CN115925812A - Zwitterionic polypeptide lipid molecule and application thereof - Google Patents

Abstract

The invention discloses a zwitterionic polypeptide lipid molecule and application thereof, belonging to the technical field of drug delivery systems. The general formula of the zwitterionic polypeptide lipid molecule is R- (E-K) _n Wherein R is a fatty acid, a diglyceride fatty acid ester derivative or a cholesterol derivative, E is glutamic acid, K is lysine, and n is an integer of 5 to 20. The zwitterionic polypeptide lipid molecule is formed by bonding fatty acid, fatty acid diglyceride derivative or cholesterol derivative and EK polypeptide, and can be used asThe substitute component of the PEG lipid in the lipid nanoparticle, the cationic lipid, the phospholipid and the cholesterol form the lipid nanoparticle, and the lipid nanoparticle has the capability of delivering mRNA to cells and animals in vivo.

Description

Zwitterionic polypeptide lipid molecule and application thereof

Technical Field

The invention relates to the technical field of drug delivery systems, in particular to a zwitterionic polypeptide lipid molecule and application thereof.

Background

Lipid Nanoparticles (LNPs) are a delivery system for hydrophobic or hydrophilic molecular drugs for drug therapy and vaccine preparation. Drugs such as small molecules, nucleic acids and proteins need to be delivered to specific locations to function effectively, for example, tumor drugs need to be delivered to tumor tissues and nucleic acid-based vaccines need to be delivered for intracellular expression. Where lipid nanoparticles have proven effective in delivering small molecule drugs, nucleic acids and proteins, for example, LNP vaccines for delivering mRNA have been extensively studied and products have been clinically approved.

Lipid nanoparticles are generally composed of cationic lipid, phospholipid, cholesterol, and polyethylene glycol lipid molecules. Among them, polyethylene glycol (PEG) is used to improve the in vivo stability of nanoparticles and prolong the in vivo circulation time, but PEG inevitably causes some problems. PEG has been shown to be somewhat immunogenic, readily cleared by the immune system and to cause allergic reactions (Polymers 2020,12, 298). Ju et al reported that LNP vaccines currently used in clinical practice against SARS-CoV-2, such as BNT162b2 and mRNA-1273, cause immune responses of the systemic system in humans, and this phenomenon is related to the PEG lipid component (ACS Nano,2022,16,8, 11769-11780), so that the development of a novel lipid molecule having low immunogenicity and reduced immune side effects, which replaces the PEG lipid component in LNP, is an urgent problem to be solved by those skilled in the art.

Studies have reported that some materials can replace PEG, such as polyoxazoline, poly (N-vinyl pyrrolidone), polyglycerol, polyacrylamide, etc. (Polymers, 2020,12,298, advanced Drug Delivery reviews,2022,180, 114079), which all have protein adsorption resistance and good biocompatibility. However, these materials have certain limitations, such as difficult and expensive synthesis of polyoxazoline (Nanoscale, 2015,7, 13671-13679), and low biodegradability of polyglycerol and polyacrylamide (Polymers, 2020,12, 298).

Zwitterionic materials can also be used as a substitute for PEG, with equal positive and negative charges providing better protein adsorption resistance and thus lower immunogenicity and longer circulation time in vivo (Nano Today,2014,9, 10-16). Common zwitterionic materials are Polycarboxybetaine (PCB), polysulfonobetaine (PSB), and zwitterionic polypeptides. Compared with the polymer, the polypeptide has definite structure and better biological safety. The polypeptide modified lipid nanoparticles mostly adopt the reaction of sulfydryl and maleimide, and the introduced maleimide has potential side effects and is not beneficial to in vivo application and clinical transformation. Therefore, how to develop a material capable of effectively replacing the PEG lipid component in the lipid nanoparticle to overcome the above problems is a problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a zwitterionic lipid molecule to replace a polyethylene glycol (PEG) component in Lipid Nanoparticles (LNPs) to prepare a drug delivery system for delivering nucleic acid molecules.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention provides a zwitterionic polypeptide lipid molecule, which has a general formula of R- (E-K) _n Wherein R is a fatty acid, a diglyceride fatty acid ester derivative or a cholesterol derivative, E is glutamic acid, K is lysine, and n is an integer of 5 to 20.

