CN118684883A - Lipidated polymer, polymer nanoparticles, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a lipidated polymer, polymer nano particles, and a preparation method and application thereof. The lipidated polymer is obtained by reacting polyethyleneimine with a compound (formula III) with active groups and aliphatic chains, wherein the active groups are groups which can react with secondary amine or primary amine of branched polyethyleneimine. The polymeric nanoparticle is made from components including lipidated polymers, steroids or derivatives thereof, helper lipids, polymer-bound lipids, and nucleic acids, and may also include functional lipid components; the helper lipid is a non-ionic lipid and/or a zwitterionic lipid; the functional lipids are cationic lipids and/or anionic lipids. The nanoparticle can greatly improve the nucleic acid delivery efficiency of the traditional cationic polymer, and can effectively deliver nucleic acid macromolecules such as mRNA, plasmid DNA and the like to cells such as designated organs, tissues, tumors and the like and play a role of gene interference or gene expression by being matched with lipid.

Description

Lipidated polymer, polymer nanoparticles, and preparation method and application thereof

Technical Field

The present invention relates to the technical field of biological carrier materials, and in particular to a lipidated polymer, polymer nanoparticles, and a preparation method and application thereof.

Background Art

Nucleic acid drugs are a general term for oligoribonucleotides (RNA) and oligodeoxyribonucleotides (DNA) and their hybrids with different functions, which play a role at the gene level. Among them, DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products or vectors. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA) and their combinations. Nucleic acid drugs have shown therapeutic potential in a series of applications such as viral vaccines, protein replacement therapy, and cancer immunotherapy. However, as nucleic acid macromolecules, how to safely and effectively deliver them to the body to play a role is a major bottleneck limiting their application. In view of the problem of in vivo delivery of nucleic acid drugs, the development and construction of effective nucleic acid delivery vectors is currently a hot topic in clinical research.

The lipid nanoparticles developed by BioNTech and Pfizer use ionizable lipids to encapsulate mRNA vaccines, which trigger effective immune responses after delivery into the body, achieving certain results. However, the in vivo delivery efficiency of polymer nucleic acid vectors is difficult to break through the bottleneck, and their in vivo expression efficiency is poor, which cannot meet the needs of clinical applications and has not yet been applied to clinical practice.

Summary of the invention

Based on this, the purpose of the present invention is to provide a lipidated polymer, which can be prepared into lipidated polymer nanoparticles with components such as steroids or their derivatives and auxiliary lipids. The nanoparticles can be used as nucleic acid delivery carriers to greatly improve the nucleic acid delivery efficiency of traditional cationic polymers.

In order to achieve the above technical objectives, the present invention includes the following technical solutions.

A lipid polymer obtained by reacting a compound of formula (II) with a compound of formula (III),

wherein each m is independently selected from: 1, 2, 3, 4, 5, 6;

n is selected from: an integer between 1 and 1000;

L is a group that can react with the secondary amine or primary amine in the compound of formula (II) or polyethyleneimine;

J is absent or is selected from: C ₁ -C ₁₂ alkylene, C ₂ -C ₁₂ unsaturated chain hydrocarbon group;

K is absent or selected from: -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S-, -S(O)-, -S(O) _2- , -SS-, -C(=O)S-, -SC(=O)-, -NRC(=O)-, -C(=O)NR-, -NRC(=O)NR-, -OC(=O)NR-, -NRC(=O)O-;

R is selected from: H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ unsaturated chain hydrocarbon group;

M is selected from the group consisting of: C ₁ -C ₂₄ alkyl, C ₂ -C ₂₄ unsaturated chain hydrocarbon group.

A lipid polymer obtained by reacting polyethyleneimine with a compound of formula (III),

Wherein, L is a group that can react with the secondary amine or primary amine in the polyethyleneimine;

J is absent or is selected from: C ₁ -C ₁₂ alkylene, C ₂ -C ₁₂ unsaturated chain hydrocarbon group;

K is absent or selected from: -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S-, -S(O)-, -S(O) _2- , -SS-, -C(=O)S-, -SC(=O)-, -NRC(=O)-, -C(=O)NR-, -NRC(=O)NR-, -OC(=O)NR-, -NRC(=O)O-;

R is selected from: H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ unsaturated chain hydrocarbon group;

M is selected from the group consisting of: C ₁ -C ₂₄ alkyl, C ₂ -C ₂₄ unsaturated chain hydrocarbon group.

The present invention also provides a method for preparing the above-mentioned lipidated polymer, including the following technical scheme.

A method for preparing a lipid polymer comprises the following steps: reacting the compound of formula (II) or polyethyleneimine with the compound of formula (III) at a certain temperature to obtain a lipid polymer.

The present invention also provides a carrier for delivering nucleic acid macromolecules, which greatly improves the gene delivery efficiency of traditional cationic polymers and includes the following technical solutions.

A carrier for delivering nucleic acid macromolecules is made of components including the lipidated polymer, steroid or its derivative, auxiliary lipid, polymer-bonded lipid; the auxiliary lipid is a nonionic lipid and/or a zwitterionic lipid.

A carrier for delivering nucleic acid macromolecules, which is made of components including the lipidated polymer, steroids or their derivatives, auxiliary lipids, functional lipids, and polymer-bonded lipids; the auxiliary lipids are non-ionic lipids and/or zwitterionic lipids; and the functional lipids are cationic lipids and/or anionic lipids.

The present invention also provides the use of the above-mentioned vector in delivering nucleic acid macromolecules in vivo or in vitro.

The present invention also provides a polymer nanoparticle having good nucleic acid delivery efficiency, which includes the following technical solutions.

A polymer nanoparticle is prepared from the carrier for delivering nucleic acid macromolecules and nucleic acid.

The present invention also provides a method for preparing the polymer nanoparticles, which includes the following technical solutions.

A method for preparing the polymer nanoparticles comprises the following steps:

The lipidated polymer, steroid or its derivative, auxiliary lipid, functional lipid and polymer-bonded lipid are dissolved in a solvent to obtain solution 1; the nucleic acid is dissolved in a buffer to obtain solution 2; the solution 1 and solution 2 are mixed evenly and dialyzed to obtain the polymer nanoparticles.

A method for preparing the polymer nanoparticles comprises the following steps:

The lipidated polymer, steroid or its derivative, auxiliary lipid, functional lipid and polymer-bonded lipid are dissolved in a solvent to obtain solution 1; solution 1 is mixed evenly with a buffer solution, dialyzed to obtain an empty polymer nanoparticle solution; the nucleic acid is added to the empty polymer nanoparticle solution to obtain the polymer nanoparticle.

