A pH-responsive PEG lipid compound based on benzoylimine bond and its application

Abstract

The present invention relates to a pH-responsive PEG lipid compound based on benzoyl imine bond and its application. The present invention provides a pH-responsive PEG lipid compound Ben-PEG2000 containing a benzoyl imine bond, and its chemical formula is shown as formula (I). The lipid nanoparticles containing the pH-responsive PEG lipid compound Ben-PEG2000 provided by the present invention have pH-responsive characteristics, can promote the endosomal escape and transfection efficiency of the mRNA loaded in the lipid nanoparticles under the weak acidic environment in the body, and at the same time have extremely high safety, and have more extensive clinical application value. (I).

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a pH-responsive PEG lipid compound based on benzoyl imine bond and its applications. Background Art

[0002] The biggest problem encountered in RNA-based gene therapy is drug delivery. First, naked RNA is a negatively charged hydrophilic macromolecule that exhibits electrostatic repulsion with cell membranes and thus is not easily able to penetrate cell membranes. Second, nuclease and hydrolase present in organisms can easily degrade exogenous RNA molecules, leading to RNA inactivation and making it difficult to accumulate in target tissues. In addition, the immune system can also recognize exogenous nucleic acids and trigger immunogenic reactions. In order to achieve safe and effective nucleic acid delivery, several RNA carriers have been researched and developed to protect nucleic acids from degradation and maximize delivery to target cells. Among them, the application of lipid nanoparticles (LNPs) in nucleic acid drugs and vaccine delivery has once again received extensive attention. LNPs consist of five parts: ionizable lipids, nucleic acids, cholesterol, phospholipids, and PEG lipids. Among them, PEG lipids, as an amphiphilic material, stabilize the LNP structure by providing a steric barrier, can drive self-assembly and prevent particle aggregation, and extend the circulation time of LNPs.

[0003] However, as a clinically approved multifunctional nucleic acid delivery platform, a major bottleneck currently faced by LNPs is the efficient in vivo expression of mRNA. Regarding "how to improve the efficient in vivo expression of mRNA", the main focus is on several strategies such as the development of novel ionizable lipids, targeted modification, and SORT.

[0004] One important factor affecting the efficient transfection or expression of mRNA is the endosomal escape of mRNA. Briefly, mRNA encapsulated in LNPs mainly undergoes three stages in cells: early endosomes, late endosomes, and lysosomes, and mRNA can only achieve its coding function and stimulate downstream processes if it successfully escapes from early or late endosomes. Research has shown that approximately 70% of mRNA is unable to achieve endosomal escape, with some being degraded by lysosomes and some being excreted. Enhancing the endosomal escape ability of mRNA can promote the effective expression of antigens. Regarding the endosomal escape theory, it includes the proton sponge effect and the membrane fusion phase transition hypothesis. Among them, the membrane fusion phase transition hypothesis states that: ionizable lipids are protonated under acidic pH conditions in endosomes and then interact with anionic lipids in the endosomal membrane to undergo membrane fusion. This fusion can induce the transformation of the endosomal membrane from a stable bilayer structure to a non-bilayer (hexagonal HII) structure, resulting in the rupture of the endosomal membrane and thus releasing effective payloads such as nucleic acids into the cytoplasm.

[0005] At present, research on pH-based endosomal escape and mRNA expression mainly focuses on ionizable lipids, and there is little research on other components in LNPs. Therefore, studying and developing pH-responsive LNP components is of great significance for improving the delivery and transfection efficiency of mRNA-LNPs to target cells. Summary of the Invention

[0006] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a pH-responsive PEG lipid compound based on benzimidamide bonds, a lipid nanoparticle containing the same, and their applications.

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

[0008] (I)

[0009] Wherein, the PEG is PEG-2000.

[0010] In another preferred example, the molecular weight of the PEG lipid compound is about 3035.69.

