CN115304756B

CN115304756B - Five-membered lipid nanoparticle as well as preparation method and application thereof

Info

Publication number: CN115304756B
Application number: CN202210114678.7A
Authority: CN
Inventors: 李剑峰; 曹燕; 何宗兴
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2022-01-30
Filing date: 2022-01-30
Publication date: 2023-05-09
Anticipated expiration: 2042-01-30
Also published as: CN115304756A

Abstract

The invention provides five-membered lipid nano particles, a preparation method and application thereof. The raw materials of the five-membered lipid nanoparticle comprise cationic lipid, polymeric (beta-amino ester), sterol, phospholipid, PEG lipid and nucleic acid molecules. The five-membered lipid nanoparticle has high stability, can be stored for a long time at 4 ℃ after freeze drying, and can be used as a nucleic acid drug delivery system (such as lung targeting and the like); has good tolerance and has a suitable surface zeta potential of approximately +5 mV.

Description

Five-membered lipid nanoparticle as well as preparation method and application thereof

Technical Field

The invention relates to the field of chemical pharmacy and biotechnology, and relates to a nucleic acid delivery system, in particular to a preparation method of high-stability lung-targeted polymer-lipid nanoparticles and application of the polymer-lipid nanoparticles in nucleic acid delivery.

Background

Outbreaks of new coronal pneumonia epidemic have led people to realize the severity of pulmonary infections. In addition to infectious diseases, other related diseases of the lung, such as lung cancer and hereditary rare diseases, lack effective therapeutic means. During new coronaries, a novel mRNA vaccine encapsulated in Lipid Nanoparticles (LNP) showed up to 95% protective efficacy, the first mRNA vaccine to obtain FDA emergency use approval. In addition, lipid Nanoparticle (LNP) -based mRNA can radically cure genetic diseases by means of coding gene editing, and can also realize immunotherapy of tumors by coding tumor antigens, immune checkpoint inhibitors, bispecific antibodies, and the like. Thus, the application of mRNA-LNP formulations to the treatment of lung-related diseases clearly has great potential and significance.

There are few studies currently suggesting strategies for lung targeting of mRNA nucleic acids, such as SORT suggesting that positively charged nanoparticles may enable lung delivery. However, the use of LNP still faces a number of problems at present, where storage stability is a major drawback limiting its wide clinical application, and low-stability LNP formulations can present a significant economic barrier to storage and transport. Instability of the LNP is therefore a major challenge. The major factors currently affecting the instability of mRNA-LNPs are as follows, namely mRNA instability and LNP instability:

1) The chemical components in mRNA are susceptible to oxidation and hydrolysis in the presence of water. For example, a 2'OH group on a phosphoester bond in mRNA attacks the p-o5' ester bond, resulting in cleavage of the mRNA strand. This process requires water, which can be catalyzed by acids or bases. 2) The force of the mRNA against the chemical lipids weakens and mRNA leaks due to degradation of the chemical material. The leaked bare mRNA is difficult to be absorbed by cells and can be rapidly degraded; 3) Lipid nanoparticles have a tendency to aggregate, fuse and leak due to insufficient repulsive forces between the nanoparticles in water.

Disclosure of Invention

To function effectively in vivo, lipid nanoparticle-mRNA needs to overcome a variety of extracellular and intracellular barriers. First, mRNA needs to be successfully encapsulated by lipid nanoparticles. Second, the lipid nanoparticle needs to reach the target tissue and then be internalized by the target cell. Most importantly, mRNA must escape from the endosome and be translated into a functional protein. In addition, the formulation is preferably stored for a long period of time at 4 ℃ for clinical use to reduce the economic burden during storage during shipping.

Overall, the physicochemical properties of the nanoparticles are the basis for subsequent evaluation of effectiveness. For example, higher nucleic acid encapsulation efficiency is a key factor in determining efficient transfection and suitable particle size; zeta potential also affects its toxicity and in vivo dispersibility to some extent. The higher the Zeta potential, the stronger the hemolytic capacity of the erythrocytes under physiological conditions and the greater the toxicity. While PDI reflects the uniformity and perfection of the prescription to some extent. The proper particle size and potential of the nano particles can reduce the probability of in vivo embolism, promote penetration of deep tissues in vivo and improve the uptake and transfection efficiency of cells.

The inventors have innovatively found that the addition of PBAE and cationic lipids can increase the stability of FNP.

For example, the present invention employs the classical cationic lipid DOTAP, which has a strong positive charge to prevent intermolecular aggregation; secondly, an auxiliary polymer poly beta-amino ester (PBAE) is firstly introduced into a traditional four-component cationic LNP formula to form a novel five-membered nano particle FNP. The PBAE is taken as a protonatable cation material containing multiple nitrogen, so that the encapsulation of mRNA is further enhanced, the leakage of mRNA is prevented, the adverse effect caused by crystallization and vacuum dehydration in the freeze-drying process is reduced, and the stability of the nanoparticle is further ensured. The addition of these two components makes the nanoparticle possess high stability, and can withstand freeze drying well, so that the influence of water on mRNA and lipid material hydrolysis can be removed through freeze drying.

The invention constructs a novel PBAE polymer library with different side chain lengths and different polymerization degrees, and further discusses the in-vivo and in-vitro structure-activity relationship. The closer the carbon chain length of the PBAE to C16, the higher the delivery efficiency; the higher the degree of polymerization of the PBAE, the higher the delivery efficiency. The FNP of the novel auxiliary polymer based on the components is screened out to obtain a specific and efficient lung targeting PBAE material in vivo, and the stabilizing time of the material at 4 ℃ after freeze drying is more than 3 months.

In addition, the invention researches the protein crown composition of FNP through mass spectrometry analysis, and further researches the lung targeting mechanism thereof. Vitronectin was found to be highly enriched in FNP. For the first time, mouse lung primary microvascular endothelial cells are adopted to verify that vitronectin passes through the receptor alpha of the lung endothelial cells _v β ₃ Binding, mediates lung-specific targeting of FNP.

The invention mainly solves the technical problems through the following technical scheme.

