Benzamide bond-based pH-responsive PEG lipid compound and application thereof

Abstract

The invention relates to a pH responsive PEG lipid compound based on a benzimide bond and application of the pH responsive PEG lipid compound. The invention provides a pH responsive PEG (Polyethylene Glycol) lipid compound Ben-PEG2000 containing a benzimide bond. The chemical formula of the pH responsive PEG lipid compound Ben-PEG2000 is shown as a formula (I). The lipid nanoparticle containing the pH responsive PEG lipid compound Ben-PEG2000 provided by the invention has a pH responsive characteristic, can promote endosome escape and transfection efficiency of mRNA loaded in the lipid nanoparticle in a pH responsive manner in an endosome weak acid environment, has extremely high safety, and has a wider clinical application value. # imgabs0 # (I).

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a benzimide bond-based pH-responsive PEG lipid compound and application thereof. Background Art

[0002] The biggest problem encountered by RNA-based gene therapy is drug delivery. First, naked RNA is a negatively charged hydrophilic macromolecule that will electrostatically repel the cell membrane, so it is not easy to pass through the cell membrane; secondly, the nucleases and hydrolases present in the body can easily degrade exogenous RNA molecules, resulting in RNA inactivation and difficulty in accumulating in target tissues; in addition, the immune system can also recognize exogenous nucleic acids to trigger immunogenic responses. In order to achieve safe and effective nucleic acid delivery, several RNA carriers have been developed to protect nucleic acids from degradation and maximize delivery to target cells. Among them, the application of lipid nanoparticles (LNPs) in nucleic acid drug and vaccine delivery has once again attracted widespread attention. LNPs are composed of 5 parts: ionizable lipids, nucleic acids, cholesterol, phospholipids, and PEG lipids. Among them, PEG lipids, as an amphiphilic material, stabilize the LNPs structure by providing a spatial barrier, which can drive self-assembly and prevent particle aggregation, thereby extending the circulation time of LNPs.

[0003] However, as a clinically approved multifunctional nucleic acid delivery platform, LNPs currently face a major bottleneck in the effective expression of mRNA in vivo. As for "how to improve the effective expression of mRNA in vivo", the main focus is on the development of new ionizable lipids, targeted modification, SORT and other strategies.

[0004] One of the important factors affecting the effective transfection or expression of mRNA is the endosomal escape of mRNA. Simply put, mRNA encapsulated in LNPs mainly goes through three stages in the cell: early endosome, late endosome and lysosome. Only when mRNA successfully escapes from the early or late endosome can it realize the coding function and stimulate the downstream. Studies have shown that about 70% of mRNA cannot escape from the endosomal part, some will be degraded by lysosomes, and some will be excreted. Improving the endosomal escape ability of mRNA can promote the effective expression of antigens. Regarding the endosomal escape theory, there are proton sponge effect and membrane fusion phase transition hypothesis. Among them, the membrane fusion phase transition hypothesis mentions that ionizable lipids are protonated under the acidic pH conditions of the endosomal membrane, and then interact with the anionic lipids of the endosomal membrane to cause membrane fusion. This fusion can induce the endosomal membrane to transform from a stable double-layer structure to a non-double-layer (hexagonal HII) structure, resulting in the rupture of the endosomal membrane, thereby releasing payloads such as nucleic acids into the cytoplasm.

[0005] At present, the research on pH-based endosomal escape and mRNA expression mainly focuses on ionizable lipids, and there is almost no research on other components in LNP. Therefore, it is of great significance to study and develop pH-responsive LNP components to improve the delivery and transfection efficiency of mRNA-LNP target cells. Summary of the invention

[0006] To address the deficiencies of the prior art, the present invention aims to provide a pH-responsive PEG lipid compound based on a benzimide bond, lipid nanoparticles comprising the same, and applications thereof.

[0007] In a first aspect of the present invention, a pH-responsive PEG lipid compound based on a benzimide bond is provided, the chemical structure of which is shown in the following formula (I): (I) Wherein, the PEG is PEG-2000.

[0008] In another preferred embodiment, the molecular weight of the PEG lipid compound is about 3035.69.

[0009] In another preferred embodiment, the PEG lipid compound is prepared by the following method: 1,2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol-400 (DMG-PEG400) and benzaldehyde-amide polyethylene glycol-2000 are reacted through a Schiff base reaction.

[0010] In another preferred embodiment, the PEG lipid compound is prepared by the following method: (1) Weigh 0.2 g of t-Boc-N-amino-PEG400-acid and dissolve it in 5 ml of dichloromethane. Add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalents of 4-dimethylaminopyridine (DMAP) under ice bath, activate under ice bath for 1 h, then add 1.1 equivalents of 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DMG) and stir at room temperature for 48 h. (2) The above system was washed with water, extracted with dichloromethane, and purified by column chromatography using a dichloromethane / methanol system to obtain intermediate-1; (3) The intermediate 1 was dissolved in dichloromethane, 15 equivalents of trifluoroacetic acid was added under ice bath conditions, and stirred at room temperature for 15 h; (4) Add saturated sodium bicarbonate solution to the system of step (3) under ice bath conditions for neutralization, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2; (5) The intermediate 2 was dissolved in an appropriate amount of N,N-dimethylformamide, and 1.5 equivalents of benzaldehyde-amide polyethylene glycol 2000 were added. The mixture was stirred at room temperature in the dark for 24 h under nitrogen protection. The system was then dialyzed using a 2500Da dialysis bag for 24 h and freeze-dried to obtain the target product, i.e., the PEG lipid compound shown in formula (I).