[0011] In another preferred example, the PEG lipid compound is prepared by the following method: obtained by Schiff base reaction of 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-400 (DMG-PEG400) and benzaldehyde-amido polyethylene glycol-2000.

[0012] In another preferred example, the PEG lipid compound is prepared by the following method:

[0013] (1) Weigh 0.2 g of N-(tert-butoxycarbonylamino) polyethylene glycol 400-carboxylic acid (t-Boc-N-amino-PEG400-acid) and dissolve it in 5 ml of dichloromethane. Under ice bath, add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalent of 4-dimethylaminopyridine (DMAP), activate at ice bath for 1 h, and then add 1.1 equivalents of , stir and react at room temperature for 48 h;

[0014] (2) Wash the above system with water, extract with dichloromethane, and purify by column chromatography using dichloromethane / methanol system to obtain intermediate-1;

[0015] (3) Dissolve the above intermediate-1 in dichloromethane, add 15 equivalents of trifluoroacetic acid under ice bath conditions, and stir at room temperature for 15 h;

[0016] (4) Under ice bath conditions, add saturated sodium bicarbonate solution to the system in step (3) above for neutralization, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2;

[0017] (5) Dissolve the above intermediate-2 in an appropriate amount of N,N-dimethylformamide, add 1.5 equivalents of benzaldehyde-amido polyethylene glycol 2000, stir at room temperature in the dark under nitrogen protection for 24 h; then dialyze the system with a 2500 Da dialysis bag for 24 h, and freeze-dry to obtain the target product, namely the PEG lipid compound shown in formula (I).

[0018] In another preferred example, the PEG lipid compound is formed by grafting through a benzimidoyl bond, has pH responsiveness, and can be reductively converted to amino and aldehyde groups in a weak acid environment.

[0019] In the second aspect of the present invention, a lipid nanoparticle is provided, and the lipid nanoparticle contains the pH-responsive PEG lipid compound based on benzimidoyl bond described in the first aspect of the present invention.

[0020] In another preferred example, the lipid nanoparticle further contains an ionizable lipid, a phospholipid, a steroid, and a PEG lipid.

[0021] In another preferred example, the molar ratio of the pH-responsive PEG lipid compound based on benzimidoyl bond, ionizable lipid, phospholipid, steroid, and PEG lipid in the lipid nanoparticle is (1.125 - 5.625):30:40:30:0.375, preferably 1.125:30:40:30:0.375.

[0022] In another preferred example, the ionizable lipid is an N1,N3,N5-tris(2-aminophenyl)benzene-1,3,5-tricarboxamide analogue (BXA).

[0023] In another preferred example, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0024] In another preferred example, the steroid is cholesterol.

[0025] In another preferred example, the PEG lipid is 1,2-dimyristoyl-propylglycerol-3-methoxypolyethylene glycol-2000-mannose (DMG-PEG2000-Man).

[0026] In another preferred example, the lipid nanoparticle contains the pH-responsive PEG lipid compound based on benzimidoyl bond, BXA, DOPE, cholesterol, and DMG-PEG2000-Man described in the first aspect of the present invention.

[0027] In another preferred example, the molar ratio of the pH-responsive PEG lipid compound based on benzimidoyl 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.

[0028] In another preferred example, the lipid nanoparticles are loaded with drugs, and the drugs include one or more of nucleic acid molecules, small molecule compounds, and proteins.

[0029] In another preferred example, the lipid nanoparticles are loaded with nucleic acid drugs.

[0030] In another preferred example, the nucleic acid drugs include RNA drugs and DNA drugs.

[0031] In another preferred example, the RNA drugs include (but are not limited to) mRNA, tRNA, rRNA, miRNA, siRNA, circRNA, sgRNA.

[0032] In another preferred example, the nucleic acid drug is mRNA.

[0033] In another preferred example, the nucleic acid drug is a therapeutic drug or a prophylactic drug, such as a vaccine.

[0034] In another preferred example, the nucleic acid drug is an mRNA vaccine.