The invention aims to provide a lung targeting nucleic acid delivery system which can effectively encapsulate nucleic acid and can quickly escape lysosomes after being efficiently taken up by cells, and finally realize the efficient expression of target genes in vivo, and meanwhile, the system has high stability and can be stored for a long time under the condition of 4 ℃ after freeze drying.

In order to achieve the purposes of effectively encapsulating nucleic acid and improving the cell uptake and lysosome escape efficiency, the invention discloses the following technical scheme: a polymer-lipid nanoparticle with the addition of an auxiliary polymer PBAE and a cationic lipid to the lipid nanoparticle, the nanoparticle consisting of the polymer PBAE, cationic lipid, sterol, phospholipid, PEG lipid and one or more nucleic acid molecules, said nucleic acid molecules being entrapped in a layer-by-layer entrapped structure formed in the lipid and polymer.

The invention introduces an auxiliary polymer poly beta-amino ester (PBAE) into a traditional four-component cation LNP formula for the first time to form a novel five-membered nano particle FNP. PBAE as a protonatable, multi-nitrogen-containing cationic material further enhances mRNA encapsulation.

The present invention provides a polymeric (beta-amino ester) (PBAE) comprising formula (I);

wherein:

R ₁ independently is a fatty chain with or without hydroxyl groups, R ₂ Independently is a terminal monomer comprising a primary, secondary or tertiary amine, R comprises a linear or branched C ₁ -C ₅₀ An alkylene chain, which may further comprise one or more heteroatoms or one or more carbocyclic, heterocyclic or aromatic groups;

m is 1-15, n is 3-45, and x is 1-13.

In the invention, the R ₁ Preferably independently of one another

The R is ₂ Preferably independently of one another

Said R is preferably independently

Or alternatively

The preparation method of the PBAE may be conventional in the art, for example, the preparation method thereof comprises: r is R ₁ 、R ₂ R and alkylamine are mixed together to perform Michael addition reaction.

The invention also provides a five-membered lipid nanoparticle, the raw materials of which comprise cationic lipid, polymerized (beta-amino ester), sterol, phospholipid and PEG lipid as described above.

When the pentad lipid nanoparticle is used as a nucleic acid delivery system, it may also comprise a nucleic acid molecule.

In the present invention, the molar ratio of the cationic lipid to the PBAE is preferably (2.5 to 40): 1.

In the present invention, the relative amounts of the cationic lipid and the PBAE mass and the nucleic acid molecule are preferably (10 to 80): 1.

In the present invention, the molar ratio of the phospholipid to the sterol is preferably (0.2 to 1): 1.

in the present invention, the molar ratio of the phospholipid to the PEG lipid is preferably (5-20): 1.

In a preferred embodiment of the invention, the ratio of n to m is 7:3; namely: the proportion of fatty chains was 30%.

In the present invention, the x is preferably 3 to 11.

In the present invention, the sterols are selected from one or more of cholesterol, campesterol, phytosterol, and carosterol; cholesterol is preferred.

In the context of the present invention, cationic lipids refer to permanently positively charged lipids, which may be selected from the list comprising: trimethyl-2, 3-dioleyloxypropyl ammonium chloride (DOTMA), trimethyl-2, 3-dioleoyloxypropyl ammonium bromide (DOTAP), dimethyl-2, 3-dioleoyloxypropyl-2- (2-argoylamino) ethyl ammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecyl ammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropyl ammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropyl ammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2, 3-dioleoyloxypropyl ammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2, 3-dioleoyloxypropyl ammonium bromide (DOHB-TTAB), dimethyl-5-hydroxypentyl-2, 3-dioleoyloxypropyl ammonium bromide (DOAB), dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropyl ammonium bromide (DORIE), dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropyl ammonium bromide (DORIE), dimethyl-3-2, 3-dioleoyloxypropyl ammonium bromide (DORIE), and DPE-2-dioleoyloxypropyl ammonium bromide (DORIE). N ' -dioctadecyl glycinamide (DOGS), 1, 2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC), 3β - [ N- (N ', N ' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol).

The term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of phosphate groups, in the context of the present invention, suitable phospholipids may be selected from the list comprising: 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DUPC), 1, 2-dioleoyl-sn-glycero-phosphocholine (DuPC), 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (OCPC), 1, 2-di-arachidonyl-sn-glycero-3-phosphorylcholine, 1, 2-di-dodecanoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-phytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-arachidonyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-dodecanoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

The term "PEG lipid" or alternatively "pegylated lipid" refers to any suitable lipid modified with PEG (polyethylene glycol) groups. May be selected from the list comprising: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. More specific examples of such PEG lipids include C14-PEG2000 (1, 2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG 2000)) and C18-PEG5000 (1, 2-distearoyl-rac-glycerol, methoxypolyethylene glycol-5000 (DSG-PEG 5000)).

In a specific embodiment, the PEG lipid is methoxypolyethylene glycol-2000 (DMG-PEG 2000).

In the present invention, the nucleic acid molecule may be RNA and/or DNA; such as mRNA, pDNA.

The preparation method of the five-membered lipid nanoparticle in a preferred embodiment of the invention comprises the following steps:

dissolving a cationic lipid, a polymeric (. Beta. -amino ester) as described above, a sterol, a phospholipid and a PEG lipid into an ethanol phase, and dissolving a nucleic acid molecule into an aqueous phase;

mixing the two phases to obtain the five-membered lipid nanoparticle;

The two phases are preferably mixed in a 1:1 ethanol to water volume ratio.

In a specific embodiment of the present invention, the five-membered lipid nanoparticle comprises DOTAP, PBAE, cholesterol, DOPE, and PEG lipid in a molar ratio of (dotap+pbae) DOPE to cholesterol to DMG-peg=50:10:38.5:1.5.

The invention also provides a pharmaceutical composition or vaccine comprising the pentad lipid nanoparticle as described above and a pharmaceutically acceptable carrier.

The invention also provides an application of the poly (beta-amino ester) in preparing five-membered lipid nano particles.