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

In order to solve the problem of effectively delivering nucleic acid macromolecules to the body and cells, the present invention designs and synthesizes a novel lipid polymer, and the polymer is prepared into lipid polymer nanoparticles with components such as steroids or their derivatives and auxiliary lipids, nucleic acids, etc., which can greatly improve the nucleic acid delivery efficiency of traditional cationic polymers. The lipid polymer provided by the present invention has good biocompatibility and high efficiency of nucleic acid delivery. It can effectively deliver nucleic acid macromolecules such as mRNA and plasmid DNA to cells such as designated organs, tissues, tumors, etc. in combination with lipids and play the role of gene interference or gene expression. In addition, whether it is intravenous injection or intramuscular injection, subcutaneous injection, intraperitoneal injection, bladder instillation, local injection in surgery, etc., good delivery effect and expression effect can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen nuclear magnetic resonance spectrum of the lipidated polymer P6-4H-90.

FIG2 shows the H NMR spectra of lipidated polymers P6-Cit-80, P6-Cit-90, P6-Cit-100, P6-Cit-120 and P6-Cit-140.

FIG. 3 is a flow cytometry diagram of plasmid delivery by polymer nanoparticles.

FIG. 4 shows the intratumoral delivery effect of polymer nanoparticles on mRNA.

FIG5 shows the effect of tail vein injection of polymer nanoparticles with different composition ratios on mRNA delivery.

FIG6 shows the distribution of luciferase mRNA expression after tail vein injection of polymer nanoparticles (left: P6-4H-90, right: P6-Cit-100).

Figure 7 shows the effect of tail vein injection of different polymer nanoparticles on mRNA delivery.

FIG8 shows the expression distribution of luciferase mRNA after subcutaneous (left) and intramuscular (right) injection of polymer nanoparticles (left: P18-Cit-100, right: P6-4H-90).

FIG. 9 shows the expression distribution of luciferase mRNA delivered by five-component polymer nanoparticles.

FIG. 10 shows the expression distribution of luciferase mRNA delivered by polymer nanoparticles prepared with different steroids.

FIG. 11 shows the intratumoral delivery effect of polymer nanoparticles prepared with different auxiliary lipids on mRNA.

FIG. 12 shows the intratumoral delivery effect of P6-4H polymer nanoparticles with different grafting rates on mRNA.

FIG. 13 shows the intratumoral delivery effect of P6-Cit polymer nanoparticles with different grafting rates on mRNA.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

In the present invention, the term "multiple" refers to two or more than two. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the associated objects are in an "or" relationship.

Unless otherwise specified, the chemical reagents and reaction raw materials used in the examples are conventional commercially available reagents or can be synthesized by simple methods according to literature reports, and the technical means used in the examples are conventional means familiar to those skilled in the art.

The present invention, in some embodiments thereof, provides a lipidated polymer, which can be obtained by reacting a compound of the general formula (II) or polyethyleneimine (e.g., branched polyethyleneimine (PEI)) with a compound (formula III) having an active group and a fatty chain, wherein the active group refers to a group that can react with the secondary amine or primary amine of the compound of the general formula (II) or polyethyleneimine, including but not limited to methyl acrylate bonds, halogens, epoxypropyl groups, carboxyl groups, thiocyanate groups, and the like.

wherein each m is independently selected from: 1, 2, 3, 4, 5, 6;

n is selected from: an integer between 1 and 1000;

L is a group that can react with secondary amine or primary amine, including but not limited to acrylate group Halogen, acyl halide, cyclopropyloxy, carboxyl, thiocyanate, etc.; J is a saturated fatty chain (alkylene) or an unsaturated fatty chain with a carbon chain length of 0-12; K does not exist or is a linking group, including but not limited to: -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S-, -S(O)-, -S(O) _2- , -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, -NRaC(=O)O-, wherein Ra is selected from H, _C1 - _C12 alkyl, or _C2 - _C12 unsaturated chain hydrocarbon group; M is selected from _C1 - _C24 alkyl, or _C2 - _C24 unsaturated chain hydrocarbon group.

According to the reaction described in the present invention, exemplary structures of the lipidated polymer described in the present invention include but are not limited to the following formula (I):

wherein each m is independently selected from: 1, 2, 3, 4, 5, 6;

n is selected from: an integer between 1 and 1000;

Each R ₁ is independently selected from: the residue after the reaction of the compound of formula III with secondary amine or primary amine, or hydrogen.

The term "alkyl" is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having a certain number of carbon atoms. For example, the definition of "C ₁ -C ₆ " in "C ₁ -C ₆ alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain or branched chain. For example, "C ₁ -C ₆ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl, etc.

The term "unsaturated chain hydrocarbon group" refers to branched and straight-chain unsaturated aliphatic hydrocarbon groups having a specific number of carbon atoms, that is, non-cyclic unsaturated chain hydrocarbon groups, and the carbon chain contains one or more carbon-carbon double bonds, or carbon-carbon triple bonds, such as: _CH2 = _CHCH2- , -( _CH2 ) ₈ (CH=CH) _CH3 , -( _CH2 ) _7CH = _CH2 , -( _CH2 ) _8CH = _CH2 , etc.

In some embodiments, in the compound of formula (III), L is selected from: Halogen, acyl halide, cyclopropyloxy, carboxyl, thiocyanate.

In some embodiments, in the compound of formula (III), L is selected from: Chloro, bromo, acyl chloride, cyclopropyloxy.

In the most preferred embodiment, in the compound of formula (III), L is selected from: In this case, lipidated polymers have better nucleic acid delivery efficiency.

In some embodiments, J is absent or is selected from: C ₁ -C ₈ alkylene, C ₂ -C ₈ unsaturated chain hydrocarbon group.

In some embodiments, J is absent or is selected from the group consisting of methylene, ethylene, propylene, butylene, pentylene, and hexylene.

In some embodiments, K is absent or is selected from: -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S-, -S(O)-, -S(O) _2- , -SS-.

In some embodiments, M is selected from: C ₆ -C ₂₀ alkyl, C ₆ -C ₂₀ unsaturated chain hydrocarbon group.

In some embodiments, M is selected from: C ₈ -C ₁₈ alkyl, C ₈ -C ₁₈ unsaturated chain hydrocarbon group.

In some embodiments, M is selected from: C ₁₂ -C ₁₄ straight chain alkyl, C ₈ -C ₁₄ branched chain alkyl, C ₁₂ -C ₁₄ unsaturated straight chain hydrocarbon, C ₈ -C ₁₄ unsaturated branched chain hydrocarbon.