[0011] In another preferred embodiment, the PEG lipid compound is formed by benzimide bond grafting, has pH responsiveness, and can be responsively reduced to amine groups and aldehyde groups in a weakly acidic environment.

[0012] In the second aspect of the present invention, a lipid nanoparticle is provided, wherein the lipid nanoparticle comprises the pH-responsive PEG lipid compound based on benzimide bond according to the first aspect of the present invention.

[0013] In another preferred embodiment, the lipid nanoparticles further comprise ionizable lipids, phospholipids, steroids and PEG lipids.

[0014] In another preferred embodiment, the molar ratio of the pH-responsive PEG lipid compound based on benzimide bonds, ionizable lipids, phospholipids, steroids and PEG lipids in the lipid nanoparticles is (1.125-5.625):30:40:30:0.375, preferably 1.125:30:40:30:0.375.

[0015] In another preferred embodiment, the ionizable lipid is N1, N3, N5 tris(2-aminophenyl)benzene-1, 3, 5-tricarboxamide analog (BXA).

[0016] In another preferred embodiment, the phospholipid is 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE).

[0017] In another preferred embodiment, the steroid is cholesterol.

[0018] In another preferred embodiment, the PEG lipid is 1,2-dimyristyl alcohol-propylglycerol-3-methoxypolyethylene glycol-2000-mannose (DMG-PEG2000-Man).

[0019] In another preferred embodiment, the lipid nanoparticles comprise the pH-responsive PEG lipid compound based on benzimide bond as described in the first aspect of the present invention, BXA, DOPE, cholesterol and DMG-PEG2000-Man.

[0020] In another preferred embodiment, the molar ratio of the pH-responsive PEG lipid compound based on benzimide bond, BXA, DOPE, cholesterol and DMG-PEG2000-Man in the lipid nanoparticles is (1.125-5.625):30:40:30:0.375; preferably 1.125:30:40:30:0.375.

[0021] In another preferred embodiment, the lipid nanoparticles are loaded with drugs, and the drugs include one or more of nucleic acid molecules, small molecule compounds, and proteins.

[0022] In another preferred embodiment, the lipid nanoparticles are loaded with nucleic acid drugs.

[0023] In another preferred embodiment, the nucleic acid drugs include RNA drugs and DNA drugs.

[0024] In another preferred embodiment, the RNA drug includes (but is not limited to) mRNA, tRNA, rRNA, miRNA, siRNA, circRNA, and sgRNA.

[0025] In another preferred embodiment, the nucleic acid drug is mRNA.

[0026] In another preferred embodiment, the nucleic acid drug is a therapeutic drug or a preventive drug, such as a vaccine.

[0027] In another preferred embodiment, the nucleic acid drug is an mRNA vaccine.

[0028] In another preferred embodiment, the nitrogen to phosphorus ratio (N / P) of the lipid nanoparticles is 3:1.

[0029] In a third aspect of the present invention, there is provided a method for preparing a pH-responsive PEG lipid compound based on a benzimide bond as described in the first aspect of the present invention, comprising the following steps: (1) Weigh 0.2 g of t-Boc-N-amino-PEG400-acid and dissolve it in 5 ml of dichloromethane. Add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalents of 4-dimethylaminopyridine (DMAP) under ice bath, activate under ice bath for 1 h, then add 1.1 equivalents of 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DMG) and stir at room temperature for 48 h. (2) The above system was washed with water, extracted with dichloromethane, and purified by column chromatography using a dichloromethane / methanol system to obtain intermediate-1; (3) The intermediate 1 was dissolved in dichloromethane, 15 equivalents of trifluoroacetic acid was added under ice bath conditions, and stirred at room temperature for 15 h; (4) Add saturated sodium bicarbonate solution to the system of step (3) under ice bath conditions for neutralization, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2; (5) The intermediate 2 was dissolved in an appropriate amount of N,N-dimethylformamide, and 1.5 equivalents of benzaldehyde-amide polyethylene glycol-2000 were added. The mixture was stirred at room temperature in the dark for 24 h under nitrogen protection. The system was then dialyzed using a 2500Da dialysis bag for 24 h and freeze-dried to obtain the target product, i.e., the PEG lipid compound shown in formula (I).

[0030] In the fourth aspect of the present invention, there is provided use of the benzimide bond-based pH-responsive PEG lipid compound as described in the first aspect of the present invention in the preparation of lipid nanoparticles.

[0031] In a fifth aspect of the present invention, a method for preparing lipid nanoparticles is provided, comprising the following steps: (S1) mixing ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzimide bond as described in the first aspect of the present invention in a uniform proportion, and dissolving in an organic solvent to prepare an organic phase; (S2) providing an aqueous phase containing a drug to be loaded; (S3) rapidly mixing the organic phase and the aqueous phase to prepare lipid nanoparticles; (S4) The obtained lipid nanoparticles were immediately dialyzed at 2-8°C for 2-4 hours.