The invention also provides a freeze-dried preparation containing the five-membered lipid nanoparticle.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

the PBAE prepared by the invention has good biocompatibility. The five-membered lipid nanoparticle prepared by the method can realize high-efficiency high-specificity in-vivo transfection, has high stability, can be stored for a long time at 4 ℃ after freeze drying, and can be used as a nucleic acid drug delivery system (such as lung targeting and the like); has good tolerance and has a suitable surface zeta potential of approximately +5 mV.

Drawings

FIGS. 1A-1C show the PBAE structure, the synthesis method and a part of the representative hydrogen spectrum.

FIG. 2 is a screen for transfection efficiency of PBAE-delivered EGFP-mRNA in 293T cells.

FIG. 3 is an evaluation of cell viability after incubation of luciferase mRNA-containing FNP with 293T cells.

FIG. 4 shows the result of screening for luciferase mRNA by FNP in cells.

FIG. 5 shows in vivo results of FNP delivery of different doses of luciferase mRNA, the expression efficiency exhibited a dose-dependent (0.15-0.45 mg/kg data expressed as mean.+ -. S.e.m, n=3).

FIG. 6 shows in vivo transfection screening of FNP. Luciferase mRNA dose was (0.3 mg/kg).

FIG. 7 shows that FNP delivers high dose mRNA (0.6 mg/kg) well tolerated in vivo; the kidney function (a, BUN, b, CREA) and liver function (c, AST, d, ALT) were measured to show no apparent toxicity.

FIG. 8 shows the results of H & E staining of each major organ. FNP delivers high dose mRNA (0.6 mg/kg) well tolerated in vivo; scale bar = 50 μm.

Fig. 9 is a cryo-electron microscope image of FNP.

FIG. 10 shows the mRNA Encapsulation Efficiency (EE) of FNP.

Fig. 11 shows the particle size, PDI and potential of FNP.

FIG. 12 shows in vivo hemolysis evaluation of FNP at pH5.0 (simulating lysosome acidic environment) and pH7.4 (simulating plasma physiological environment).

FIG. 13 is cellular uptake and lysosomal escape; 4, 6-diamino-2-phenylindole (DAPI) stained nuclei (blue), lysoTracker stained lysosomes (green).

FIG. 14 shows the uptake of FNP in 293T cells.

FIG. 15 shows the lysosomal escape efficiency of FNP in 293T cells.

Fig. 16 is a comparison of the effect of cell transfection after conventional LNP and introduction of positive and negative charges and before and after lyophilization of the PBAE formulation alone.

Fig. 17 is a comparison of particle size, PDI and potential for conventional LNP and after introduction of positive and negative charges and before and after lyophilization of PBAE alone.

FIG. 18 shows that FNP after lyophilization (left panel) can maintain stable cell transfection effect at 4℃for a long period of time, while FNP transfection effect without lyophilization decreases rapidly (right panel).

Fig. 19 shows that the lyophilized FNP can maintain stable particle size, PDI and electric potential at 4℃for a long period of time.

FIG. 20 shows that FNP induces specific tdTom fluorescent expression in the lung of Ai9 mice.

FIG. 21 is a fluorescence microscopy image showing tdTomato positive cells of lung after FNP administration, which included cre mRNA; scale bar: 50 μm.

Fig. 22 is a graph of the percentage of tdtom+ cells in a particular cell type in the lung quantified using flow cytometry (FACS).

FIG. 23 is an organ distribution of FNP; a) Image of organ distribution of FNP entrapped with Cy5-mRNA, b) detection of mRNA distribution of each organ by qPCR.

FIG. 24 is vitronectin-enriched protein crown mediated α _v β ₃ Receptor-related lung-specific expression; a) Proteomic analysis of coronal proteins isolated from 11C18E1 (n=23.64 FNP, control, dlin-MC3 LNP) (n=4/group), b) incubation of FNP entrapped EGFP-mRNA with different amounts of vitronectin for pairs α _v β ₃ Receptor positive U87 cell line and mouse primary pulmonary microvascular endothelial cells and alpha _v β ₃ The receptor-negative HepG2 cell line was transfected with a scale of 50 μm, c) quantitative analysis of the proportion of eGFP-positive cells for each group of panel b.

Fig. 25 is a PBAE delivery pDNA screening for transfection efficiency at 293T cells.

FIG. 26 is a diagram of FNP that can effectively encapsulate pDNA; particle size, PDI and Zeta potential (a) were measured using Dynamic Light Scattering (DLS), and agarose gel electrophoresis results showed that FNP was effective in encapsulating pDNA (b).

Fig. 27 is that FNP (11c18e1n=23.64) can effectively deliver pDNA (0.3 mg/kg) to the mouse lung.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The invention researches the cell transfection effect before and after the freeze-drying of the prescription with different surface potentials. Firstly, the transfection effect of the traditional prescription of C12-200 and Dlin-MC3 is obviously reduced after freeze-drying and reconstitution, which indicates that the traditional liver targeting nanoparticle is difficult to achieve; the process of lyophilization is tolerated. According to our previous hypothesis, the addition of cationic lipids to the nanoparticles increased the interparticle rejection by positively charging the surface, decreased particle agglomeration during lyophilization and remained stable, thus 50% DOTAP was added to the formulations of C12-200 and Dlin-MC3 to form positively surface nanoparticles, and the results indicated that both formulations remained essentially unchanged after lyophilization. The further achieved results indicate that 50%18: after lyophilization, the transfection effect of the nanoparticles with negative charges on the two surfaces of PA is obviously reduced.

In addition, the present invention devised a series of PBAE materials with different hydrophobic chain lengths, and based on the results of previous screening, a group (12e2n=24.11) was selected in which the in vitro and in vivo effects were best studied whether the individual PBAE formulations could maintain the transfection effect after lyophilization by promoting encapsulation of nucleic acids. The results show that PBAE can effectively increase the stability of nanoparticles.

Combining the results of the above two aspects, it is predicted that the prescription stability can be effectively increased by adding the polymer PBAE and the cationic lipid to LNP and that the transfection effect can be maintained for a long time after lyophilization. The invention further carries out stability study on five FNP prescriptions with the best in vivo effect, and the cell transfection effect of the FNP prescriptions at different time points after freeze-drying is measured, and the results show that the stability of the first four groups of FNPs is better, while the fifth group has the highest surface potential, the capability of transfecting cells is reduced along with the time, the poor stability is shown, the stability is possibly related to the fact that the hydrophobic chain length of PBAE is shorter than that of the other four groups, and the result also suggests that the stability of nanoparticles is better when the surface potential is higher, and the PBAE plays an extremely important role.