The invention increases zwitterionic polypeptide EK by modifying fatty acid, diglyceride fatty acid ester derivative or cholesterol derivative _n The prepared zwitterionic polypeptide lipid molecule can be self-assembled with other lipid molecules to form lipid nanoparticles, wherein the zwitterionic polypeptide EK _n Stably modifying the surface of the nano-particles and endowing the nano-particles with protein adsorption resistance.

The zwitterionic polypeptide EK _n Consists of glutamic acid (E) with negative charge and lysine (K) with positive charge which are arranged alternately. Preferably, n =8.

Further, the fatty acid diglyceride derivative or the cholesterol derivative is connected with the N end of the polypeptide molecule by a chemical coupling method.

Preferably, the fatty acid is a fatty acid having 1 to 21 carbon atoms.

Preferably, the fatty acid is a saturated fatty acid or an unsaturated fatty acid having 10 to 21 carbon atoms. More preferably, the fatty acid is stearic acid or oleic acid.

The diglyceride fatty acid ester derivative is a compound which is derived from diglyceride fatty acid ester to form a group capable of being chemically coupled with the N terminal of the polypeptide molecule, and can be but is not limited to diglyceride succinic acid monoester.

The diglyceride fatty acid ester is generated by esterifying two molecules of fatty acid and glycerol. The fatty acid for producing the diglyceride fatty acid ester may be a fatty acid having 1 to 21 carbon atoms, and preferably, the fatty acid is a saturated fatty acid or an unsaturated fatty acid having 10 to 21 carbon atoms; more preferably, oleic acid is used as the fatty acid.

The cholesterol derivative is a compound which is derived from cholesterol to form a group capable of being chemically coupled with the N end of a polypeptide molecule, and can be but is not limited to cholesterol succinate monoester.

In particular, carboxyl terminal of fatty acid, diglyceride succinic acid monoester or cholesterol succinic acid monoester and polypeptide molecule EK _n Is bonded through an amide bond.

Specifically, the structural formula of the zwitterionic polypeptide lipid molecule is any one of formulas (I) to (IV),

the invention provides a method for preparing the zwitterionic polypeptide lipid molecule by a solid-phase synthesis method, which comprises the following steps: first, a solid-phase synthesis method is used to synthesize a polypeptide (E-K) on a solid-phase resin _n And n is 5-20, adding fatty acid, diglyceride succinic acid monoester or cholesterol succinic acid monoester to terminate the polypeptide, and adding cracking liquid to remove solid phase resin and side chain protecting groups to prepare the zwitterionic polypeptide lipid molecule.

The invention also provides application of the zwitterionic polypeptide lipid molecule in preparation of a lipid nanoparticle carrier.

The invention provides a lipid nanoparticle carrier, which comprises cationic lipid molecules, phospholipid, cholesterol and the zwitterionic polypeptide lipid molecules.

The cationic lipid molecule may be, but is not limited to: 1-octylnonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate (trade name SM 102), ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (trade name ALC-0315), (2, 3-dioleyloxypropyl) trimethylammonium chloride (trade name DOTAP), 4- (N, N-dimethylamino) butyric acid (6Z, 9Z,28Z, 31Z) -heptatriacontane-6, 9,28, 31-tetralin-19-yl ester (trade name Dlin-MC 3-DMA).

The phospholipid may be, but is not limited to: distearoyl phosphatidylcholine (DSPC), 1, 2-dioleoyl lecithin (DOPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE).

Preferably, the cationic lipid molecule is SM102.

Preferably, the phospholipid is DSPC.

Preferably, wherein the molar ratio of cationic lipid molecules, phospholipids, cholesterol and zwitterionic lipid molecules is 50:10:38.5:1.5.

the invention provides application of the lipid nanoparticle carrier in preparation of nucleic acid drugs.

The application comprises the following steps: adding cationic lipid molecules, phospholipid, cholesterol and zwitterionic polypeptide lipid molecules into an acidic buffer solution containing nucleic acid, and self-assembling to form lipid nanoparticles carrying nucleic acid, thus preparing the nucleic acid medicament.

Specifically, cationic lipid, phospholipid, cholesterol and zwitterionic polypeptide lipid molecules were solubilized with ethanol, and nucleic acids were solubilized with 40mM sodium acetate buffer. Mixing the ethanol solution and the sodium acetate buffer solution according to a volume ratio of 1.