[0032] In another preferred embodiment, the molar ratio of the ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on the benzimide bond as described in the first aspect of the present invention in the step (S1) is 30:40:30:0.375:(1.125-5.625), preferably, 30:40:30:0.375:1.125.

[0033] In another preferred embodiment, in the step (S1), the organic solvent is anhydrous ethanol.

[0034] In another preferred embodiment, in the step (S2), the drug to be loaded (such as mRNA) is diluted to a target volume with DEPC water and then mixed with trisodium citrate solution to prepare an aqueous phase.

[0035] In another preferred embodiment, the concentration of sodium citrate in the final aqueous phase system is 50 mM-100 mM, preferably 50 mM.

[0036] In another preferred embodiment, in the step (S3), the volume ratio of the organic phase to the aqueous phase is 1:3.

[0037] In another preferred embodiment, in the step (S4), the obtained lipid nanoparticles are dialyzed in 1×PBS or HEPES solution, more preferably, dialyzed in HEPES solution.

[0038] In the sixth aspect of the present invention, there is provided use of the lipid nanoparticles according to the second aspect of the present invention in preparing a pharmaceutical composition.

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figures 1 and 2 are the H NMR spectra and mass spectra of the intermediate compounds, a is the H NMR spectrum of intermediate-1, b is the H NMR spectrum of intermediate-2, and c is the mass spectrum of intermediate-2.

[0041] Figure 2 This is the Ben-PEG2000 H NMR spectrum.

[0042] Figure 3 This is the hydrolysis rate diagram of Ben-PEG2000 under different pH conditions.

[0043] Figure 4 These are the Ben-PEG2000 grafting rate test results of Ben-LNP when the molar ratio of Ben-PEG2000 grafted substance is 1.125%, 2.625% and 5.625% respectively.

[0044] Figure 5 These are the cytotoxicity test results of Ben-LNP when the molar ratio of Ben-PEG2000 grafted substance is 1.125%, 2.625% and 5.625% respectively.

[0045] Figure 6 The transfection efficiency of Ben-LNP in DC2.4 and 293T cells was 1.125%, 2.625% and 5.625% respectively when the molar ratio of Ben-PEG2000 grafted substance was 1.125%, 2.625% and 5.625%.

[0046] Figure 7 The expression levels of pro-inflammatory factors TNFα and IL6 activated when Ben-LNP was loaded with STING mRNA.

[0047] Figure 8The changes in tumor volume after different intervention methods in pancreatic cancer (Panc02) tumor-bearing mice.

[0048] Fig. 9 Schematic diagram of body weight detection of pancreatic cancer (Panc02) tumor-bearing mice.

[0049] Fig.10 Schematic diagram of histological staining of mouse heart, liver, spleen, lung and kidney. DETAILED DESCRIPTION

[0050] After extensive and in-depth research, the inventors unexpectedly obtained a pH-responsive PEG lipid compound based on a benzimide bond for the first time (as shown in formula (I)). The pH-responsive PEG lipid compound of the present invention can be used as a lipid nanoparticle component for preparing lipid nanoparticles, which can be responsively reduced in a weak acid environment, and the ionizable lipids in the lipid nanoparticles can be protonated in the weak acid environment of the endosomal body, and further fuse with the negatively charged endosomal membrane, fully destroy the endosomal membrane structure, promote the endosomal escape of the loaded drug (such as mRNA), and improve the delivery efficiency. Therefore, the nucleic acid lipid nanoparticles prepared by the present invention using the pH-responsive PEG lipid compound can improve the overall biocompatibility of LNP and the delivery efficiency of the loaded drug, and at the same time have extremely high safety, can be applied to tumor vaccines, cell therapy, gene editing and other fields, and have a wide range of clinical application value.

[0051] On this basis, the present invention has been completed.

[0052] pH-responsive PEG lipid compounds based on benzimide bonds The present invention provides a pH-responsive PEG lipid compound based on a benzimide bond, the chemical structure of which is shown in the following formula (I): (I) Wherein, the molecular weight of the pH-responsive PEG lipid is about 3035.69.

[0054] In a specific embodiment of the present invention, the pH-responsive PEG lipid compound (Ben-PEG2000) based on benzimide bond as shown in the chemical formula (I) is obtained by 1,2-dimyristol-sn-glycerol-3-methoxypolyethylene glycol-400 (DMG-PEG400) and benzaldehyde-amide polyethylene glycol-2000 through Schiff base reaction. Its structure contains benzimide bond, and this specific structure can be responsively reduced to amine and aldehyde under the stimulation of the acidic environment of the tumor, resulting in the release of the long-chain part benzaldehyde-PEG2000 in Ben-PEG2000, while the short-chain part intermediate-2 remaining on the surface of the lipid nanoparticle exposes free primary amine functional groups, which can cooperate with the ionizable lipid to be protonated in the weak acid environment of the endosomal body, further fuse with the negatively charged endosomal membrane, destroy the endosomal membrane structure, promote the endosomal escape of mRNA, and improve its transfection and expression.