[0035] In another preferred example, the nitrogen-to-phosphorus ratio (N / P) of the lipid nanoparticles is 3:1.

[0036] In the third aspect of the present invention, a preparation method of the pH-responsive PEG lipid compound based on benzimidoyl bond as described in the first aspect of the present invention is provided, including the following steps:

[0037] (1) Weigh 0.2 g of N-(tert-butoxycarbonylamino) polyethylene glycol 400-carboxylic acid (t-Boc-N-amino-PEG400-acid) and dissolve it in 5 ml of dichloromethane. Under ice bath, add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalent of 4-dimethylaminopyridine (DMAP), and activate it under ice bath for 1 h. Then add 1.1 equivalents of , and stir and react at room temperature for 48 h;

[0038] (2) Wash the above system with water, extract with dichloromethane, and purify by column chromatography using a dichloromethane / methanol system to obtain intermediate-1;

[0039] (3) The above intermediate-1 was dissolved in dichloromethane, and 15 equivalents of trifluoroacetic acid were added under an ice bath condition, followed by stirring at room temperature for 15 h;

[0040] (4) Under an ice bath condition, saturated sodium bicarbonate solution was added to the system of step (3) above for neutralization, stirred for 15 min, diluted and extracted with dichloromethane, and rotary evaporated to obtain intermediate-2;

[0041] (5) The above intermediate-2 was dissolved in an appropriate amount of N,N-dimethylformamide, 1.5 equivalents of benzaldehyde-amido polyethylene glycol-2000 were added, and the mixture was stirred at room temperature in the dark under nitrogen protection for 24 h; then the system was dialyzed for 24 h using a 2500 Da dialysis bag and freeze-dried to obtain the target product, namely the PEG lipid compound as shown in formula (I).

[0042] In the fourth aspect of the present invention, there is provided the use of the pH-responsive PEG lipid compound based on benzimidoyl bond as described in the first aspect of the present invention in the preparation of lipid nanoparticles.

[0043] In the fifth aspect of the present invention, there is provided a method for preparing lipid nanoparticles, comprising the following steps:

[0044] (S1) The ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzimidoyl bond as described in the first aspect of the present invention were mixed evenly in proportion and dissolved in an organic solvent to prepare an organic phase;

[0045] (S2) An aqueous phase containing the drug to be loaded was provided;

[0046] (S3) The organic phase and the aqueous phase were rapidly mixed to prepare lipid nanoparticles;

[0047] (S4) The obtained lipid nanoparticles were immediately dialyzed at 2 - 8 °C for 2 - 4 hours.

[0048] In another preferred example, in step (S1), the molar ratio of the ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzimidoyl bond as described in the first aspect of the present invention is 30:40:30:0.375:(1.125 - 5.625), preferably 30:40:30:0.375:1.125.

[0049] In another preferred example, in step (S1), the organic solvent is absolute ethanol.

[0050] In another preferred example, in step (S2), the drug to be loaded (such as mRNA) was diluted to the target volume with DEPC water and mixed with trisodium citrate solution to prepare an aqueous phase.

[0051] In another preferred example, the concentration of sodium citrate in the final aqueous phase system is 50 mM - 100 mM, preferably 50 mM.

[0052] In another preferred example, in the step (S3), the volume ratio of the organic phase to the aqueous phase during mixing is 1:3.

[0053] In another preferred example, in the step (S4), the obtained lipid nanoparticles are dialyzed in 1×PBS or HEPES solution, and more preferably, dialyzed in HEPES solution.

[0054] In the sixth aspect of the present invention, there is provided the use of the lipid nanoparticles as described in the second aspect of the present invention in the preparation of a pharmaceutical composition.

[0055] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0056] Figure 1 1H NMR and mass spectrometry diagrams of the intermediate compounds, where a is the 1H NMR diagram of intermediate - 1, b is the 1H NMR diagram of intermediate - 2, and c is the mass spectrometry diagram of intermediate - 2.