The nucleic acid medicament prepared by the invention can be used for delivering exogenous nucleic acid molecules into cells, so that the exogenous nucleic acid molecules can be translated and expressed in the cells; can also be used to deliver exogenous nucleic acid molecules into experimental animals so that the exogenous nucleic acid molecules can be expressed translationally in the animal.

The invention has the beneficial effects that:

(1) The invention provides a zwitterionic polypeptide lipid molecule which is formed by bonding fatty acid, diglyceride fatty acid ester derivative or cholesterol derivative and EK polypeptide. Zwitterionic polypeptide lipid molecule R-EK _n After the lipid nanoparticles are formed with cationic lipid, phospholipid and cholesterol, the lipid nanoparticles have good particle size and particle size distribution, and the lipid nanoparticles have the capability of delivering mRNA to cells and animals, so that the zwitterionic polypeptide lipid molecule R-EK _n Can be used as a substitute component of PEG lipid in lipid nanoparticles to overcome the problems of PEG.

(2) Experiments prove that compared with PEG lipid, the cell transfection capacity of the lipid nanoparticles formed by the zwitterionic polypeptide lipid molecules in vivo and in vitro is remarkably improved, the delivery efficiency of the lipid nanoparticles is improved, and no obvious cytotoxicity exists.

Drawings

FIG. 1 is a mass spectrum of EK peptide lipid molecules prepared in examples 1-4, (a) Ste-EK8; (b) Ole-EK8; (c) Chol-EK8; (d) DOG-EK8.

FIG. 2 shows the cytotoxicity of EK peptide lipid molecules at various concentrations.

FIG. 3 is a graph showing the particle size of GFP-mRNA-loaded lipid nanoparticles.

FIG. 4 shows the results of transfection of cells carrying GFP-mRNA lipid nanoparticles, (a) fluorescence microscopy of the cells after transfection; (b) efficiency of cell transfection and activity of the transfected cells.

FIG. 5 is a particle size diagram of Luci-mRNA loaded lipid nanoparticles.

FIG. 6 shows the particle size stability of Luci-mRNA-loaded lipid nanoparticles in serum.

FIG. 7 shows the results of transfection in vivo in mice loaded with Luci-mRNA lipid nanoparticles, (a) in vivo fluorescence imaging photographs after cell transfection; and (b) fluorescence quantification.

Detailed Description

The present invention is further illustrated by the following examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and nature of the invention.

The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The compounds referred to in the examples:

Fmoc-Lys (Boc) -Wang Resin (loading =0.348 mmol/g), fmoc-Glu (OtBu) -OH (CAS: 71989-18-9), fmoc-Lys (Boc) -OH (CAS: 71989-26-9) were purchased from Gill biochemicals.

Stearic acid (CAS: 57-11-4), oleic acid (CAS: 112-80-1), glycerol dioleate (CAS: 25637-84-7), cholesterol succinic acid monoester (CAS: 1510-21-0), cholesterol (CAS: 57-88-5) were purchased from Annagi chemical.

SM102 was purchased from Sonopont, DSPC, DMG-PEG-2k from Kekay technology.

The compounds referred to in the examples are described by the following abbreviations in English:

DMF: n, N-dimethylformamide, HOBT: 1-hydroxybenzotriazole, HBTU: o-benzotriazole-tetramethylurea hexafluorophosphate.

Example 1: preparation of stearic acid modified EK peptide lipid molecule

Stearic acid-modified EK peptides were synthesized according to solid phase synthesis in the literature (angelw. Chem. Int.ed.,2020,59, 22378-22381). Specifically, 200mg of Fmoc-lys (boc) -Wang Resin was first added to a solid phase synthesis tube, and then anhydrous DMF was added to swell the tube for 1 hour. After draining off DMF, a deprotection agent (piperidine: DMF =1 = 4) was added to remove the Fmoc group at the N-terminus and the ninhydrin test was used to check whether deprotection was successful. Fmoc-Glu (OtBu) -OH (0.6966 mmol,296 mg), HBTU (0.6966 mmol, 264mg) and HOBT (0.6966 mmol, 94mg) were then dissolved in a coupling reagent (DMF: N-methylmorpholine = 95) and added to a solid phase synthesis tube for 2h reaction. The above coupling and deprotection steps were repeated, fmoc-Lys (Boc) -OH and Fmoc-Glu (OtBu) -OH were added sequentially, 7 lysines and 8 glutamic acids were connected sequentially, and finally stearic acid (0.6966 mmol, 198mg) was added for capping. After the coupling is completed, a cracking solution (water, triisopropylsilane, trifluoroacetic acid) is added

= 2.5.