Example 1

The invention designs and synthesizes a series of PBAE materials, the skeleton structure of which is 11, and the materials are combined with figures 1A to 1C: the first 1 refers to the structure of back bone (B), i.e., B1; the second 1 refers to the structure of the side chain (S), S1. To further investigate the effect of the end-capping groups of various PBAEs on their efficiency, we selected two sets of end-capping groups, E1 and E2. Based on the hypothesis that alkyl side chains of PBAE can promote mRNA encapsulation, lysosome escape, we designed the lengths of the different carbon chain alkyl side chains, 8, 10, 12, 14, 16 and 18 respectively, the addition of hydrophobic chains of different lengths helped the PBAE to form a more stable structure for encapsulation of nucleic acids, allowing it to undergo the freeze drying process. Since it has been reported that the degree of polymerization of PBAE has a great influence on the transfection efficiency, 3 main polymerization degree types (hereinafter, n; hereinafter, unless otherwise specified, the ratio of fatty chains is 30%) are set as one of the variables, 4 to 7, 9 to 12, 17 to 26, respectively. A series of PBAEs were synthesized by Michael (Michael) addition reactions as shown in fig. 1A-1C: the diacrylate backbone (B) monomer was mixed with the total side chain (S) monomer in a molar ratio of 1.1:1 (30% of the total side chain monomer being alkyl chain monomer) with an excess of B to ensure that the BS-based polymer was diacrylate terminated. Stirring at 90℃for different times gave PBAE of different molecular weights. The resulting polymer base was dissolved in tetrahydrofuran or Dimethylsulfoxide (DMSO), then 10-fold equivalent of the terminal group (E) molecule was added, and stirred at room temperature for 2 hours. The resulting polymer was then precipitated from the solution by the addition of diethyl ether, washed twice with diethyl ether and dried in vacuo. The resulting polymer was dissolved in CDCl3 or d6-DMSO and its molecular weight was measured by 1H-NMR (Bruker 400 MHz).

EXAMPLE 2 transfection Effect of different PBAE materials on 293T cells with FNP prepared at different prescription proportions

1. Preparation of FNP nanoparticles entrapping EGFP mRNA

FNP is prepared, which comprises PBAE, DOTAP, chol, DOPE, DMG-PEG and a nucleic acid molecule, has an average diameter of 60-160nm and a surface potential of 3-20 mV.

The preparation method comprises the following steps: DOTAP, PBAE, DOPE, chol and ethanol phase were dissolved in absolute ethanol at a specific molar ratio, (DOTAP+PBAE)/DOPE/Chol/DMG-PEG molar ratio fixed at 50/10/38.5/1.5. The molar ratio of DOTAP to PBAE varies between 2.5/1 and 40/1 depending on the type of PBAE. The aqueous phase was 100mM citrate buffer (pH=3) containing nucleic acid. The mass ratio of DOTAP+PBAE to mRNA was 20. The two phases were mixed rapidly at a volume ratio of ethanol to water of 1/1. After 24 hours the expression level was observed under a fluorescence microscope.

2. MTT method for researching influence of FNP on 293T cell in-vitro survival rate

To evaluate the transfection efficiency of PBAE in vitro, 293T cells were selected as a model in the present invention. The PBAE is taken as a protonatable cationic material, and the optimal proportion is different from DOTAP due to the different structures. Thus, to determine the optimal ratio, EGFP-mRNA was coated with different DOTAP-PBAE ratios of FNP (DOTAP: PBAE=2.5:1, 5:1, 10:1), and 293T cells in complete medium were transfected. Thereafter, the most appropriate DOTAP/PBAE ratio for each PBAE was determined and will continue for subsequent in vivo and in vitro experiments (FIG. 2).

For in vitro cell transfection, fresh FNP was directly diluted in DMEM complete medium; at the time of in vivo delivery, the prepared FNP was dialyzed against 3500MWCO dialysis membrane in PBS solution at 4℃for 2 hours.

MTT method was used to study the effect of FNP on the in vitro survival rate of 293T cells, and after 24h of transfection with FNP nanoparticles (relative amount of FNP nanoparticles and cells was 150ng mRNA/well; seeding density of cells in 96 well plates was 10) ⁴ MTT (5 mg/mL in PBS,20ul, beyotime) was added and incubated with cells at 37℃for 4h. After the incubation period was completed, the cell culture medium was removed, 150 μl DMSO (solabio) was added, and absorbance was measured at 490nm using a microplate reader (spectromax i3, MD). Cell viability (%) was calculated using the treated cells compared to untreated control cells.

As shown in fig. 3. Most of these PBAEs were not significantly cytotoxic. 77.77% of PBAE cells had an activity of greater than 80%.

Example 3 evaluation of dose-dependence of the transfection effects of FNP prescriptions of several different PBAE Material compositions on luciferase mRNA transfected in mice

To further explore the structure-activity relationship of PBAE, 293T cells were transfected with luciferase (Fluc) mRNA and luciferase activity was quantified, as shown in FIG. 4, with substantially the same transfection tendencies of EGFP-mRNA and Fluc-mRNA. According to the results, E1 end-capping had better transfection effect than E2 end-capping. The higher the degree of polymerization, the better the transfection effect, the closer the carbon chain length is to 16, and the better the transfection effect.

Based on the mRNA transfection results of the cells, the first three materials 11 c14e1n=17.41, 1c14e2n=17.41, 1c18e1n=23.64 were selected for the best transfection effect to investigate if the dose dependency of the FNP consisting of PBAE was present in the body (as in example 1, the first 1 refers to the structure of back bone (B), i.e. B1; the second 1 refers to the structure of side chain (S), i.e. S1; n is the degree of polymerization of PBAE calculated by hydrogen profile. C14 or C18 represents the length of the carbon chain alkyl side chain).