In some embodiments, M is selected from:

When M is a hydrocarbon group of the above-mentioned specific chain length, especially a branched hydrocarbon group of the specific chain length, the lipidated polymer has a very excellent nucleic acid delivery effect.

Specific examples of compounds of formula (III) include, but are not limited to, the following compounds:

In some embodiments, the polyethyleneimine used to prepare the lipidated polymer of the present invention is preferably a branched polyethyleneimine; its molecular weight is preferably 300-10000, more preferably 400-2000, more preferably 500-1800, more preferably 500-700, more preferably 600. The lipidated polymer prepared by the branched polyethyleneimine with this preferred molecular weight has better nucleic acid delivery efficiency when combined with lipids.

In some embodiments, the reaction feed ratio of the compound of formula (II) or polyethyleneimine and the compound of formula (III) is 0.4-1.8mmoL, preferably 40mg: 0.7-1.5mmoL, more preferably 40mg: 0.8-0.9mmoL, or more preferably 40mg: 1.3-1.4mmoL. For example, when the compound of formula (III) is tetrahydrogeraniol acrylate, the optimal reaction feed ratio of the compound of formula (II) or polyethyleneimine and the compound of formula (III) is 40mg: 0.8-0.9mmoL, and at this grafting rate, the resulting lipid polymer has a better nucleic acid delivery effect. For another example, when the compound of formula (III) is citronellol acrylate, the optimal reaction feed ratio of the compound of formula (II) or polyethyleneimine and the compound of formula (III) is 40mg: 1.3-1.4mmoL, and at this grafting rate, the resulting lipid polymer has a better nucleic acid delivery effect.

In some embodiments, the present invention also provides a method for preparing the lipidated polymer, comprising the following steps: reacting the compound of formula (II) or polyethyleneimine with the compound of formula (III) at a certain temperature to obtain.

The reaction can be carried out without adding a solvent or in a solvent selected from dichloromethane, chloroform, methanol, acetonitrile, N'N'-dimethylformamide, dimethyl sulfoxide, benzene and toluene.

In some embodiments, the reaction temperature is 4°C-200°C, and the reaction time is 10h-120h.

When L is selected from: When halogen or cyclopropyloxy is used, the reaction temperature is preferably 60°C-90°C, more preferably 70°C-80°C; the reaction time is preferably 60h-84h, more preferably 68h-75h.

When L is selected from: acyl halide, the reaction temperature is preferably 4°C-10°C; the reaction time is preferably 6h-18h, more preferably 10h-15h.

In some embodiments, the present invention also provides a carrier for delivering nucleic acid macromolecules, which is made of components including the lipidated polymer, steroid or its derivatives, auxiliary lipids, and polymer-bonded lipids; or is made of components including the lipidated polymer, steroid or its derivatives, auxiliary lipids, functional lipids, and polymer-bonded lipids.

Among them, steroids or their derivatives include but are not limited to compounds containing structural formula (IV), such as cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, bile acid, cholesterol and steroid hormones, etc.

Auxiliary lipids refer to lipids that exist in an uncharged or neutral zwitterionic form at a selected pH value (i.e., nonionic lipids and/or zwitterionic lipids), including but not limited to 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingosine (SM), etc.

Functional lipids refer to lipids that exist in a positively or negatively charged form at a selected pH value (i.e., cationic lipids and/or anionic lipids), including but not limited to 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyloctadecyl ammonium (DDAB), 1,2-dimyristoyl-glycero-3-phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18PG), etc.

A polymer-bound lipid refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a pegylated lipid. A pegylated lipid refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxypolyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG) and the like.

The effect of vector delivery of nucleic acid can be further optimized by adjusting the ratio of each component.

In some of the embodiments, the mass ratio of the lipidated polymer, steroid or its derivative, auxiliary lipid, functional lipid and polymer-bonded lipid is 16:1-20:1-16:0-45:1-8, preferably 16:4-16:2-12:0-40:1-6; preferably 16:4-16:2-12:0-30:1-6.

In some of the embodiments, the carrier for delivering nucleic acid macromolecules is made of the lipidated polymer, steroid or its derivative, auxiliary lipid and 1-(monomethoxypolyethylene glycol)-2,3-dimyristyl glycerol;

The steroid or its derivative is cholesterol, lanosterol and/or stigmasterol;

The auxiliary lipid is 1,2-distearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and/or 1,2-dioleoyl-sn-glycerol-3-phosphocholine;

The mass ratio of the lipidated polymer, steroid or its derivative, auxiliary lipid and 1-(monomethoxypolyethylene glycol)-2,3-dimyristyl glycerol is 16:4-14:2-12:1-6; preferably 16:12-14:8:1, or 16:4:8:1-4, or 16:8:4-6:1, or 16:8-12:2:1, or 16:10:12:1-6, or 16:12:8-10:1.

In some of the embodiments, the carrier for delivering nucleic acid macromolecules is made of the lipidated polymer, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane and 1-(monomethoxypolyethylene glycol)-2,3-dimyristyl glycerol; the mass ratio of the lipidated polymer, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane and 1-(monomethoxypolyethylene glycol)-2,3-dimyristyl glycerol is 16:8:4:18-22:1.

The above-mentioned vector provided by the present invention can be further prepared into nanoparticles with nucleic acids to effectively deliver nucleic acid macromolecules to specific locations in vivo or in vitro and express them efficiently. Delivery refers to the transportation of nucleic acid substances to designated organs, tissues, tumors and the exertion of gene interference or gene expression. Gene interference refers to the reduction of the content of specific genes in cells, and gene expression refers to the translation of nucleic acids into encoded proteins. The methods of administration include but are not limited to intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, bladder instillation, local injection during surgery, etc.

In some embodiments, the present invention further provides a polymer nanoparticle, which is prepared from the carrier for delivering nucleic acid macromolecules and nucleic acid.

Wherein, nucleic acid refers to a polymer containing at least two or two ribonucleotides or deoxyribonucleotides in single-stranded or double-stranded form, including DNA, RNA and hybrids thereof. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products or vectors. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA) and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified main chain residues or connections, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphates, phosphate esters, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides and peptide nucleic acids (PNAs).

In some embodiments, the nucleic acid includes luciferase mRNA, Cas9 mRNA, sgRNA, EGFP mRNA, and PX458 plasmid.

In some embodiments, the mass ratio of the lipidated polymer in the carrier to the nucleic acid is 10:0.2-2, preferably 10:0.5-1.