The mass spectrum characterization chart of Ste-EK8 is shown in FIG. 1 (a), and the m/z =2343.414 of the polypeptide lipid molecule Ste-EK8 is shown according to the mass spectrum detection result, and the result is in line with the expectation.

Example 2: preparation of oleic acid modified EK peptide lipid molecules

Oleic acid-modified EK peptides were synthesized according to the solid phase synthesis in the literature (angelw. Chem. Int.ed.,2020,59, 22378-22381). Specifically, 200mg of Fmoc-lys (boc) -Wang Resin was first added to a solid phase synthesis tube, and anhydrous DMF was added to swell for 2h. After draining off DMF, a deprotection agent (piperidine: DMF =1 = 4) was added to remove the Fmoc group at the N-terminus and the ninhydrin test was used to check whether deprotection was successful. Fmoc-Glu (OtBu) -OH (0.696mmol, 296mg), HBTU (0.696mmol, 264mg) and HOBT (0.696mmol, 94mg) were dissolved in a coupling agent (DMF: N-methylmorpholine = 95) and added to a solid phase synthesis tube for 1h. The above coupling and deprotection steps were repeated, fmoc-Lys (Boc) -OH and Fmoc-Glu (OtBu) -OH were added sequentially, 7 lysines and 8 glutamic acids were connected sequentially, and oleic acid (0.6966 mmol, 195mg) was added for capping. And finally, shrinking the resin by using methanol, adding a lysis solution (water: triisopropylsilane: trifluoroacetic acid = 2.5).

The mass spectrum characterization chart of Ole-EK8 is shown in fig. 1 (b), and according to the mass spectrum detection result, the m/z =2340.678 of the polypeptide lipid molecule Ole-EK8 is as expected.

Example 3: preparation of cholesterol derivative modified EK peptide lipid molecule

Cholesterol derivative modified EK peptides were synthesized according to solid phase synthesis in the literature (angelw. Chem. Int. Ed.,2020,59, 22378-22381). Specifically, 200mg of Fmoc-lys (boc) -Wang Resin was first added to a solid phase synthesis tube, and anhydrous DMF was added to swell for 2h. After draining off DMF, a deprotection agent (piperidine: DMF =1 = 4) was added to remove the Fmoc group at the N-terminus and the ninhydrin test was used to check whether deprotection was successful. Fmoc-Glu (OtBu) -OH (0.696mmol, 296mg), HBTU (0.696mmol, 264mg) and HOBT (0.696mmol, 94mg) were dissolved in a coupling agent (DMF: N-methylmorpholine = 95) and added to a solid phase synthesis tube for 1h. Repeating the coupling and deprotection steps, adding Fmoc-Lys (Boc) -OH and

Fmoc-Glu (OtBu) -OH, 7 lysines and 8 glutamic acids were connected in sequence, capped by the addition of cholesteryl succinate (0.6966 mmol, 339mg). And finally, shrinking the resin by using methanol, adding a lysis solution (water: triisopropylsilane: trifluoroacetic acid = 2.5.

The mass spectrum characterization chart of Chol-EK8 is shown in FIG. 1 (c), and according to the mass spectrum detection result, the m/z =2545.458 of the polypeptide lipid molecule Chol-EK8 is in line with the expected result.

Example 4: preparation of EK peptide lipid molecule modified by glycerol dioleate derivative

1. Firstly, glycerol dioleate succinic acid monoester is synthesized by the following specific synthesis method:

after 5g of glycerol dioleate (8.05 mmol), 1.61g of succinic anhydride (16.1 mmol) and 2.46g of 4-dimethylaminopyridine (16.1 mmol) were dissolved in 50mL of dichloromethane and reacted overnight, the mixture was extracted with 1M dilute hydrochloric acid and saturated sodium chloride in this order, the organic phase was collected and the organic solvent was removed by rotary evaporation to give a crude glycerol dioleate succinic acid monoester product, which was purified by silica gel chromatography (eluent n-hexane: ethyl acetate = 20) to give a pure glycerol dioleate succinic acid monoester product.