The nanoparticle preparation method was the same as in the above example, and after the nanoparticle preparation was completed, the prepared FNP was dialyzed with 3500mwco dialysis membrane in PBS solution, and dialyzed at 4℃for 2 hours. Then, the mixture is injected into a mouse body through tail vein, 30mg/mL of potassium luciferase is injected into the abdominal cavity according to the dosage of 150mg/kg after six hours of administration, and the expression of the luciferase is detected about ten minutes after injection.

As shown in FIG. 5, these three materials all had lung specificity and dose sensitivity in the range of 0.15mg/kg to 0.45mg/kg of Fluc mRNA. Subsequent evaluation of all PBAEs in vivo will be at a dose of 0.3 mg/kg.

PBAEs with luciferase activity (RLU/Well) greater than 100,000 and various aggregation levels of 11C10E1 were selected for screening in vivo as shown in fig. 6 a. As can be seen from b in fig. 6, the 4 most efficient PBAE bioluminescence signals (12c16e2n=24.11, 12c18e1n=23.64, 1c16e1n=11.85, 1c14e2n=17.41) are all distributed mainly in the lung, wherein the fluorescence efficiency of the best material 11 c16e2n=24.11 can reach as high as 10 ⁸ . In addition, the in vivo structure-activity relationship of PBAE is substantially consistent with in vitro. From the viewpoint of the degree of polymerization, 11c10e1n=5.13, 11c10e1n=9.66, 1c10e1n=20.58, and the transfection efficiency increases with the increase in the degree of polymerization. Of the top 5 PBAEs with highest transfection efficiency in vivo, 3/5 belonged to group E1 for the different end capping groups. In terms of carbon chain length, comparing 11C10E1, 11C12E1/11C14E1, 11C16E1 and 11C18E1 in groups n=9-12 or 17-26, it follows that the conclusion that the better the transfection effect is still applicable the closer the carbon chain length is to 16.

Example 4 evaluation of toxicity of FNP formulation in mice

Excellent formulations not only require high transfection efficiency but also good biocompatibility. For this, the in vivo toxicity of the first 5 PBAEs was subsequently assessed using a higher mRNA dose (0.6 mg/kg).

Male C57BL/6 mice weighing 15-20g were randomly grouped, 4 per group. The prepared FNP preparation was administered via tail vein at a luciferase mRNA dose of 0.6 mg/kg. The positive control group was intraperitoneally injected with lipopolysaccharide (5 mg/kg), and the negative control group was intravenously injected with PBS. After 48 hours, whole blood was collected and serum was centrifuged. The mice were sectioned for organs (heart, liver, spleen, lung, kidney) and H & E stained. The liver (AST, ALT) and kidney (BUN, CREA) functions were then tested using the kit.

FNP with the first 5 PBAE had no change in renal function and liver function, as shown in FIG. 7, and FIG. 8 showed no sign of histologically bad lesions. The above results indicate that FNP has good tolerability.

Example 5

FNP nanoparticles were prepared for cryo-electron imaging, and after negative staining with 2% uranyl acetate, FNP was imaged with a Tecnai Spirit transmission electron microscope (FEI, hillsboro, OR). The results of the electron microscopy (FIG. 9) showed that FNP consisting of 11C18E1n=23.64 had a rose-like structure and had a particle diameter of about 70nm.

The encapsulation efficiency of the mRNA by the different FNPs was evaluated using a modified Quant-iT riboGreen RNA analysis (Invitrogen) kit.

The preparation of FNP formulations of the different PBAE materials was performed by the method of the above example, and after completion of the preparation, the FNP was characterized by diluting it 40 times in DPBS solution, and the size, polydispersity (PDI) and Zeta potential were measured using a NanoZS Zetasizer (Malvern).

As shown in fig. 10 and 11, the most effective groups of PBAEs have the smallest particle size, the smallest PDI, and the packing efficiency is nearly 80%, and all four best-performing FNPs have a surface zeta potential of nearly +5mV, neither too high nor too low.

Example 6 evaluation of hemolysis of different FNPs

Male C57BL/6 mice weighing 15-20g were selected, whole blood was collected, and blood cells were isolated by centrifugation (10000 g,5 min). The blood cells were then resuspended separately using PBS solutions pH7.4 and Ph5.0, to which different FNP materials were added, incubated at 37℃for 30min, and 10000g centrifuged for 5min, the supernatant was taken and its absorbance at 540nm was determined, with the group without FNP nanoparticles as negative control and Triton X-100 solution (1 wt.%) as positive control.

The membrane-disrupting activity of FNP was evaluated by a hemolysis test, and as shown in FIG. 12, PBAE with a good transfection effect showed a high hemolytic ability at pH5.0, indicating a high escape ability in an acidic lysosome environment.

Example 7 assessment of FNP uptake and escape in 293T cells

293T cells were seeded on 8-well glass plates (CellVis, mountain View, calif., cat.C8-1.5H-N). When the cell density reached 60-70%, 375ng of cy 5-labeled mRNA-coated FNP was added per well and imaged on a Nikon confocal microscope (Ti-E+A1RSI) with a 60-fold oil immersion objective after 4 hours. Nuclei and lysosomes were labeled with DAPI and LysoTracker Red DND-99, respectively.

Lysosomes and mRNAs are respectively marked by utilizing LysoTracker and cy5, the ingestion condition of the mRNAs is determined by semi-quantitative analysis of cy5 signals in single cells through imageJ, and the endosome escape capacity is semi-quantitatively analyzed through the dispersion degree of cy5 and LysoTracker.

Cell uptake assays were performed using ImageJ (FIJI). The area where the single cell is located is selected from the image, and the fluorescence intensity of cy5 in the area is calculated to represent the uptake of FNP by the single cell. Co-localization analysis was performed using the Coloc 2 insert of ImageJ (FIJI) and lysosomal escape was indicated by calculation of the Person correlation coefficient for the far red (cy 5-mRNA) channel and red (endosomal marker). Wherein, the closer the pierce correlation coefficient is to 0, the lower the two-channel correlation is, and the higher the lysosome escape efficiency is.

As shown in FIGS. 13-15, FNP with good transfection effect on cells and in vivo has high uptake effect and good lysosomal escape ability.