The present invention provides two methods for preparing the polymer nanoparticles. In some embodiments, the method for preparing the polymer nanoparticles comprises the following steps:

The lipidated polymer, steroid and its derivatives, auxiliary lipids, functional lipids and polymer-bonded lipids are dissolved in a solvent to obtain solution 1; the nucleic acid is dissolved in a buffer to obtain solution 2; the solution 1 and solution 2 are mixed evenly and dialyzed to obtain the polymer nanoparticles.

In other embodiments, the method for preparing the polymer nanoparticles comprises the following steps:

The lipidated polymer, steroid or its derivative, auxiliary lipid, functional lipid and polymer-bonded lipid are dissolved in a solvent to obtain solution 1; solution 1 is evenly mixed with a buffer solution, dialyzed to obtain an empty polymer nanoparticle solution; the nucleic acid is added to the empty polymer nanoparticle solution to obtain the polymer nanoparticle.

Wherein, the mixing can be performed by dropwise addition or microfluidics; the solvent includes but is not limited to methanol, ethanol, dimethyl sulfoxide, N'N'-dimethylformamide, etc.; the buffer refers to an ion pair solution that can maintain a specific pH value, including but not limited to phosphate buffer, citrate buffer, sodium acetate buffer, sodium carbonate buffer, etc.

The present invention is further described in detail below with reference to specific embodiments.

The structural formula of PEI used in the following examples is shown in formula (II-a):

(i) In the following Examples 1-22, the structure of the compound of formula (III) is shown in formula (III-a), wherein Ra is a saturated fatty carbon chain, an unsaturated fatty carbon chain, or a fatty carbon chain containing heteroatoms (such as S, O, N, etc.).

Example 1: Preparation of polymer P6-4H-50

Weigh 40 mg of PEI600 (number average molecular weight 600, the same below) and 101 mg of tetrahydrogeraniol acrylate (structural formula as follows), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 17%.

Example 2: Preparation of polymer P6-4H-60

Weigh 40 mg of PEI600 and 121 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 24%.

Example 3: Preparation of polymer P6-4H-70

Weigh 40 mg of PEI600 and 141 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 25%.

Example 4: Preparation of polymer P6-4H-80

Weigh 40 mg of PEI600 and 162 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 34%.

Example 5: Preparation of polymer P6-4H-90

Weigh 40 mg PEI600 and 182 mg tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 36%. The H NMR spectrum of the obtained lipid polymer P6-4H-90 is shown in Figure 1. Near 0.8 ppm is the methyl hydrogen at the end of the fatty chain, 4.1 ppm is the methylene hydrogen adjacent to the ester bond, and 2.4-3.0 ppm is the methylene hydrogen of polyacetimide. Its H NMR spectrum can prove its successful synthesis.

Example 6: Preparation of polymer P6-4H-100

Weigh 40 mg of PEI600 and 202 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 43%.

Example 7: Preparation of polymer P6-4H-120

Weigh 40 mg of PEI600 and 242 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 57%.

Example 8: Preparation of polymer P6-4H-140

Weigh 40 mg of PEI600 and 282 mg of tetrahydrogeraniol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 60%.

Example 9: Preparation of polymers P6-Cit-80, P6-Cit-90, P6-Cit-100, P6-Cit-120, P6-Cit-140

Weigh 40 mg of PEI600 and 202 mg of citronellol acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography to obtain P6-Cit-100 with a yield of 39%.

Weigh 40 mg of PEI600 and 162 mg of citronellol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography to obtain P6-Cit-80. The yield is 21%.

Weigh 40 mg of PEI600 and 182 mg of citronellol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography to obtain P6-Cit-90. The yield is 25%.

Weigh 40 mg PEI600 and 242 mg citronellol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography to obtain P6-Cit-120 with a yield of 38%.

Example 8: Preparation of polymer P6-4H-140

Weigh 40 mg PEI600 and 282 mg citronellol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography to obtain P6-Cit-140 with a yield of 35%.

The hydrogen nuclear magnetic resonance spectra of the obtained lipidated polymers P6-Cit-80, P6-Cit-90, P6-Cit-100, P6-Cit-120 and P6-Cit-140 are shown in FIG2 , wherein the proportion of secondary amine groups at around 2.9 ppm gradually decreases with the increase of the feed ratio of citronellol acrylate.

Example 10: Preparation of polymer P18-Cit-100

Weigh 40 mg PEI1800 (number average molecular weight 1800) and 202 mg citronellol acrylate, heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 27%.

Example 11: Preparation of polymer P100-Cit-100

Weigh 40 mg of PEI10000 (number average molecular weight 10000) and 202 mg of citronellol acrylate, heat to 75°C, and stir for 3 days. The product is separated by column chromatography. The yield is 9%.

Example 12: Preparation of polymer P6-C8-100

Weigh 40 mg of PEI600 and 175 mg of octyl acrylate (structural formula shown below), heat to 75° C., and stir to react for 3 days.

The product was isolated by column chromatography with a yield of 42%.

Example 13: Preparation of polymer P6-C10-100

Weigh 40 mg of PEI600 and 202 mg of decyl acrylate (structural formula shown below), heat to 75° C., and stir to react for 3 days.

The product was isolated by column chromatography with a yield of 32%.

Example 14: Preparation of polymer P6-C12-100

Weigh 40 mg of PEI600 and 228 mg of dodecyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 42%.

Example 15: Preparation of polymer P6-C14-100

Weigh 40 mg of PEI600 and 256 mg of tetradecyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 32%.

Example 16: Preparation of polymer P6-C16-100

Weigh 40 mg of PEI600 and 281 mg of hexadecyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 21%.

Example 17: Preparation of polymer P6-C18-100

Weigh 40 mg of PEI600 and 308 mg of octadecyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 23%.

Example 18: Preparation of polymer P6-IC10-100

Weigh 40 mg of PEI600 and 202 mg of isodecyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 31%.

Example 19: Preparation of polymer P6-5EC8-100

Weigh 40 mg of PEI600 and 175 mg of cis-5-octen-1-ol acrylic acid ester (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 34%.

Example 20: Preparation of polymer P6-2YC8-100

Weigh 40 mg of PEI600 and 175 mg of 2-ethylhexyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 29%.

Example 21: Preparation of polymer P6-IC8-100

Weigh 40 mg of PEI600 and 175 mg of isooctyl acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 27%.

Example 22: Preparation of polymer P6-C12B-100

Weigh 40 mg of PEI600 and 262 mg of 3,4-dithiododecanol acrylate (structural formula shown below), heat to 75°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 30%.

