organic phase); the aqueous phase was extracted with a further 1mL of ethyl acetate and the procedure repeated until the aqueous phase was free of product. Combining the organic phases, adding saturated saline, shaking thoroughly, separating the organic phases, and adding anhydrous Na ₂ SO ₄ Drying, and finally, the solvent was removed under reduced pressure from the organic phase to obtain 41mg of a yellow oily substance, which was dried in a vacuum oven for 24 hours to obtain 24mg of a pale yellow solid. The product was tested by MALDI-TOF and HPLC, and its MS value was [ M+18 ] by MALDI-TOF] ⁺ See fig. 27A. The product was tested by HPLC, and its purity was about 93% by HPLC (method: column chromatography: ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm), mobile phase: 25% of A water, 75% of B methanol, flow rate: 1ml/min, column temperature: 30 ℃ C., test wavelength: 220nm, sample injection amount: 20. Mu.l), see FIG. 27B. Thus obtaining the CGYYK-ergosterol coupling product.

EXAMPLE 14 CGYKK-Cholesterol coupler Liposome preparation

Liposomes were prepared using the CGYKK-cholesterol conjugate from example 7 as the major lipid material; the operation is as follows: firstly, 14.96mg of DOTAP, 14.88mg of DOPE and 7mg of CGYKK-cholesterol coupling are weighed and dissolved in 3mL of chloroform, the organic solvent is removed by reduced pressure rotary evaporation at 37 ℃ and a layer of film is formed on the bottle wall; then, 2.99mL nuclease-free water was added and hydrated for 20min at 55 ℃; then transferring the liposome into a penicillin bottle, performing ultrasonic treatment on the liposome for 5min by using an ultrasonic cell grinder, and filtering with a 0.22 mu m filter membrane to obtain the cationic liposome. The cationic liposome is mixed with EGFP mRNA in a mass ratio of 8:1, and incubated for 20min, so that the liposome containing EGFP mRNA can be prepared.

EXAMPLE 15 preparation of CGYRR-Cholesterol conjugate Liposome

Preparing liposome by using CGYRR-cholesterol coupling obtained in example 8 as main lipid material; the operation is as follows: firstly, weighing 19.26mg of DOTAP, 5.129mg of DOPE, 5.06mg of DPPC and 7mg of CGYRR-cholesterol coupling, adding 3.14mg of coumarin-6 to dissolve in 3mL of chloroform, and removing the organic solvent by rotary evaporation under reduced pressure at 37 ℃ to form a layer of film on the bottle wall; then, 5.16mL nuclease-free water was added and hydrated for 20min at 55 ℃; then transferring the liposome into a penicillin bottle, ultrasonically treating the liposome for 5min by using an ultrasonic cell grinder probe, and filtering with a 0.22 mu m filter membrane to obtain the cationic liposome.

EXAMPLE 16 preparation of CGYYK-Cholesterol conjugate Liposome

Liposomes were prepared using the CGYYK-cholesterol conjugate obtained in example 9 as the primary lipid material; the operation is as follows: firstly, 18.87mg of DOTAP, 5.129mg of DOPE, 4.95mg of DPPC and 7mg of CGYYK-cholesterol coupling are weighed, then 3.59mg of coumarin-6 is added and dissolved in 3mL of chloroform together, the organic solvent is removed by rotary evaporation under reduced pressure at 37 ℃, and a layer of film is formed on the bottle wall; then, 5.07mL nuclease-free water was added and hydrated for 20min at 55 ℃; then transferring the liposome into a penicillin bottle, ultrasonically treating the liposome for 5min by using an ultrasonic cell grinder probe, and filtering with a 0.22 mu m filter membrane to obtain the cationic liposome.

Comparative example 1

A commonly used transfection reagent Lipo2000 is commercially available.

Experimental example 1

The molecular weight of CGYKK, CGYRR, CGYYK obtained in example 1, example 2 and example 3 was confirmed by MALDI TOF-MS, and the results are shown in Table 1.

TABLE 1

As can be seen from Table 1, MALDI TOF-MS is [ M-1 ]] ^- The peptide chain MS was determined to be correct.

Experimental example 2

The CGYKK-cholesterol conjugate, CGYRR-cholesterol conjugate and CGYYK-cholesterol conjugate obtained in example 7, example 8 and example 9 were used to confirm the molecular weight by MALDI TOF-MS, and the results are shown in Table 2.

TABLE 2

Experimental example 3

The CGYKK-ergosterol conjugates, CGYRR-ergosterol conjugates, and CGYYK-ergosterol conjugates obtained in example 11, example 12, and example 13 were used to confirm the molecular weight by MALDI TOF-MS, and the results are shown in Table 3.