[0055] Lipid Nanoparticles (LNPs) As used herein, the term "lipid nanoparticle", "lipid nanoparticle" or "LNP" refers to a particle with a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticle contains one or more active agents (biologically active substances, or become the drug carried). In some embodiments, the lipid nanoparticle includes nucleic acids. In some embodiments, the nucleic acid is condensed with ionizable lipids, polymers or multivalent small molecules inside the nanoparticle and an external lipid coating that interacts with the biological environment. Due to the repulsive forces between phosphate groups, nucleic acids are naturally rigid polymers that prefer elongated configurations. In cells, in order to cope with volume constraints, DNA can package itself with the help of ions and other molecules under appropriate solution conditions. Generally, DNA condensation is defined as the collapse of an extended DNA chain into compact, ordered particles containing only one or a few molecules. By binding to phosphate groups, ionizable lipids can condense DNA by neutralizing the phosphate charge and causing it to be tightly packed.

[0056] In some embodiments, the bioactive substance is encapsulated into LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, nucleoproteins containing DNA, such as complete or partially deproteinized virus particles (virions), oligomeric and polymeric anionic compounds other than DNA (such as acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0057] Lipid nanoparticles used for mRNA delivery are mainly composed of four parts: cationic lipids / ionizable lipids, auxiliary phospholipids, cholesterol and PEGylated lipids. For example, Modern uses ionizable cationic lipids SM-102 and DSPC, cholesterol, and DMG-PEG200 as the main components to prepare LNPs to deliver mRNA encoding the S antigen of the SARS-CoV-2 virus for the prevention of the SARS-CoV-2 virus, and achieves an ideal delivery effect.

[0058] The present invention also provides a lipid nanoparticle (Ben-LNP) and a preparation method thereof. The lipid nanoparticle comprises the above-mentioned benzimide bond-based pH-responsive PEG lipid compound Ben-PEG2000.

[0059] The lipid nanoparticles provided by the present invention also contain ionizable lipids, phospholipids, cholesterol, and PEG lipids.

[0060] In some specific embodiments of the present invention, in the lipid nanoparticles, the molar ratio (molar ratio) of ionizable lipid: phospholipid: cholesterol: PEG lipid: pH-responsive PEG lipid compound Ben-PEG2000 based on benzimide bond is 30:40:30:0.375:(1.125-5.625); more preferably, the molar ratio of ionizable lipid: cholesterol: phospholipid: PEG lipid: Ben-PEG2000 is 30:40:30:0.375:1.125.

[0061] The "ionizable lipid" generally refers to a class of organic lipid molecules, which are composed of a hydrophilic head group, a linker and a hydrophobic tail. The hydrophilic head group contains a tertiary amine group, the linker contains an ester group, the number of hydrophobic tails is 3 to 4, and it has 1 to 2 unsaturated carbon chains of 8 to 18. Ionizable lipids are neutral at physiological pH values. At acidic pH values, the hydrophilic head group is ionized with a positive charge (+), and the drug is loaded through electrostatic action. Exemplary examples include 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLin-MC3-DMA, also known as MC3), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA, also known as KC2), and 1,2-dioleoyl-sn-glycero-3-methyl-4-aminopropane (also known as SM-102).

[0062] In a specific embodiment of the present invention, the ionizable lipid is N1,N3,N5 tris(2-aminophenyl)benzene-1,3,5-tricarboxamide analog (also known as BXA).

[0063] The "phospholipid" is used to form a phospholipid bilayer structure in LNP, and exemplary ones include 1,2-dioleoyl-sn-glycerol 3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol 3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycerol 3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol 3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycerol 3-phosphocholine (DSPC), etc.

[0064] In a specific embodiment of the present invention, the phospholipid is 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE).

[0065] The "PEG lipid" generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds. PEGylated lipids in LNP can increase the stability of the system, prevent particle aggregation, and reduce unnecessary serum protein adsorption. Including but not limited to PEG-modified phospholipids and derived lipids, exemplary such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol One or more combinations.

[0066] In a specific embodiment of the present invention, the PEG lipid is 1,2-dimyristyl alcohol-propylglycerol-3-methoxypolyethylene glycol-2000-mannose (DMG-PEG2000-Man).

[0067] application The present invention also provides the use of the lipid nanoparticles (Ben-LNP) in preparing a pharmaceutical composition, wherein the pharmaceutical composition further comprises the carried drug and a pharmaceutically acceptable excipient.

[0068] The "carried drug" described in the present invention includes but is not limited to any one of nucleic acids, small molecules, proteins or a combination of multiple thereof.

[0069] The "nucleic acid" of the present invention can be a nucleotide polymer of any length, including but not limited to single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (IncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide or biological custom oligonucleotide one or more combinations thereof.

[0070] In certain embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information and can guide protein synthesis. The mRNA can be a monocistronic mRNA or a polycistronic mRNA.

[0071] The "small molecule" of the present invention refers to a compound that is not a protein or nucleic acid molecule. The small molecule can be a small molecule of a therapeutic agent and / or a preventive agent, such as an antibiotic, an anti-inflammatory drug, an anticancer drug, an antiviral drug, an immunosuppressant, an analgesic, an antifungal drug, an antiparasitic drug, an anticonvulsant, an antidepressant, etc.

[0072] The "protein" of the present invention refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary and / or quaternary structures. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, etc.