(ii) In the following Examples 23-24, the structure of the compound of formula (III) is shown in formula (III-b), wherein Rb is a saturated fatty carbon chain, an unsaturated fatty carbon chain, or a fatty carbon chain containing heteroatoms (such as S, O, N, etc.).

Example 23: Preparation of polymer P6-C6A1-100

Weigh 40 mg PEI600 and 400 mg C6A1 (structural formula shown below), heat to 75°C, and stir for 3 days. The product is separated by column chromatography. The yield is 33%.

Example 24: Preparation of polymer P6-C6A2-100

Weigh 40 mg PEI600 and 344 mg C6A2 (structural formula shown below), heat to 75°C, and stir for 3 days. The product is separated by column chromatography. The yield is 32%.

(III) In the following Examples 25-27, the structure of the compound of formula (III) is shown in formula (III-c), wherein Rc is a saturated fatty carbon chain, an unsaturated fatty carbon chain, or a fatty carbon chain containing heteroatoms (such as S, O, N, etc.).

Example 25: Preparation of polymer P6-A12-100

Weigh 40 mg PEI600 and 220 mg dodecanoyl chloride (structural formula shown below) and react at 4°C for 12 h. The product is separated by column chromatography. The yield is 22%.

Example 26: Preparation of polymer P6-A14-100

Weigh 40 mg of PEI600 and 240 mg of tetradecanoyl chloride (structural formula shown below) and react at 4°C for 12 h. The product is separated by column chromatography. The yield is 25%.

Example 27: Preparation of polymer P6-A16-100

Weigh 40 mg of PEI600 and 270 mg of hexadecanoyl chloride (structural formula shown below) and react at 4°C for 12 h. The product is separated by column chromatography. The yield is 21%.

(IV) In the following Examples 28-30, the structure of the compound of formula (III) is shown in formula (III-d), wherein Rd is a saturated fatty carbon chain, an unsaturated fatty carbon chain, or a fatty carbon chain containing heteroatoms (such as S, O, N, etc.).

Example 28: Preparation of polymer P6-EC12-100

Weigh 40 mg of PEI600 and 175 mg of 1,2-epoxydodecane (structural formula shown below), heat to 80°C, and stir for 3 days. The product is separated by column chromatography. The yield is 31%.

Example 29: Preparation of polymer P6-EC14-100

Weigh 40 mg of PEI600 and 200 mg of 1,2-epoxytetradecane (structural formula shown below), heat to 80°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 34%.

Example 30: Preparation of polymer P6-EC16-100

Weigh 40 mg of PEI600 and 228 mg of 1,2-epoxyhexadecane (structural formula shown below), heat to 80°C, and stir to react for 3 days. The product is separated by column chromatography. The yield is 27%.

(V) Taking P6-4H-90 as an example, Examples 31 to 86 are examples of preparing polymer nanoparticles, and the methods for preparing polymer nanoparticles with other polymers are the same. In the following examples, the molecular weight of PEG in PEG-DMG is 2000.

Example 31: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 32: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DOPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 33: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 34: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DMPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DMPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 35: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg P6-4H-90, dissolve it in 1 mL ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL lanosterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of lanosterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 36: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of sitosterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of sitosterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 37: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of stigmasterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of stigmasterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 38: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of ergosterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of ergosterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 39: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of bile acid solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of bile acid solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 40: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 41: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 6 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 42: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 43: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 10 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 44: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 12 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 45: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 14 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 46: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 16 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 47: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 4 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 48: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 6 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 49: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 50: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 10 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 51: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 12 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 52: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 14 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 53: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 16 uL of DSPC solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 54: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 2 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 55: Preparation of four-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 56: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 6 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 57: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 10 uL of cholesterol solution, 12 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 58: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 10 uL of cholesterol solution, 12 uL of DOPE solution, and 2 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 59: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 10 uL of cholesterol solution, 12 uL of DOPE solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 60: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 10 uL of cholesterol solution, 12 uL of DOPE solution, and 6 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 61: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 2 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 62: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 4 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 63: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 6 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 64: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 12 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 65: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 2 uL of DOPE solution, and 2 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 66: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 2 uL of DOPE solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 67: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 2 uL of DOPE solution, and 6 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 68: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 2 uL of DOPE solution, and 8 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 69: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 2 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 70: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 6 uL of cholesterol solution, 2 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 71: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 12 uL of cholesterol solution, 2 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 72: Preparation of Four-Component P6-4H-90 Lipid Nanoparticles

Weigh 10 mg of P6-4H-90 and dissolve it in 1 mL of ethanol to prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 16 uL of cholesterol solution, 2 uL of DOPE solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 73: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg P6-4H-90, dissolve it in 1 mL ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL lipidated polymer solution, 4 uL cholesterol solution, 8 uL DSPC solution, 4 uL DOTAP solution, and 4 uL PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 74: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 8 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 75: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 12 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 76: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 16 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution evenly with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 77: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 20 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution evenly with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 78: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 24 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 79: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 28 uL of DOTAP solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution evenly with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 80: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, 18PG solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, 28 uL of 18PG solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 81: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg P6-4H-90, dissolve it in 1 mL ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL cholesterol solution, DSPC solution, DDAB solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL lipidated polymer solution, 4 uL cholesterol solution, 8 uL DSPC solution, 28 uL DDAB solution, and 4 uL PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 82: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg P6-4H-90, dissolve it in 1 mL ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL cholesterol solution, DSPC solution, DOPE solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL lipidated polymer solution, 4 uL cholesterol solution, 8 uL DSPC solution, 28 uL DOPE solution, and 4 uL PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 83: Preparation of five-component P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, DOTAP solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of sodium acetate buffer solution (pH=5.2, 25 mM) of luciferase mRNA. Take 16 uL of lipidated polymer solution, 8 uL of cholesterol solution, 4 uL of DSPC solution, 20 uL of DOTAP solution, and 1 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution evenly with 80 uL of the above-mentioned sodium acetate buffer solution of luciferase mRNA. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 84: Preparation of gene-edited P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare a sodium acetate buffer solution (pH = 5.2, 25 mM) containing Cas9 mRNA (0.2 mg/mL) and sgRNA (0.2 mg/mL). Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution evenly with 80 uL of the above-mentioned sodium acetate buffer solution of Cas9 mRNA and sgRNA. Dialyze and collect through a dialysis bag with a molecular weight cutoff of 3500. Concentrate by ultrafiltration centrifugation and set aside.