Example 8 evaluation of stability of FNP nanoparticles from the change of cell transfection ability with time

Preparation of FNP As in the above examples, sucrose was added to a final concentration of 10% (w/v) after completion of the preparation, and a freeze-dryer (Labconco, USA) was used after pre-freezing at-80 ℃) Freeze-drying for 12 hours until the water is completely evaporated. The lyophilized FNP was stored at 4℃and was then subjected to ddH at the original volume at the selected time point ₂ O is redissolved. FNP containing 150ng of nucleic acid was mixed with 100uL of DMEM (High glucose) Complete Medium (10% FBS), added to 293T cells of 80% -90% density and replaced with the original Medium. Luciferase expression was measured after 24 hours.

FIGS. 16-17 show that the transfection effect was significantly reduced for the conventional formulations of C12-200 and Dlin-MC3 after lyophilization and was maintained substantially unchanged after lyophilization by adding 50% DOTAP to the formulations of C12-200 and Dlin-MC3 to render them surface positively charged nanoparticles, while incorporation of 50%18: after the nanoparticles with negative charges on the surfaces of the PA are freeze-dried, the transfection effect is obviously reduced; whether a separate PBAE formulation can maintain transfection effects after lyophilization by facilitating encapsulation of nucleic acids. This indicates that the addition of DOTAP as well as PBAE can effectively increase the lyophilization stability of nanoparticles.

Figure 18 shows that the FNP nanoparticles were freeze-dried and stored at 4℃for a long period of time and their cell transfection effect was measured at selected time points, among the 5 best PBAEs, the first 4 PBAEs were all better in stability, while the 5 th PBAE was slightly less stable, and their ability to transfect cells was decreased with time. The effect of FNP transfection was rapidly decreased when stored directly at 4℃without lyophilization, as compared to the lyophilized treatment group.

In addition, FNP particle size and polydispersity index (PDI) changes were measured at selected time points after lyophilization using Zetasizer Nano-ZS (Malvern, UK) Dynamic Light Scattering (DLS). The measurements for each sample were repeated 3 times.

The results show that: the particle size, PDI and zeta potential of the FNP nanoparticles also remained substantially stable over time, with the FNP having good stability compared to the control group, and being stable for a long period of time after lyophilization, since the addition of cationic lipids to the FNP increased its surface charge, increasing its interparticle repulsive force, while the addition of polymer PBAE made its encapsulation of the nucleic acid tighter, better tolerating the lyophilization process (fig. 19).

Example 9 evaluation of edit efficiency of cre mRNA delivery in Ai9 mice

To detect and quantify the cell type and proportion of FNP entering the lung, we used a transgenic mouse (Ai 9) with tdTom reporter gene with loxP flanking, ai9 mouse is a transgenic mouse with STOP sequence designed with loxP flanking inserted at Gt (ROSA) 26Sor locus to prevent transcription of red fluorescent protein (tdTomato) driven by CAG promoter. Ai9 mice express strong tdmamio fluorescence after Cre-mediated recombination. The cre mRNA (0.3 mg/kg) was entrapped by tail vein injection and tdTomato red fluorescent protein expression was detected by in vivo imaging after 48 hours.

Example 10 evaluation of distribution of FNP in various types of cells in the lung

FNP was injected into Ai9 mice by tail vein and the mice were sacrificed after 48 hours, 1mL of dispese (15U/mL) was injected into the lungs dissected through the trachea, the lungs were automatically collapsed, the lungs were removed and placed in PBS solution, the lobes were separated and the tracheal tissue removed, the lung tissue was digested in dispese (15U/mL) for 45min, after which the lung tissue was triturated using a pipette until no significant tissue mass was seen, 10ml DMEM+2%FBS+1%P/S+0.33U/mL DNaseI was added, after shaking with a pipette gun for 10min after shaking with force at 37 ℃, cells were collected by 100 μm,70 μm and 40 μm in succession, centrifugation at 1200rpm for 5min, 1mL of red blood cell lysate was added to resuspend and lyse red blood cells, after which cells were collected again by centrifugation, DMEM+2% FBS+P/S was added to resuspend cells at 37℃for at least 20 min, cell plates were used to count cells to 10 density ⁷ /mL. Cell staining was then performed using antibodies Pacific Blue (BioLegend, 157212) against mouse CD45, alexa Fluor 488 against mouse CD31 (BioLegend, 102414) and Alexa Fluor 647 against mouse CD326 (Ep-CAM) (BioLegend, 118212). Ghost Dye Red 780 (cell signaling, 18452S) was used to identify living cells. Lung cells were analyzed with LSRForessa SORP.

The type and proportion of cells edited by Cre recombinase can be detected by delivering FNP that encapsulates Cre mRNA. Cre mRNA was delivered with the 5 most potent FNPs, and fluorescent tissues were evident in lung tissue 2 days after dosing, see figure 20. We note that the fluorescent signal of FNP consisting of 11c16e2n=24.11 was 6-9 times higher in its lungs than in PBS group. In addition, tdTom positive cells were readily seen under a tissue section fluorescence microscope, as shown in FIG. 21. To estimate 11C16E2 transmissible cell types (n=24.11), we quantified the specific cell types delivered into the lung using flow cytometry after delivery of Cre mRNA. 11c16 e2n=24.11 FNP transfected about 22% of all endothelial cells, about 6% of epithelial cells and about 8% of immune cells, as shown in fig. 22.

If one were to optimize this formulation, to achieve more surprising delivery efficiency, and lay the foundation for expanding the FNP platform to more accurate cell targeting, one would have to study the mechanism factors that determine FNP lung targeting characteristics.

Example 11 evaluation of FNP distribution in mice

For lung targeting, the nanoparticles first need to be specifically distributed to the lungs. Therefore, we want to verify if FNP is fully distributed in the lung. FNP consisting of 11C18E1 n-23.64 and cy5-EGFP mRNA was injected into mice via tail vein, and after 6 hours, cy5 fluorescence distribution and EGFP mRNA content in each organ were detected using IVIS Lumina system and RT-qPCR, respectively.