[0073] The pharmaceutical composition of the present invention also includes pharmaceutically acceptable excipients. Generally, these substances can be configured in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7.

[0074] The "pharmaceutically acceptable excipients" of the present invention should be compatible with the active ingredients and should not produce adverse, allergic or other untoward reactions when the drug is properly administered to animals or humans.

[0075] Compared with the prior art, the present invention has the following excellent effects: (1) The pH-responsive PEG lipid compound (Ben-PEG2000) provided by the present invention has a benzimide bond, which can be responsively reduced to amine and aldehyde in a weak acid environment, resulting in the release of the long-chain portion benzaldehyde-amide polyethylene glycol 2000 in Ben-PEG2000, while the short-chain portion intermediate-2 remaining on the surface of the lipid nanoparticles exposes free primary amine functional groups, which can cooperate with the ionizable lipid to be protonated in the weak acid environment of the endosomal body, further fuse with the negatively charged endosomal membrane, destroy the endosomal membrane structure, promote the endosomal escape of mRNA, and improve its transfection and expression.

[0076] (2) Compared with commercial lipid nanoparticles (e.g., MC3-LNP), the lipid nanoparticles (Ben-LNP) prepared using the pH-responsive PEG lipid compound (Ben-PEG2000) have a 3-4-fold increase in mRNA transfection efficiency, and their safety both in vivo and in vitro is greatly improved.

[0077] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0078] Example 1: Synthesis and characterization of a pH-responsive PEG lipid compound based on benzimide bonds (Ben-PEG2000) The synthesis route of a pH-responsive PEG lipid compound (Ben-PEG2000) based on a benzimide bond is as follows:

[0079] The specific steps of synthesis are as follows: (1) Weigh 0.2 g of t-Boc-N-amino-PEG400-acid (purchased from Xi'an Qiangqiang Biotechnology Co., Ltd., catalog number: K-MP-2262) and dissolve it in 5 ml of dichloromethane. Add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalents of 4-dimethylaminopyridine (DMAP) under ice bath, and activate it under ice bath for 1 h. Then add 1.1 equivalents of 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DMG) (purchased from Xi'an Qiangqiang Biotechnology Co., Ltd., catalog number: K-AM-11354) and stir at room temperature for 48 h.

[0080] (2) The above system was washed with water, extracted with dichloromethane, and purified by column chromatography using a dichloromethane / methanol system to obtain intermediate-1.

[0081] (3) The intermediate 1 was dissolved in an appropriate amount of dichloromethane, and 15 equivalents of trifluoroacetic acid were added under ice bath conditions, and stirred at room temperature for 15 h.

[0082] (3) Add saturated sodium bicarbonate solution to the above system under ice bath conditions for neutralization, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2.

[0083] (4) The intermediate-2 was dissolved in an appropriate amount of N,N-dimethylformamide, and 1.5 equivalents of benzaldehyde-amide polyethylene glycol-2000 (purchased from Xi'an Ruixi Biotechnology Co., Ltd., product number: R-BJQPE-2K) was added. The mixture was stirred at room temperature in the dark for 24 hours under nitrogen protection. The system was then dialyzed with a 2500Da dialysis bag for 24 hours and freeze-dried to obtain the target product Ben-PEG2000.

[0084] The prepared Ben-PEG2000 compound was subjected to structural characterization and pH responsiveness verification, as described below.

[0085] Structural characterization of Ben-PEG2000 and its intermediates Nuclear magnetic resonance spectroscopy (1H NMR) and mass spectrometry (MS) were used to detect the structural formula and molecular weight of the compound to verify whether the compound was successfully synthesized.

[0086] The NMR images of intermediate-1 and intermediate-2 are as follows Figure 1 As shown in a and b, the chemical shift of 1.42 ppm belongs to the tert-butyloxycarbonyl group (boc group, Figure 1 The peak c in a) and Figure 1 In b, this peak is significantly weakened or disappears, indicating that the reaction from intermediate-1 to intermediate-2 is successful. Figure 1 Figure c is the mass spectrometry characterization of intermediate-2. The detected mass is the same as the target mass 935.69, indicating that intermediate-2 was successfully synthesized.

[0087] The NMR hydrogen spectrum of Ben-PEG2000 is as follows Figure 2 As shown, peak a is attributed to methyl hydrogen with a chemical shift of 0.88 ppm, and peaks b and c are attributed to benzene ring hydrogen with chemical shifts of 7.86 and 8.08 ppm, respectively. The H-NMR spectrum shows that Ben-PEG2000 was successfully synthesized.

[0088] Ben-PEG2000 pH responsiveness verification Take 0.03g (0.01 mmol) Ben-PEG2000, dissolve in 3mL citric acid buffer solution (pH5.0), MES buffer solution (pH6.5), PBS buffer solution (pH7.4), incubate at 37℃, 100rpm, take 100µL samples at fixed points (0, 10min, 20min, 30min, 60min, 90min, 120min), add 200µL borate buffer solution (pH10), then add 100µL TNBS (0.1%), incubate at room temperature for 1h, and detect the absorbance at 420nm with an enzyme marker. The intermediate-2 with the same amount of substance was used as the positive control group, and the blank control group was used without adding Ben-PEG2000.