Example 85: Preparation of green fluorescence reporter P6-4H-90 lipid nanoparticles

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare 0.2 mg/mL of green fluorescent protein (EGFP) mRNA sodium acetate buffer solution (pH=5.2, 25 mM). Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned EGFP mRNA sodium acetate buffer solution. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

Example 86: Preparation of P6-4H-90 lipid nanoparticles for plasmid delivery

Weigh 10 mg of P6-4H-90, dissolve it in 1 mL of ethanol, and prepare a 10 mg/mL lipidated polymer solution. Using ethanol as the solvent, prepare 10 mg/mL of cholesterol solution, DSPC solution, and PEG-DMG solution respectively. Prepare a 0.2 mg/mL sodium acetate buffer solution (pH=5.2, 25 mM) of PX458 plasmid. Take 16 uL of lipidated polymer solution, 4 uL of cholesterol solution, 8 uL of DSPC solution, and 4 uL of PEG-DMG solution, and mix them evenly. Then mix the resulting mixed solution with 80 uL of the above-mentioned sodium acetate buffer solution of PX458 plasmid. Dialyze through a dialysis bag with a molecular weight cutoff of 3500 and collect. Concentrate by ultrafiltration centrifugation and set aside.

(vi) The following are examples of applications of polymer nanoparticles.

Example 87: Delivery of PX458 plasmid by P6-Cit-100 polymer nanoparticles

The experimental steps are as follows: melanoma cells B16F10 were seeded into a 48-well plate, with 20,000 cells per well. After the cells adhered to the wall, the prepared P6-cit-100 polymer nanoparticles encapsulating the PX458 plasmid (the carrier in the nanoparticles contains 4 components, namely, lipid polymer (P6-Cit-100, Example 11), cholesterol, DOPE, and PEG-DMG) were added to the culture medium, and the concentration of PX458 plasmid per well was 200 ng. After 72 hours, trypsin was added to digest and collect the cells, and the expression level of green fluorescent protein was detected by flow cytometry.

The results show ( FIG. 3 ) that the polymer nanoparticles of the present invention are capable of delivering DNA into cells and expressing the DNA.

Example 88: Intratumoral delivery of mRNA by P6-4H-90 polymer nanoparticles

The experimental steps are as follows: Take 4-6 week old C57BL/c mice. Each mouse is inoculated with 500,000 B16F10 cells by subcutaneous injection. Each mouse is inoculated with 2 tumors (located in the upper and lower parts of the body respectively) or 1 tumor. When the tumor volume grows to ^200-300mm3 , P6-4H-90 nanoparticles with different dosage ratios encapsulating luciferase mRNA are injected into the tumor (the carrier in the nanoparticles contains 4 components, namely lipid polymer (P6-4H-90, Example 5), cholesterol, DSPC, and PEG-DMG), and each mouse is injected with 2ug mRNA per tumor. After 6 hours, 100uL 15mg/mL luciferin is injected intraperitoneally, and 10 minutes later, the location and intensity of luciferase expression are observed by small animal in vivo imaging.

The results show ( FIG. 4 ) that the nanoparticles of the present invention can effectively deliver mRNA into the tumor for expression, and the ratio of the components in the nanoparticles has a great influence on the in vivo delivery.

Example 89: Delivery of mRNA by P6-4H-90 polymer nanoparticles via tail vein injection

The experimental steps are as follows: Babl/c mice aged 4-6 weeks were taken. Each mouse was injected with nanoparticles carrying luciferase mRNA in different ratios (the carrier in the nanoparticles contains 4 components, namely lipid polymer (P6-4H-90, Example 5), cholesterol, DSPC, and DMG-PEG) through the tail vein, and the mRNA content injected into each mouse was 2ug. After 6 hours, 100uL 15mg/mL luciferin was injected intraperitoneally, and 10 minutes later, the location and intensity of luciferase expression were observed by small animal live imaging.

The results show ( FIG. 5 ): the nanoparticles of the present invention can well deliver mRNA into mice via tail vein injection and express it, and the expression effect is excellent; and the ratio of each component in the nanoparticles has a great influence on the in vivo delivery.

Example 90: Delivery of mRNA by tail vein injection using different polymer nanoparticles

The experimental steps are as follows: Babl/c mice aged 4-6 weeks were taken. Each mouse was injected with nanoparticles loaded with luciferase mRNA prepared by different polymers through the tail vein (the carrier in the nanoparticles contains 4 components, and the mass ratio of lipid polymer: cholesterol: DSPC: DMG-PEG is 16:8:4:1), and the mRNA content injected into each mouse was 2ug. After 6 hours, 100uL 15mg/mL luciferin was injected intraperitoneally, and 10 minutes later, the location and intensity of luciferase expression were observed by small animal live imaging.

The results show ( FIG. 6 and FIG. 7 ) that the structure of the lipidated polymer in the present invention has a great influence on the mRNA delivery effect.

Example 91: Delivery of mRNA by polymer nanoparticles via intramuscular and subcutaneous injection

The experimental steps are as follows: Babl/c mice aged 4-6 weeks were taken. Each mouse was injected subcutaneously and intramuscularly with nanoparticles loaded with luciferase mRNA (the carrier in the nanoparticles contains 4 components, lipid polymer (P6-4H-90 or P18-Cit-100): cholesterol: DSPC: DMG-PEG mass ratio is 16:8:4:1), and the mRNA content injected into each mouse was 2ug. After 6 hours, 100uL 15mg/mL luciferin was injected intraperitoneally, and 10 minutes later, the location and intensity of luciferase expression were observed by small animal live imaging.

The results show ( FIG. 8 ) that the nanoparticles of the present invention can effectively deliver mRNA to mice via intramuscular and subcutaneous injections for expression.

Example 92: Five-component polymer nanoparticles delivering mRNA

The experimental steps are as follows: Babl/c mice aged 4-6 weeks were taken. Each mouse was injected with nanoparticles loaded with luciferase mRNA through the tail vein (the carrier in the nanoparticles contains 5 components, P6-4H-90: cholesterol: DSPC: DOTAP: DMG-PEG mass ratio is 16:8:4:20:1), and the mRNA content injected into each mouse was 2ug. After 6 hours, 100uL 15mg/mL luciferin was injected intraperitoneally, and 10 minutes later, the location and intensity of luciferase expression were observed by small animal live imaging.

The results showed (Figure 9): After the addition of the fifth component, the location of mRNA expression in the mouse body changed significantly, the mRNA expression ratio in the mouse liver decreased, while the mRNA expression ratio in the mouse lung and spleen increased. The results indicate that the functional lipids in the nanoparticle dosage form of the present invention have a regulatory effect on its organ distribution.