C57BL/6 mice weighing 15-20 g were respectively injected intravenously with FNP and PBS solution encapsulating EGFP mRNA (0.3 mg/kg), 3 in each group. After 6 hours, the mice were sacrificed and organs were removed and total RNA was extracted using Vazyme Cat.RC112-01. cDNA was synthesized by Vazyme Cat.R223-01. qPCR detection was then performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Cat. Q711-02). The RT-qPCR primer is GAPDH:5'-CGACTTCAACAGCAACTCCCACTCTTCC-3' (forward), primer 5'-TGGGTGGTCCAGGGTTTCTTACTCCTT-3' (reverse); EGFP:5'-CGACCACTACCAGCAGAACA-3' (Forward), EGFP primer was 5'-TCTCGTTGGGGTCTTTGCTC-3' (reverse). PBS group is negative control group and GAPDH is endogenous control.

FIG. 23a shows that cy5 fluorescence signals are distributed predominantly in the lung, with only a small amount of fluorescence signals scattered in the liver, kidney, spleen (the intrapulmonary fluorescence signals are about 2 times that of the liver, kidney, spleen). As shown in FIG. 23b, the RT-qPCR results showed that cy5-EGFP mRNA was distributed mainly in the lung, much higher than in other organs (fluorescence signal of lung was at least 10 times higher than that of liver, kidney, spleen). The two experimental results show that the distribution ratio of mRNA in lung and other organs is different, and the fluorescence imaging result shows that a small part of cy5 signal on EGFP mRNA is distributed in liver, kidney and spleen, however, the content of mRNA of each organ is detected to be very low in non-lung organ by RT-qPCR, which is probably due to degradation metabolism.

This explains to some extent why the nanoparticles are specifically expressed only in the lung, but to truly understand their mechanism, it is also necessary to understand why these nanoparticles will be specifically expressed in the lung and why they promote their absorption by the lung.

Example 12 analysis of the composition of the protein corona bound to FNP and verification of the Effect of the target protein on the promotion of Primary cell transfection in the lung

Conventional four-component LNPs can deliver functional RNA to hepatocytes by adsorbing ApoE in serum and binding to low density lipoprotein receptor (LDL-R) on hepatocytes. We therefore hypothesize that FNP is also a specific lung endothelial cell target achieved by binding to proteins in serum.

The FNP was incubated with mouse serum at 37℃to simulate the formation of protein crowns in vivo, the FNP after binding to the protein crowns was separated by centrifugation, and 6 volumes of cold acetone was added to the protein crowns separated from the FNP to remove lipid content and precipitate out the protein. 16000g are centrifuged at 4℃for 15min and the resulting protein mixture is resuspended in 1M urea/50 mM NH ₄ HCO ₃ In this, 1M DTT was added to give a final DTT concentration of 5mM and incubated at 37℃for 1h. 500mM IAM was then added to give a final IAM concentration of 10mM and incubated for 45min at room temperature. The protein was digested by adding trypsin. The resulting polypeptide mixture was resuspended in 0.1% formic acid and analyzed with Q exact HF (thermo Fisher).

The composition of the protein corona was formed by proteomic analysis, as shown in fig. 24 a. Vitronectin is the most abundant plasma protein in FNP compared to the nanoparticle formulation composed of control Dlin-MC 3. In contrast to ApoE, different functions of vitronectin may be formingFNP has a role in pulmonary target recognition. Vitronectin promotes cell attachment, diffusion, and migration of a variety of cell types. At least four known integrin receptors are capable of recognizing vitronectin, including alpha _v β ₃ ，α _v β ₅ ，α _v β ₁ And alpha _Ⅱb β ₃ . Integrin alpha _v β ₃ Is a major vitronectin receptor and can be combined with vitronectin in blood plasma. In fact, integrin alpha _v β ₃ Are present in many cells, such as endothelial cells, chondrocytes, fibroblasts, and blasts. Alpha _v β ₁ Distributed among limited cell types such as fibroblasts and certain tumor cell lines. Alpha _v β ₅ There is also a distribution in fibroblasts and tumors. And alpha is _Ⅱb β ₃ Mainly distributed in platelets and megakaryocytes, and require activation to bind to vitronectin. The nanoparticles are injected by tail vein, and then return to heart through systemic circulation, then enter pulmonary circulation rapidly, exchange alveolar capillary network around air alveoli, then are injected into left atrium and left ventricle to pass through pulmonary veins of each level, finally flow through aorta and several branches of artery generated by the aorta, and blood is conveyed to corresponding organs for substance exchange.

Thus, combining the two main aspects, we assume that FNP adsorbs vitronectin once it enters the blood, rapidly passes through the heart to perform pulmonary circulation, and is highly expressed with pulmonary endothelial cells _v β ₃ Receptor interactions. Thus, most of the nanoparticles will be intercepted in the lung, thereby greatly reducing the distribution of the nanoparticles in the liver and kidney parts.

To verify the hypothesis, FNP harboring eGFP mRNA and different doses of vitronectin were incubated for respective transfection of α _v β ₃ Receptor positive U87 cell line, mouse lung primary microvascular endothelial cells, and alpha _v β ₃ The receptor negative HepG2 cell line is shown in figures 24b,24 c. At alpha _v β ₃ In the receptor positive U87 cell line and the mouse lung primary microvascular endothelial cells, the transfection efficiency is increased along with the increase of vitronectinHas obvious improvement. And at alpha _v β ₃ In the receptor-negative HepG2 cell line, there was no significant increase in transfection efficiency with increasing vitronectin. This description alpha _v β ₃ Receptors are critical contributors to cell transfection of fibronectin-enriched FNP. In addition, we incubated FNP with varying doses of bovine serum albumin for transfection of alpha _v β ₃ The receptor positive U87 cell line did not significantly increase transfection efficiency with increasing bovine serum albumin. This demonstrates that vitronectin is mediated at FNP _v β ₃ The same plays a critical role in the transfection of receptor positive cells.

In conclusion, after FNP enters the body, vitronectin in blood is rapidly enriched and passes through and lung alpha _v β ₃ Receptor interactions mediate lung-specific transfection.