[0089] like Figure 3 As shown in the data, under pH 5.5, 6.5 and 7.4, the hydrolysis rates of Ben-PEG2000 were 85.62±5.73%, 44.85±4.46% and 27.19±3.14% after 2 h, respectively, and the hydrolysis rates under different pH conditions were significantly different, indicating that Ben-PEG2000 has pH response characteristics and the hydrolysis rate under acidic conditions is faster than that under neutral conditions.

[0090] Example 2: Optimization of the preparation process of nucleic acid lipid nanoparticles (Ben-LNP) The present invention provides a novel nucleic acid lipid nanoparticle (Ben-LNP) and a preparation method thereof, and the preparation process optimization process thereof is specifically described in the following comparative example.

[0091] Comparative Example 1: Effect of different raw material feed ratios The PEG lipid compound (Ben-PEG2000), ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man prepared in Example 1 were dissolved in anhydrous ethanol at a molar ratio of 1.125 / 2.625 / 5.625:30:40:30:0.375 to form an organic phase, and the specific groups are as follows: Table 1 Raw material ratios in the preparation process of lipid nanoparticles

[0092] The original mRNA solution was diluted to the target volume with DEPC water and mixed with trisodium citrate solution to prepare the aqueous phase. The final trisodium citrate system was 50mM. The volume ratio of the organic phase to the aqueous phase was 1:3, and the nitrogen-phosphorus ratio (N / P) was 3:1. The organic phase and the aqueous phase were quickly mixed using a microfluidic system to prepare lipid nanoparticles.

[0093] The obtained lipid nanoparticle solution was allowed to stand at room temperature for 20 min, and then dialyzed in PBS (1×) through a 14000 MWCO dialysis bag at 4°C for 2 h to obtain the Ben-LNP solution.

[0094] The Ben-LNP solution obtained in Comparative Example 1 was subjected to the following experiments I-III respectively.

[0095] The results showed that in the lipid nanoparticle preparation process, when the feed ratio (molar ratio) of Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man was 1.125:30:40:30:0.375, the Ben-PEG2000 grafting rate and mRNA transfection efficiency were significantly higher than those of other groups, and the transfection efficiency of mCherry in DC2.4 and 293T cell lines was approximately 4 times and 3 times that of the commercial formula MC3-LNP, respectively; when the working concentration of Ben-LNP prepared in the three groups was below 1 μg / mL, the cell survival rate was as high as over 90%, indicating that Ben-LNP has good biosafety. Therefore, in the preparation process of lipid nanoparticles (Ben-LNP), the preferred feed ratio (molar ratio) of Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man is 1.125:30:40:30:0.375.

[0096] (I) Evaluation of Ben-PEG2000 grafting rate in nucleic acid lipid nanoparticles (Ben-LNP) prepared under different Ben-PEG2000 feed ratios Take 200µL of LNP grafted with different amounts of Ben-PEG2000, add 200µL borate buffer solution (0.1M, pH9.5), 100µL 10% (W / V) Triton X-100, let stand at room temperature for 20min, add 200µL TNBS (0.1%), incubate at room temperature for 1h, and measure the absorbance at 420nm (A1). LNP prepared by replacing Ben-PEG2000 with an equal molar ratio of intermediate-2 as the positive control group, and PBS (0.01M) as the negative control group, the operation method is the same as above, where the absorbance of the positive control group is A2, and the absorbance of the negative control group is A0. The final grafting rate of Ben-PEG2000 on LNP is calculated according to the following formula: Grafting rate (%) = 1-(A1-A0) / (A2-A0).

[0097] The results showed that the final Ben-PEG2000 grafting rate in LNP was detected by TNBS method (see Figure 4), the final grafting rates of Ben-PEG2000 with feed molar ratios of 1.125, 2.625, and 5.625 were 44.53%, 11.07%, and 23.59%, respectively. Among them, the final grafting rate of Ben-PEG2000 in group 1 with a feed molar ratio of Ben-PEG2000 of 1.125 was the highest.

[0098] Table 2 Effect of Ben-PEG2000 feed ratio on grafting rate of lipid nanoparticles (Ben-LNP)

[0099] (II) Evaluation of cytotoxicity of nucleic acid lipid nanoparticles (Ben-LNP) prepared under different Ben-PEG2000 feed ratios The cell viability of DC2.4 cells was verified after treatment with Ben-LNP at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1 μg / mL for 24 hours. The results showed (see Figure 5 ): The Ben-LNP prepared in Group 1-Group 3 had little effect on cell viability. When the working concentration was below 1 μg / mL, the cell survival rate was as high as over 80%, indicating that Ben-LNP had good biosafety.

[0100] (III) Evaluation of transfection efficiency of DC2.3 and 293T cells prepared by Ben-PEG2000 feed ratios using nucleic acid lipid nanoparticles (Ben-LNP) DC2.4 and 293T cells were diluted to 1×10 5 / mL, spread in a 24-well plate, and inoculate 1mL per well. The positive control group was the MC3-LNP group, and the experimental group was the Ben-LNP. Each well was transfected with 400ng mCherry mRNA. After 24h of transfection, the culture medium was discarded, 500µL of cell lysis buffer was added to each well, and the cell was lysed at 1000rpm for 20min. The supernatant was aspirated, and the total protein content (BCA method) and the fluorescence intensity of mCherry (Ex = 590nm, Em = 645nm) were detected respectively. The cell transfection efficiency was detected by calculating the unit fluorescence intensity of mCherry.