Example 93: Delivery of mRNA by polymer nanoparticles prepared from different steroids

The experimental steps are as follows: Babl/c mice aged 4-6 weeks were selected. Each mouse was injected with nanoparticles loaded with luciferase mRNA through the tail vein. The carriers were (1) P6-4H-90: lanosterol: DSPC: DMG-PEG with a mass ratio of 16:8:4:1, (2) P6-4H-90: β-sitosterol: DSPC: DMG-PEG with a mass ratio of 16:8:4:1; (3) P6-4H-90: stigmasterol: DSPC: DMG-PEG with a mass ratio of 16:8:4:1. Each mouse was injected with 2ug of mRNA. After 6 hours, 100uL of 15mg/mL luciferin was injected intraperitoneally. After 10 minutes, the location and intensity of luciferase expression were observed by small animal live imaging.

The results showed ( FIG. 10 ): the mRNA expression intensity in mice was significantly changed, indicating that the steroid component in the nanoparticles of the present invention has a significant effect on the delivery effect of mRNA.

Example 94: Intratumoral delivery of mRNA by polymer nanoparticles prepared with different auxiliary lipids

The experimental steps are as follows: Take 4-6 week old C57BL/6 mice. Each mouse is inoculated with 500,000 B16F10 cells by subcutaneous injection. When the tumor volume grows to ^200-300mm3 , each mouse is injected intratumorally with nanoparticles loaded with luciferase mRNA. The polymer molecule in the lipid nanoparticles used is P6-4H-90. The weight ratio used is polymer molecule: cholesterol: auxiliary lipid: PEG-DMG = 16:12:8:1. The auxiliary lipids selected are DSPC, DOPE, and DOPC. The mRNA content injected into each mouse is 2ug. After 6 hours, 100uL 15mg/mL luciferin is injected intraperitoneally. After 10 minutes, the location and intensity of luciferase expression are observed by small animal in vivo imaging.

The results show (Figure 11): the mRNA expression in mouse tumors changes significantly, and the mRNA expression in mouse tumors of nanoparticles containing DSPC is significantly higher than that of nanoparticles containing DOPE and DOPC. The results show that the auxiliary lipid component in the polymer nanoparticles of the present invention has a great influence on mRNA transfection.

Example 95: Intratumoral delivery of mRNA by polymer nanoparticles prepared with polymers having different grafting ratios

The experimental steps are as follows: Take 4-6 week old C57BL/6 mice. Each mouse is inoculated with 500,000 B16F10 cells by subcutaneous injection. When the tumor volume grows to ^200-300mm3 , each mouse is injected intratumorally with nanoparticles loaded with luciferase mRNA (the carrier in the nanoparticles contains 4 components, the mass ratio of polymer: cholesterol: DSPC: DMG-PEG is 16:12:8:1, and the polymers are P6-4H-80, P6-4H-90, P6-4H-120, P6-Cit-80, P6-Cit-100, P6-Cit-120, P6-Cit-140). Each mouse is injected with 2ug of mRNA. After 6 hours, 100uL of 15mg/mL luciferin is injected intraperitoneally. After 10 minutes, the location and intensity of luciferase expression are observed by small animal live imaging.

The results show (Figures 12 and 13): for P6-4H polymer, nanoparticles prepared with P6-4H-90 have the strongest mRNA expression in mouse tumors (Figure 12); for P6-Cit polymer, nanoparticles prepared with P6-Cit-140 have the strongest mRNA expression in mouse tumors (Figure 13). The results show that the grafting rate of the polymer component in the polymer nanoparticles of the present invention has a certain effect on mRNA transfection.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (30)

1. A lipidated polymer, characterized in that it is obtained by reacting a compound of formula (II) or polyethyleneimine with a compound of formula (III),

Wherein each m is independently selected from: 1.2, 3, 4, 5, 6;

n is selected from: an integer between 1 and 1000;

L is a group reactive with a secondary or primary amine in the compound of formula (II) or polyethyleneimine;

J is absent, or selected from: c ₁-C₁₂ alkylene, C ₂-C₁₂ unsaturated chain hydrocarbon group;

K is absent, or selected from ：-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-O-、-S-、-S(O)-、-S(O)₂-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRC(＝O)-、-C(＝O)NR-、-NRC(＝O)NR-、-OC(＝O)NR-、-NRC(＝O)O-;

R is selected from: H. c ₁-C₁₂ alkyl, C ₂-C₁₂ unsaturated chain hydrocarbon group;

M is selected from: c ₁-C₂₄ alkyl, C ₂-C₂₄ unsaturated chain alkyl.

2. The lipidated polymer of claim 1, wherein L in the compound of formula (III) is selected from: Halogen, acyl halide, cyclopropyloxy, carboxyl, thiocyanate.

3. The lipidated polymer of claim 2, wherein L in the compound of formula (III) is selected from: Chlorine, bromine, acyl chloride, cyclopropyloxy.

4. The lipidated polymer of claim 1, wherein J is absent or selected from the group consisting of: c ₁-C₈ alkylene group, C ₂-C₈ unsaturated chain hydrocarbon group.

5. The lipidated polymer of claim 4, wherein J is absent or selected from the group consisting of: methylene, ethylene, propylene, butylene, pentylene, and hexylene.

6. The lipidated polymer of claim 1, wherein K is absent or selected from the group consisting of:

-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-O-、-S-、-S(O)-、-S(O)₂-、-S-S-。

7. the lipidated polymer of claim 1, wherein M is selected from the group consisting of: c ₆-C₂₀ alkyl, C ₆-C₂₀ unsaturated chain alkyl.

8. The lipidated polymer of claim 7, wherein M is selected from the group consisting of: c ₈-C₁₈ alkyl, C ₈-C₁₈ unsaturated chain alkyl.

9. The lipidated polymer of claim 8, wherein M is selected from the group consisting of: c ₁₂-C₁₄ straight-chain alkyl, C ₈-C₁₄ branched-chain alkyl, C ₁₂-C₁₄ unsaturated straight-chain alkyl, and C ₈-C₁₄ unsaturated branched-chain alkyl.

10. The lipidated polymer of claim 9, wherein M is selected from the group consisting of:

11. the lipidated polymer according to claim 1, wherein the compound of formula (III) is selected from:

12. The lipidated polymer according to any one of claims 1 to 11, wherein it is obtained by reacting a polyethyleneimine with a compound of formula (III), said polyethyleneimine being a branched polyethyleneimine.