TABLE 3 Table 3

Experimental example 4 particle size potential measurement

The prepared example 15 was diluted by an appropriate factor, and the particle size and potential of the liposome were analyzed under room temperature conditions using a Malvern Nano-ZS 90 laser particle sizer. As shown in FIG. 28, the particle size is about 110nm, the particle sizes are all less than 200nm, and the polydispersity is less than 0.2, which indicates that the dispersity is good.

The potential was about +40mv, which proves that the liposome is positively charged and is a cationic liposome.

Experimental example 5 Transmission Electron microscope test

The liposome prepared in example 14 was added dropwise onto a copper mesh, and the mixture was blotted with filter paper, followed by the addition of phosphotungstic acid and air-drying. The compound liposome morphology was determined by transmission electron microscopy, TEM. From fig. 29, it can be seen that the liposomes were uniformly dispersed in a spherical shape.

Experimental example 6 cell uptake experiment

HepG2 cells were cultured to 5X 10 ⁴ The culture was performed in an incubator for 36 hours by plating/ml on a 6-well plate. Sucking out original culture medium, adding 1ml PBS, washing, adding 1ml lysosome (lso-track-red) dye, and 1ml HoechThe est staining solution was added simultaneously with 60. Mu.l of each of example 15 and example 16 in a cell culture plate, and after culturing in a cell culture incubator at 37℃for 30-45min, the staining solution was aspirated, washed once every 5min with 1ml PBS, and after washing twice, the PBS solution was left for inverted fluorescence microscopy. As can be seen from fig. 30, example 15 and example 16 can enter cells through cell membranes, indicating good uptake capacity.

Experimental example 7 transfection efficiency experiment

HEK-293 cells were cultured at a cell density of 1X 10 ⁵ After plating 6-well plates per ml and culturing in a cell incubator for 24 hours. 1ml of comparative example 1 was diluted 10-fold and added to the first well of the cell culture plate, followed by 60. Mu.l of the liposome prepared in example 14 to the second well of the cell culture plate. Following 24h incubation, the assay was performed by flow cytometry. As can be seen from fig. 31, the transfection efficiency of example 14 was as high as 66.70%, and the transfection efficiency of comparative example 1 was 48.30%, demonstrating that example 14 has a higher transfection efficiency.

Experimental example 8 cytotoxicity experiment of Compounds

HepG2 cells were cultured at a cell density of 1X 10 ^5/ The cells were cultured in 96-well plates and cell culture boxes for 24 hours in ml, and then cytotoxicity MTT assay was performed at the same dose for each of example 7, example 8, example 9, example 11, example 12 and example 13, and the cell viability assay results are shown in FIG. 32. From the results, it can be seen that the compounds are less toxic and the cell viability is high.

Experimental example 9 Liposome toxicity experiment

HEK-293 cells were cultured at a cell density of 1X 10 ⁵ The cells were cultured in a 96-well plate/ml incubator for 24 hours, and then the cytotoxicity MTT assay was performed at the same dose as in comparative example 1 and example 15 and example 16, respectively, and the results are shown in FIG. 33. And it is obvious that the cell survival rate is high and the toxicity is low.

In summary, adding CGYKK-cholesterol coupling, CGYRR-cholesterol coupling and CGYYK-cholesterol coupling in the liposome formulation can improve the transfection efficiency of carrier cells, reduce cytotoxicity, and is a high-quality novel lipid material which can be added in nano-drug delivery systems.

Claims

1. A cell penetrating peptide-sterol conjugate, characterized by: the structure of the cell penetrating peptide is selected from any one of CGYKK, CGYRR, CGYYK;

the sterol is cholesterol or ergosterol or beta-sitosterol or stigmasterol or lanosterol;

the sterols are linked to the cysteine at the N-terminus of the peptide chain.

2. The use of a cell penetrating peptide-sterol conjugate according to claim 1 in the preparation of a nanocarrier.

3. Use of a cell penetrating peptide-sterol conjugate according to claim 2 for the preparation of a nanocarrier, said nanocarrier being a lipid nanoparticle, a liposome, an exosome, a micelle.

4. Use of a cell penetrating peptide-sterol conjugate according to claim 2 for the preparation of nanocarriers, wherein: mixing the cell penetrating peptide-sterol coupling with a phospholipid material, dissolving the mixture by an organic solvent, and preparing the nano-carrier with the entrapment delivery function by a film dispersion method, an anti-solvent volatilization method or a microfluidic method.

5. The use of a cell penetrating peptide-sterol conjugate according to any one of claims 1-4 in the preparation of a pharmaceutical product, a health product, a cosmetic product.