[0101] Results Figure 6 : Figure 6Figures a and b show that, based on the intracellular unit fluorescence intensity (RFU), the unit fluorescence intensity of group 1, group 2, and group 3 in DC2.4 cells were 168481, 133295, and 94261, respectively; the unit fluorescence intensity of group 1, group 2, and group 3 in 293T cells were 259837, 234245, and 169534, respectively, indicating that the transfection efficiency of group 1 was higher. Ben-LNP prepared using the group 1 preparation scheme was further compared with the commercial formula MC3-LNP. Figure 6 Middle c shows that the transfection efficiency of mCherry by group 1 Ben-LNP in DC2.4 and 293T cell lines is approximately 4 times and 3 times that of MC3-LNP, respectively.

[0102] Comparative Example 2: Effect of different nitrogen-phosphorus ratios (N / P) (1) The Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, DMG-PEG2000-Man, and PEG lipid compound (Ben-PEG2000) prepared in Example 1 were dissolved in anhydrous ethanol at a molar ratio of (1.125:30:40:30:0.375) to prepare an organic phase.

[0103] (2) The original mRNA solution was diluted to the target volume with DEPC water and then mixed with trisodium citrate solution to prepare the aqueous phase. The final trisodium citrate system was 50 mM. The volume ratio of the organic phase to the aqueous phase was 1:3, and the nitrogen-phosphorus ratio (N / P) was 3:1, 4:1, and 5:1, respectively. The organic phase and the aqueous phase were quickly mixed using a microfluidic system to prepare lipid nanoparticles.

[0104] Table 3 Different nitrogen-phosphorus ratios (N / P) in lipid nanoparticle preparation process

[0105] (3) The obtained lipid nanoparticle solution was allowed to stand at room temperature for 20 min and then dialyzed in PBS (1×) using a 14000 MWCO dialysis bag at 4°C for 2 h to obtain the Ben-LNP solution.

[0106] In Comparative Example 3, the nucleic acid liposome nanoparticles (Ben-LNP) obtained under different nitrogen-phosphorus ratios (N / P) were filtered and sterilized, and the average particle size and PDI (polydispersity index) of Ben-LNP were characterized using Malvern Zetasizer Nano ZS, and the mRNA encapsulation efficiency was determined using Ribogreen RNA quantitative detection kit. The results are shown in Table 4: Table 4 Characterization data of nucleic acid lipid nanoparticles prepared under different nitrogen-phosphorus ratios (N / P)

[0107] The results showed that when the nitrogen-phosphorus ratio (N / P) of the prepared nucleic acid lipid nanoparticles (Ben-LNP) was 3:1, the average particle size was about 95.6 nm, the PDI was the lowest, and the encapsulation efficiency was the highest. Therefore, the preferred nitrogen-phosphorus ratio (N / P) was 3:1.

[0108] Example 3: In vitro efficacy evaluation of nucleic acid lipid nanoparticles (Ben-LNP) (1) Nucleic acid lipid nanoparticles (Ben-LNP) encapsulating STING mRNA were prepared according to the optimized conditions of Example 2.

[0109] (2) Dilute the induced mouse bone marrow-derived dendritic cells (BMDC) to 5×10 5 / mL, spread in a 24-well plate, 1mL per well. The negative control group was the PBS group, the positive control group was the MC3-LNP group, and the experimental group was the Ben-LNP. After 24 hours of drug stimulation, the content of TNFα and IL6 in the BMDC supernatant was detected according to the elabscience ELISA instructions.

[0110] The experimental results show (see Figure 7 ): The average expression levels of pro-inflammatory factors TNFα and IL6 in Ben-LNP encapsulated with STING mRNA were as high as 649.04pg / mL and 5591.49pg / mL, respectively, which were 158.07% and 2245.88% higher than those stimulated by MC3-LNP of the same concentration. Therefore, Ben-LNP can more effectively activate immune cells to produce TNFα and IL6, thereby triggering a series of immune responses.

[0111] Example 4: In vivo efficacy and safety evaluation of nucleic acid lipid nanoparticles (Ben-LNP) (1) Nucleic acid lipid nanoparticles (Ben-LNP) encapsulating STING mRNA were prepared according to the optimized conditions of Example 2.

[0112] (2) Construction of pancreatic cancer model: The right hind limbs and back of C57BL / 6 mice were shaved, and 2×10 6 Panc02 cells were used, and 8 days later, a subcutaneous mouse pancreatic cancer model was successfully established and can be used for subsequent experiments.

[0113] (3) Group administration: Group I was injected with PBS only; Group II received intraperitoneal injection of PD-L1 (100 μg / mouse) on days 0, 5, 10, and 15; Group III received intratumoral injection of Ben-LNP (10 μg / mouse) on days 0, 5, 10, and 15; Group IV received intratumoral injection of MC3-LNP (10 μg / mouse) on days 0, 5, 10, and 15; Group V received intratumoral injection of Ben-LNP (10 μg / mouse) on days 0, 5, 10, and 15, and intraperitoneal injection of PD-L1 (100 μg / mouse) on days 1, 6, 11, and 16. Tumor volume and body weight were recorded every 4 days for 20 days.