2. Glycerol dioleate derivative modified EK peptides were synthesized according to solid phase synthesis methods in the literature (angelw. Chem. Int. Ed.,2020,59, 22378-22381). Specifically, 200mg of Fmoc-lys (boc) -Wang Resin was first added to a solid phase synthesis tube, and anhydrous DMF was added to swell for 2h. After draining off DMF, a deprotection agent (piperidine: DMF =1 = 4) was added to remove the Fmoc group at the N-terminus and the ninhydrin test was used to check whether deprotection was successful. Fmoc-Glu (OtBu) -OH (0.6966 mmol,296 mg), HBTU (0.6966 mmol, 264mg) and HOBT (0.6966 mmol, 94mg) were dissolved in a coupling agent (DMF: N-methylmorpholine = 95) and added to a solid phase synthesis tube for 1h. The above coupling and deprotection steps were repeated, fmoc-Lys (Boc) -OH and Fmoc-Glu (OtBu) -OH were added sequentially, 7 lysines and 8 glutamic acids were connected sequentially, and glycerol dioleate succinic acid monoester (0.6966 mmol, 502mg) was added for capping. And finally, shrinking the resin by using methanol, adding a lysis solution (water: triisopropylsilane: trifluoroacetic acid =2.5 = 2.95) to remove a protecting group, filtering an organic phase, adding glacial ethyl ether to separate out polypeptide lipid molecules, and centrifuging to remove the ethyl ether to obtain the solid powder of the glycerol dioleate derivative modified EK peptide (DOG-EK 8).

3. The mass spectrum characterization chart of DOG-EK8 is shown in FIG. 1 (d), and the m/z =2800.953 of the polypeptide lipid molecule DOG-EK8 is determined according to the mass spectrum detection result, which is in line with the expectation. Test example 1: cytotoxicity of EK peptide lipid molecules

Add 1X 10 per well in 96-well plates ⁴ After 24 hours, 2. Mu.M, 10. Mu.M, 50. Mu.M, 250. Mu.M of DMG-PEG2k, ste-EK8, ole-EK8 or Chol-EK8 was added to each BHK cell. After 24h incubation, the medium was removed and fresh medium containing 10% CCK8 was added. After incubation for 1.5h at 37 ℃, absorbance in each well was measured using a microplate reader at a wavelength of 450nm, and cell viability was obtained by calculating the ratio of absorbance of the dosed wells to that of the blank control.

As shown in FIG. 2, ste-EK8 and Ole-EK8 have no obvious cytotoxicity with the increase of the material concentration, and Chol-EK8 has obvious cytotoxicity with the concentration higher than 50. Mu.M. At concentrations above 250. Mu.M, DMG-PEG2k was significantly cytotoxic. Therefore, ste-EK8 and Ole-EK8 have better safety than DMG-PEG2k and are more suitable for large-dose injection.

Test example 2: preparation of GFP-mRNA-loaded lipid nanoparticles

1. Method for preparing lipid nanoparticles

SM-102, DSPC, cholesterol and polypeptide lipid molecules (Ste-EK 8, ole-EK8 or Chol-EK 8) were solubilized in ethanol at a molar ratio of 50.10. mRNA encoding Green Fluorescent Protein (GFP) was dissolved with 40mM sodium acetate buffer. Mixing the ethanol solution and sodium acetate buffer solution at a volume ratio of 1.

Meanwhile, a control group was set, and SM-102, DSPC, cholesterol and DMG-PEG2k were dissolved in ethanol as a solvent at a molar ratio of 50. mRNA encoding GFP was dissolved with 40mM sodium acetate buffer. Lipid nanoparticles were prepared using the above method to obtain lipid nanoparticles containing a PEG lipid component (PEG-LNP/mGFP).