Example 13 evaluation of Properties after FNP coating with pDNA

The effect of FNP inclusion on pDNA was tested, and the preparation method of FNP formulation was the same as when mRNA was encapsulated.

The variation in FNP particle size and polydispersity index (PDI) after lyophilization, which was maintained for a long period of time, was measured using Zetasizer Nano-ZS (Malvern, UK) Dynamic Light Scattering (DLS). Measurements for each sample were repeated 3 times; the encapsulation of FNP-encapsulated pDNA was analyzed by agarose gel electrophoresis.

Particle size, PDI and Zeta potential of FNP-pDNA were measured using Dynamic Light Scattering (DLS), and fig. 26a shows that FNP has no significant difference in nanoparticle size and potential for encapsulated pDNA and mRNA. FIG. 26b shows that FNP can effectively encapsulate pDNA.

The transfection effect of the FNP formulation on the transfection of luciferase pDNA in mice was evaluated. The nanoparticle preparation method is the same as in the third example. Then, the mixture is injected into a mouse body through tail vein, 30mg/mL of potassium luciferase is injected into the abdominal cavity according to the dosage of 150mg/kg after six hours of administration, and the expression of the luciferase is detected about ten minutes after injection.

Finally, the transfection effect of the FNP prescription on the luciferase pDNA transfected in mice was verified. FIG. 27 shows that lung specific luciferase expression can be efficiently achieved at a plasmid dose of 0.3 mg/kg.

Example 14 evaluation of transfection effects of different PBAE materials on FNP prepared at different prescription proportions on 293T cells

FNP nanoparticles entrapping EGFP pDNA were prepared by rapidly mixing FNP with an aqueous phase containing nucleic acid (100 mM sodium citrate buffer solution) at a volume ratio of 1:1 in ethanol at a molar ratio of (DOTAP+PBAE)/DOPE/Chol/DMG-PEG of 50/10/38.5/1.5. The prepared nanoparticles are directly added into a complete DMEM culture medium to transfect cells, and the expression level is observed under a fluorescence microscope after 24 hours. The molar ratio of DOTAP to PBAE varies from 5/1 to 20/1 depending on the type of PBAE, and the optimal ratio of each PBAE material is selected depending on the transfection efficiency.

For in vitro cell transfection, fresh FNP was directly diluted in DMEM complete medium; at the time of in vivo delivery, the prepared FNP was dialyzed against 3500mwco dialysis membrane in PBS solution at 4℃for 2 hours. Fig. 25 shows the transfection effect of different PBAE materials on 293T cells at different ratios.

Claims

1. A polymeric (β -amino ester), characterized in that it comprises formula (I);

wherein:

R ₁ independently a fatty chain with or without hydroxyl groups;

R ₂ independently is a terminal monomer comprising a primary, secondary or tertiary amine;

r comprises straight-chain or branched C ₁ -C ₅₀ An alkylene chain, which may further comprise one or more heteroatoms or one or more carbocyclic, heterocyclic or aromatic groups;

m is 1-15, n is 3-45, x is 1-13; x is an integer;

the raw material of (2) is->

The raw material of (2) is->

The raw materials of (1) are

The preparation method of the polymerized (beta-amino ester) comprises the following steps: will be

Raw materials of->

Raw materials of (a),

Mixing together the raw materials and alkylamine to perform Michael addition reaction.

2. Five-membered lipid nanoparticle, characterized in that its raw material comprises cationic lipids, polymeric (β -amino esters) according to claim 1, sterols, phospholipids, PEG lipids and nucleic acid molecules.

3. The five-membered lipid nanoparticle according to claim 2, wherein the molar ratio of the cationic lipid to the polymeric (β -amino ester) is (2.5-40): 1;

and/or the relative amount of the cationic lipid and the polymeric (. Beta. -amino ester) mass to the nucleic acid molecule is (10-80): 1;

And/or the mole ratio of the phospholipid, the sterol and the PEG lipid is phospholipid sterol= (0.2-1) 1; phospholipid: PEG lipid= (5-20): 1;

and/or, the ratio of n to m is 7:3;

and/or x is 3 to 11.

4. The five-membered lipid nanoparticle of claim 2, wherein the cationic lipid is selected from one or more of DOTAP, DOTMA, DODAB, DDAB and dmriie;

and/or the sterol is selected from one or more of cholesterol, campesterol, trehalose sterol, and carrot sterol;

and/or the phospholipid is selected from one or more of DOPE, DOPC, DOPS, POPS, DOPG, DOPI, DSPC, HSPC, eggPC, DPPC, POPC, POPE and DLPC;

and/or the PEG lipid is selected from one or more of DMG-PEG2000, DMG-PEG5000, DSPE-PEG2000 and DSPE-PEG 5000;

and/or, the nucleic acid molecule is RNA and/or DNA.

5. The five-membered lipid nanoparticle of claim 4, wherein the cationic lipid is DOTAP;

and/or, the sterol is cholesterol;

and/or, the phospholipid is DOPE.

6. The five-membered lipid nanoparticle according to claim 2, wherein the preparation method thereof comprises:

Dissolving a cationic lipid, a polymeric (β -amino ester) according to claim 1, a sterol, a phospholipid and a PEG lipid into an ethanol phase, and dissolving a nucleic acid molecule into an aqueous phase;

and mixing the two phases to obtain the five-membered lipid nanoparticle.

7. The five-membered lipid nanoparticle of claim 6, wherein the two phases are mixed in a 1:1 ratio of ethanol to water.

8. The five-membered lipid nanoparticle according to any one of claims 2 to 7, wherein the starting material comprises DOTAP, polymeric (β -amino ester), cholesterol, DOPE and PEG lipid in a molar ratio (dotap+pbae): DOPE: cholesterol: DMG-peg=50:10:38.5:1.5.

9. A pharmaceutical composition or vaccine comprising the pentad lipid nanoparticle of any one of claims 2-8 and a pharmaceutically acceptable carrier.

10. Use of a polymeric (β -amino ester) according to claim 1 for the preparation of five-membered lipid nanoparticles.

11. A lyophilized formulation comprising the pentad lipid nanoparticle of any one of claims 2-8.