[0114] Table 5 Grouping of pancreatic cancer-bearing mice

[0115] (4) HE staining detection: After the end of treatment (20 days after administration), the mice were euthanized, and their tumor tissues and major organs, including the heart, liver, spleen, lungs, and kidneys, were dissected and collected. Subsequently, the tumor tissues and organs of the mice were immersed in 10% formalin for fixation for 2 days, and then embedded to prepare tissue wax blocks. The wax blocks were cut into tissue sections with a thickness of 2 μm using a freezing microtome, and then the cell nuclei and cytoplasm were stained with hematoxylin and eosin (HE staining), respectively. Finally, the morphological changes of tumor tissues and tissue cells of various organs were observed under a microscope.

[0116] result: (I) Weight evaluation according to Fig. 9 : During the administration period, the body weight of mice in all groups showed a steady growth trend, reflecting good biosafety.

[0117] (II) Tumor size evaluation according to Figure 8 : During the medication period, the tumor volume of the PBS group, MC3 group and PD-L1 group gradually increased. The Ben-LNP group and Ben-LNP+PD-L1 group effectively inhibited the growth of pancreatic cancer, and the therapeutic effect of the Ben-LNP+PD-L1 group was better than that of the Ben-LNP group. The therapeutic effects of the two were better than those of the PBS group, MC3 group and PD-L1 group.

[0118] (III) Safety evaluation of HE chromosomes according to Fig.10After 20 days of administration, HE staining histological examination of tumor tissues in each group showed that the cell nuclear density of the Ben-LNP+PD-L1 group was sparse and the nuclear shape was abnormal, indicating cell necrosis or apoptosis. HE staining histological observation of major organs (heart, liver, spleen, lung, and kidney) showed that there were no obvious physiological morphological changes or tissue damage to mice in the control group or the treatment group during the administration period, and its acute pathological toxicity and adverse reactions were negligible. Ben-LNP+PD-L1 treatment had no obvious side effects on mice and had good biosafety.

[0119] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A pH-responsive PEG lipid compound based on a benzimide bond, the chemical structure of which is shown in the following formula (I): （I） in, The PEG is PEG-2000.

2. The PEG lipid compound of claim 1, wherein The molecular weight of the PEG lipid compound is 3035.

69.

3. A lipid nanoparticle, characterized in that: The lipid nanoparticles comprise the benzimide bond-based pH-responsive PEG lipid compound as claimed in claim 1.

4. The lipid nanoparticle according to claim 3, characterized in that The lipid nanoparticles also contain ionizable lipids, phospholipids, steroids and PEG lipids.

5. The lipid nanoparticle according to claim 4, characterized in that The molar ratio of the pH-responsive PEG lipid compound based on benzimide bonds, ionizable lipids, phospholipids, steroids and PEG lipids in the lipid nanoparticles is (1.125-5.625):30:40:30:0.

375.

6. A method for preparing a pH-responsive PEG lipid compound based on a benzimide bond as claimed in claim 1, comprising the following steps: (1) Weigh 0.2 g of t-Boc-N-amino-PEG400-acid and dissolve it in 5 ml of dichloromethane. Add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalents of 4-dimethylaminopyridine (DMAP) under ice bath, activate under ice bath for 1 h, then add 1.1 equivalents of 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DMG) and stir at room temperature for 48 h. (2) washing the system obtained in step (1) with water, extracting with dichloromethane, and purifying by column chromatography using a dichloromethane / methanol system to obtain intermediate-1; (3) The intermediate 1 was dissolved in dichloromethane, 15 equivalents of trifluoroacetic acid was added under ice bath conditions, and stirred at room temperature for 15 h; (4) Add saturated sodium bicarbonate solution to the system of step (3) under ice bath conditions for neutralization, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2; (5) The intermediate 2 was dissolved in an appropriate amount of N,N-dimethylformamide, and 1.5 equivalents of benzaldehyde-amide polyethylene glycol-2000 were added. The mixture was stirred at room temperature in the dark for 24 h under nitrogen protection. The system was then dialyzed using a 2500Da dialysis bag for 24 h and freeze-dried to obtain the target product, i.e., the PEG lipid compound shown in formula (I).

7. Use of the pH-responsive PEG lipid compound based on benzimide bonds as claimed in claim 1 in the preparation of lipid nanoparticles.

8. A method for preparing lipid nanoparticles, comprising the following steps: (S1) mixing ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzimide bond as claimed in claim 1 in a uniform proportion, and dissolving them in an organic solvent to prepare an organic phase; (S2) providing an aqueous phase containing a drug to be loaded; (S3) rapidly mixing the organic phase and the aqueous phase to prepare lipid nanoparticles; (S4) The obtained lipid nanoparticles were immediately dialyzed at 2-8°C for 2-4 hours.

9. The method according to claim 8, characterized in that In the step (S1), the molar ratio of the ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzimide bond as described in claim 1 is 30:40:30:0.375:(1.125-5.625).

10. Use of the lipid nanoparticles according to any one of claims 3 to 5 in the preparation of a pharmaceutical composition.

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