[0057] Figure 2 1H NMR diagram of Ben - PEG2000.

[0058] Figure 3 Hydrolysis rate diagrams of Ben - PEG2000 under different pH conditions.

[0059] Figure 4 Detection results of the grafting rate of Ben - PEG2000 on Ben - LNP when the molar ratio of the grafted Ben - PEG2000 is 1.125%, 2.625%, and 5.625% respectively.

[0060] Figure 5 Cytotoxicity detection results of Ben - LNP when the molar ratio of the grafted Ben - PEG2000 is 1.125%, 2.625%, and 5.625% respectively.

[0061] Figure 6 Transfection efficiency of Ben - LNP in transfecting DC2.4 and 293T cells when the molar ratio of the grafted Ben - PEG2000 is 1.125%, 2.625%, and 5.625% respectively.

[0062] Figure 7The expression levels of pro-inflammatory factors TNFα and IL6 when Ben-LNP loaded STING mRNA.

[0063] Figure 8 The changes in tumor volume of tumor-bearing mice with pancreatic cancer (Panc02) after different interventions.

[0064] Figure 9 Schematic diagram of body weight detection of tumor-bearing mice with pancreatic cancer (Panc02).

[0065] Figure 10 Schematic diagram of histological staining of the heart, liver, spleen, lungs, and kidneys of mice. Detailed implementation mode

[0066] After extensive and in-depth research, the present inventors unexpectedly obtained for the first time a pH-responsive PEG lipid compound based on benzimidamide bond (as shown in formula (I)). The pH-responsive PEG lipid compound of the present invention can be used as a component of lipid nanoparticles for preparing lipid nanoparticles, which can be reductively responsive under weak acid conditions, cooperate with the ionizable lipid in the lipid nanoparticles to be protonated under weak acid conditions in the endosome, further fuse with the negatively charged endosomal membrane, fully disrupt 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 using the pH-responsive PEG lipid compound of the present invention can overall improve the biocompatibility of LNP and the delivery efficiency of the loaded drug, and at the same time have extremely high safety, and can be applied to fields such as tumor vaccines, cell therapy, and gene editing, and have broad clinical application value.

[0067] On this basis, the present invention was completed.

[0068] pH-responsive PEG lipid compound based on benzimidamide bond

[0069] The present invention provides a pH-responsive PEG lipid compound based on benzimidamide bond, and its chemical structural formula is shown as the following formula (I):

[0070]

[0071] (I)

[0072] Among them, the molecular weight of the pH-responsive PEG lipid is about 3035.69.

[0073] In a specific embodiment of the present invention, the pH-responsive PEG lipid compound (Ben-PEG2000) based on benzoyl imine bond shown by the chemical formula (I) is obtained by the Schiff base reaction of 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-400 (DMG-PEG400) and benzaldehyde-amido polyethylene glycol-2000. Its structure contains a benzoyl imine bond. This specific structure can be reductively responsive to amines and aldehydes under the stimulation of the acidic environment of tumors, 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 nanoparticles exposes free primary amine functional groups, which can cooperate with ionizable lipids to be protonated in the weak acidic environment inside the endosome, further fuse with the negatively charged endosomal membrane, disrupt the endosomal membrane structure, promote mRNA endosomal escape, and improve its transfection and expression.

[0074] Lipid nanoparticles (LNP)

[0075] As used herein, the terms "lipid nanoparticles", "lipid nanoparticles" or "LNP" refer to particles with a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances, or the loaded drugs). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed inside the nanoparticles with ionizable lipids, polymers or multivalent small molecules and an external lipid coating that interacts with the biological environment. Due to the repulsive force between phosphate groups, nucleic acids are natural rigid polymers and tend to be in an elongated configuration. In cells, in order to cope with volume limitations, 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 extended DNA strands into compact and 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 packing it tightly.

[0076] In some embodiments, the bioactive substances are encapsulated into the LNP. In some embodiments, the bioactive substances 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, DNA-containing nucleoproteins, such as intact 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 substances can be mixed with adjuvants.