2. Particle size characterization of GFP-mRNA loaded lipid nanoparticles

LNP samples (Chol-EK-LNP/mGFP, ste-EK-LNP/mGFP, or Ole-EK-LNP/mGFP) were diluted with phosphate buffer to 0.16mg/mL, and 1mL of the sample was placed in a sample cell dedicated to a particle sizer, and dynamic light scattering test was performed using a particle sizer to measure the hydration kinetic particle size and particle size distribution of LNP, as shown in FIG. 3. The experiment was repeated three times to obtain an average value, and the results of the particle size and the particle size distribution data are shown in table 1.

TABLE 1 particle size and particle size distribution of GFP-mRNA-loaded lipid nanoparticles

Lipid nanoparticles	Particle size (nm)	Particle size distribution PDI
			Chol-EK-LNP/mGFP	249.4±13.8	0.16
Ste-EK-LNP/mGFP	159.9±3.9	0.14
			Ole-EK-LNP/mGFP	143.2±1.2	0.17

The results show that LNP has a particle size of 100-300nm and can be used for in vivo and in vitro drug delivery.

3. Validation of the Effect of lipid nanoparticles on mRNA delivery at cellular level

Adding 2.5X 10 of the mixture into each hole of a 48-hole plate ⁴ For each BHK cell, 24 hours later, PEG-LNP/mGFP, ste-EK-LNP/mGFP, ole-EK-LNP/mGFP or Chol-EK-LNP/mGFP containing 2. Mu.g of GFP-mRNA was added to 300uL of serum-free DMEM medium, replacing the cell culture medium in the 48-well plate. GFP expression was observed after 24h and transfection efficiency was determined using flow-through experiments.

LNP/mGFP cell transfection results and transfection efficiencies As shown in FIG. 4, LNP containing zwitterionic polypeptide lipids was shown to be more efficient in transfection than the conventional PEG fraction, and not significantly cytotoxic.

Test example 3: preparation of Luci-mRNA-loaded lipid nanoparticles

1. Method for preparing lipid nanoparticles

Dissolving SM-102, DSPC, cholesterol and polypeptide lipid molecules (Ste-EK 8, ole-EK8 or Chol-EK 8) in ethanol as a solvent at a molar ratio of 50.10. The mRNA encoding luciferase (Luci) was dissolved with 40mM sodium acetate buffer. Mixing the ethanol solution and sodium acetate buffer solution at a volume ratio of 1.

Meanwhile, a control group was set, and SM-102, DSPC, cholesterol and DMG-PEG were dissolved in ethanol as a solvent at a molar ratio of 50. The mRNA encoding Luci was solubilized with 40mM sodium acetate buffer. Lipid nanoparticles were prepared using the above method to obtain lipid nanoparticles containing a PEG lipid component (PEG-LNP/mLuci).

2. Particle size characterization of Luci-mRNA-loaded lipid nanoparticles

LNP samples (Chol-EK-LNP/mLuci, ste-EK-LNP/mLuci, or Ole-EK-LNP/mLuci) were diluted with phosphate buffer to 0.16mg/mL, and 1mL of the sample was placed in a sample cell dedicated to a particle sizer, and dynamic light scattering test was performed using a particle sizer to measure the hydration kinetic particle size and particle size distribution of LNP, as shown in FIG. 5. The experiment was repeated three times to obtain an average value, and the results of the particle size and the particle size distribution data are shown in table 2.

TABLE 2 particle size and particle size distribution of Luci-mRNA loaded lipid nanoparticles

The results show that the particle size of LNP is less than 300nm, and can be used for in vivo or in vitro drug delivery.

3. Particle size stability of Luci-mRNA-loaded lipid nanoparticles

Luci-mRNA loaded LNP samples (PEG-LNP/mLuci, chol-EK-LNP/mLuci, ste-EK-LNP/mLuci, or Ole-EK-LNP/mLuci) were diluted with complete medium containing 10% fetal bovine serum and placed in a 37 ℃ shaking incubator to simulate in vivo environment, and dynamic light scattering measurements were performed with a particle sizer at 0h, 2h, 4h, 8h, 18h, 24h, and 48h, respectively, to monitor the hydration kinetic particle size changes of LNP, as shown in FIG. 6.

The results show that LNP can maintain the particle size stability within 48h of incubation in the culture medium containing serum, which indicates that the EK peptide lipid molecules are still better in stability after replacing PEG lipid.