13. The lipidated polymer according to claim 12, wherein the branched polyethylenimine has a molecular weight of 300-10000, preferably 400-2000, more preferably 500-1800, more preferably 500-700, more preferably 600.

14. The lipidated polymer according to any one of claims 1 to 11, wherein the reaction feed ratio of the compound of formula (II) or polyethyleneimine and the compound of formula (III) is 40mg:0.4-1.8mmoL, preferably 40mg:0.7-1.5mmoL, more preferably 40mg:0.8-0.9mmoL, or 40mg:1.3-1.4mmoL.

15. A method of preparing a lipidated polymer according to any one of claims 1 to 14, comprising the steps of: the compound of the formula (II) or polyethyleneimine reacts with the compound of the formula (III) at a certain temperature to obtain the compound.

16. The method according to claim 15, wherein the reaction temperature is 4 ℃ to 200 ℃ and the reaction time is 10h to 120h.

17. The method of claim 15, wherein the reaction is performed without the addition of a solvent; or the reaction is carried out in a solvent selected from at least one of dichloromethane, chloroform, methanol, acetonitrile, N' -dimethylformamide, dimethyl sulfoxide, benzene and toluene.

18. A carrier for delivering a nucleic acid macromolecule, characterized by being made of a component comprising a lipidated polymer, a steroid or derivative thereof, a helper lipid, a polymer-bound lipid;

The lipidated polymer being the lipidated polymer of any one of claims 1-14; the helper lipid is a non-ionic lipid and/or a zwitterionic lipid.

19. The carrier for delivering a nucleic acid macromolecule of claim 18, made of a component comprising a lipidated polymer, a steroid or derivative thereof, a helper lipid, a functional lipid, a polymer-bonded lipid; the functional lipids are cationic lipids and/or anionic lipids.

20. The carrier for delivering a nucleic acid macromolecule according to claim 18 or 19, wherein the steroid or derivative thereof is selected from at least one of cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol, bile acid, bile alcohol and steroid hormone; and/or the number of the groups of groups,

The auxiliary lipid is at least one selected from 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine and phosphatidylethanolamine; and/or the number of the groups of groups,

The functional lipid is at least one selected from 1, 2-dioleoyl-3-trimethylammonium-propane, dimethyl octadecyl ammonium 1, 2-dicarboxyl acyl-glycerol-3-phosphoric acid and 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphoric acid- (1' -rac-glycerol); and/or the number of the groups of groups,

The polymer-bound lipid is a pegylated lipid.

21. The carrier for delivering a nucleic acid macromolecule according to claim 20, wherein the pegylated lipid is 1- (monomethoxy polyethylene glycol) -2, 3-dimyristoyl glycerol; and/or the phosphatidylethanolamine is 1, 2-dioleoyl-sn-glycerol-3-phosphorylethanolamine and/or sphingosine.

22. The carrier for delivering a nucleic acid macromolecule according to claim 18 or 19, wherein the mass ratio of lipidated polymer, steroid or derivative thereof, helper lipid, functional lipid and polymer-bonded lipid is 16:1-20:1-16:0-45:1-8, preferably 16:4-16:2-12:0-40:1-6; preferably 16:4-16:2-12:0-30:1-6.

23. The carrier for delivering a nucleic acid macromolecule of claim 22, wherein the carrier is made from the lipidated polymer, a steroid or derivative thereof, a helper lipid and 1- (monomethoxypolyethylene glycol) -2, 3-dimyristoyl glycerol;

The steroid or derivative thereof is cholesterol, lanosterol and/or stigmasterol;

The auxiliary lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine and/or 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine;

The mass ratio of the lipidation polymer, the steroid or the derivative thereof, the auxiliary lipid and the 1- (monomethoxy polyethylene glycol) -2, 3-dimyristoyl myristicin is 16:4-14:2-12:1-6; preferably 16:12-14:8:1, or 16:4:8:1-4, or 16:8:4-6:1, or 16:8-12:2:1, or 16:10:12:1-6, or 16:12:8-10:1.

24. The carrier for delivering a nucleic acid macromolecule of claim 22, wherein the carrier is made from the lipidated polymer, cholesterol, 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, 1, 2-dioleoyl-3-trimethylammonium-propane, and 1- (monomethoxy polyethylene glycol) -2, 3-dimyristoyl-glycerol; the mass ratio of the lipidation polymer to cholesterol to 1, 2-distearoyl-sn-glycero-3-phosphorylcholine to 1, 2-dioleoyl-3-trimethylammonium-propane to 1- (monomethoxy polyethylene glycol) -2, 3-dimyristoyl-glycerol is 16:8:4:18-22:1.

25. Use of the vector of any one of claims 18-24 for in vivo or in vitro delivery of a nucleic acid macromolecule.

26. A polymeric nanoparticle prepared from the nucleic acid macromolecule delivery vehicle and the nucleic acid of any one of claims 18-24.

27. The polymeric nanoparticle of claim 26, wherein the nucleic acid comprises luciferase mRNA, cas9 mRNA, sgRNA, EGFP mRNA, PX458 plasmid.

28. The polymeric nanoparticle of claim 26 or 27, wherein the mass ratio of lipidated polymer to the nucleic acid in the carrier is 10:0.2-2, preferably 10:0.5-1.

29. A method of preparing the polymeric nanoparticle of any one of claims 26-28, comprising the steps of:

Dissolving the lipidated polymer, steroid or derivative thereof, auxiliary lipid, functional lipid and polymer-bonded lipid in a solvent to obtain a solution 1; dissolving the nucleic acid in a buffer solution to obtain a solution 2; uniformly mixing the solution 1 and the solution 2, and dialyzing to obtain the polymer nano particles;

or the preparation method of the polymer nano-particles comprises the following steps:

Dissolving the lipidated polymer, steroid or derivative thereof, auxiliary lipid, functional lipid and polymer-bonded lipid in a solvent to obtain a solution 1; uniformly mixing the solution 1 with a buffer solution, and dialyzing to obtain an empty polymer nanoparticle solution; and adding the nucleic acid into the empty polymer nanoparticle solution to obtain the polymer nanoparticle.

30. The method of preparing polymer nanoparticles of claim 29, wherein the solvent is selected from the group consisting of methanol, ethanol, dimethyl sulfoxide, N' -dimethylformamide; and/or the number of the groups of groups,

The buffer solution is selected from phosphate buffer solution, citrate buffer solution, sodium acetate buffer solution and sodium carbonate buffer solution.