4. Validation of the Effect of lipid nanoparticles on mRNA delivery at the animal level

6-8 week-old Balb/C mice were randomly divided into 4 groups of 3 mice each, and injected with PEG-LNP/mLuci, chol-EK-LNP/mLuci, ste-EK-LNP/mLuci, or Ole-EK-LNP/mLuci, respectively, via the tail vein, at a dose of 5 μ g Luci-mRNA per mouse. Luciferase expression in vivo was observed by small animal live imaging 6h after injection and quantified by fluorescence. In vivo transfection results are shown in fig. 7, compared with the conventional PEG component, the in vivo transfection effect of LNP containing zwitterionic polypeptide lipid is improved, wherein the luciferase expression of Ste-EK8-LNP is improved by 1.62 times, and the luciferase expression of Ole-EK8-LNP is improved by 1.23 times, which indicates that the zwitterionic polypeptide lipid is capable of promoting the ability of delivering mRNA molecules into animals after replacing the PEG lipid component of lipid nanoparticles.

In summary, the present invention provides a zwitterionic polypeptide lipid molecule, wherein the lipid molecule is an EK polypeptide modified by a fatty acid, a diglyceride fatty acid ester derivative or a cholesterol derivative, and the lipid molecule can be used as a replacement molecule for a polyethylene glycol component in a lipid nanoparticle composition, and can form a lipid nanoparticle together with a cationic lipid, a phospholipid and cholesterol, so as to deliver a nucleic acid molecule into an animal body or a cell. Experiments prove that the lipid nanoparticles formed by the zwitterionic polypeptide lipid molecules have uniform and stable particle size, compared with PEG lipid, the cell transfection capacity of the lipid nanoparticles in vivo and in vitro is improved to a certain extent, and no obvious cytotoxicity exists, so that the zwitterionic polypeptide lipid molecules can be used for preparing the lipid nanoparticles together with cationic lipid, phospholipid and cholesterol, and the in vivo or in vitro delivery of nucleic acid molecules is promoted.

Claims (10)

1. AA zwitterionic polypeptide lipid molecule, wherein the zwitterionic polypeptide lipid molecule has a general formula of R- (E-K) _n Wherein R is a fatty acid, a diglyceride fatty acid ester derivative or a cholesterol derivative, E is glutamic acid, K is lysine, and n is an integer of 5 to 20.

2. The zwitterionic polypeptide lipid molecule of claim 1, wherein said fatty acid, di-fatty acid ester derivative or cholesterol derivative and polypeptide EK _n The N-terminal of (2) is connected by a chemical coupling method.

3. The zwitterionic polypeptide lipid molecule of claim 1, wherein said fatty acid or fatty acid used to form the diglyceride fatty acid ester derivative is a fatty acid having from 1 to 21 carbon atoms.

4. The zwitterionic polypeptide lipid molecule according to claim 3, wherein the fatty acid is a saturated or unsaturated fatty acid with 10-21 carbon atoms.

5. The zwitterionic polypeptide lipid molecule of claim 1, wherein the zwitterionic polypeptide lipid molecule has a structural formula selected from any one of formulas (I) - (IV),

6. a method of preparing the zwitterionic polypeptide lipid molecule of claim 1, comprising: first, a polypeptide (E-K) is synthesized on a solid resin by a solid phase synthesis method _n N is 5-20, then adding fatty acid and glycerinAnd (3) end-capping the polypeptide by diester succinate monoester or cholesterol succinate monoester, and then adding a cracking solution to remove the solid phase resin and the side chain protecting group to prepare the zwitterionic polypeptide lipid molecule.

7. Use of the zwitterionic polypeptide lipid molecule of any one of claims 1-5 in the preparation of a lipid nanoparticle carrier.

8. A lipid nanoparticle carrier, comprising a cationic lipid molecule, a phospholipid, cholesterol, and the zwitterionic polypeptide lipid molecule according to any one of claims 1-5.

9. Use of the lipid nanoparticle carrier of claim 8 for the preparation of a nucleic acid drug.

10. The application of claim 9, wherein the application comprises: adding cationic lipid molecules, phospholipids, cholesterol and zwitterionic polypeptide lipid molecules into an acidic buffer solution containing nucleic acid, and self-assembling to form lipid nanoparticles carrying nucleic acid, thereby preparing the nucleic acid medicament.

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