[0077] Lipid nanoparticles for mRNA delivery mainly consist of four parts: cationic lipids / ionizable lipids, helper phospholipids, cholesterol, and PEGylated lipids. For example, Moderna prepares LNPs with ionizable cationic lipid SM-102, DSPC, cholesterol, and DMG-PEG200 as the main components 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.

[0078] The present invention also provides a lipid nanoparticle (Ben-LNP) and a preparation method thereof, and the lipid nanoparticle contains the above-mentioned pH-responsive PEG lipid compound Ben-PEG2000 based on benzimidamide bond.

[0079] The lipid nanoparticles provided by the present invention further contain ionizable lipids, phospholipids, cholesterol, and PEG lipids.

[0080] In some specific embodiments of the present invention, in the lipid nanoparticles, the molar ratio of ionizable lipid: phospholipid: cholesterol: PEG lipid: pH-responsive PEG lipid compound Ben-PEG2000 based on benzimidamide 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.

[0081] The "ionizable lipid" generally refers to a class of organic lipid molecules 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 - 4, and it has 1 - 2 unsaturations and an 8 - 18 carbon chain. The ionizable lipid is neutral at physiological pH values, and at acidic pH values, the hydrophilic head group is ionized to carry a positive charge (+) and loads drugs through electrostatic interaction. Exemplary ones are 1,2-dilinoleyloxy-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), 1,2-dioleoyl-sn-glycero-3-methyl-4-aminopropane (also known as SM-102).

[0082] 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).

[0083] The "phospholipid" is used in the LNP to form a phospholipid bilayer structure, such as, for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), etc.

[0084] In a specific embodiment of the present invention, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0085] The "PEG lipid" generally refers to a conjugate formed by chemically linking PEG (polyethylene glycol) with a lipid molecule. The PEGylated lipid in the LNP can increase the stability of the system, prevent particle aggregation, and reduce unnecessary serum protein adsorption. It includes, but is not limited to, PEG-modified phospholipids and derived lipids, such as, for example, one or a combination of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0086] In a specific embodiment of the present invention, the PEG lipid is 1,2-dimyristoyl-propylglycerol-3-methoxypolyethylene glycol-2000-mannose (DMG-PEG2000-Man).

[0087] Application

[0088] The present invention also provides the use of the lipid nanoparticles (Ben-LNP) in the preparation of a pharmaceutical composition, and the pharmaceutical composition further comprises the loaded drug and a pharmaceutically acceptable excipient.

[0089] The "loaded drug" described in the present invention includes, but is not limited to, any one or a combination of nucleic acids, small molecules, and proteins.

[0090] The "nucleic acid" in 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 isoforms, mRNA, tRNA, rRNA, long non-coding RNA (IncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA, small nuclear RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozyme guide RNA (gRNA), small guide RNA (sgRNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotides, morpholino oligonucleotides or a combination of one or more of bio-customized oligonucleotides.

[0091] In certain embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to guide protein synthesis. The mRNA can be monocistronic mRNA or polycistronic mRNA.

[0092] The "small molecule" in the present invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecules of therapeutic and / or prophylactic agents, such as antibiotics, anti-inflammatory drugs, anti-cancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungal drugs, anti-parasitic drugs, anti-convulsants, antidepressants, etc.

[0093] The "protein" in the present invention refers to a molecule or complex containing one or more polypeptides with 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.

[0094] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 4-8, preferably about 5-7.

[0095] The "pharmaceutically acceptable excipient" in the present invention should be compatible with the active ingredient and will not produce adverse, allergic or other adverse reactions when the drug is properly administered to animals or humans.

[0096] Compared with the prior art, the present invention has the following excellent effects:

[0097] (1) The pH-responsive PEG lipid compound (Ben-PEG2000) provided by the present invention has a benzimidoyl bond, which can be reductively responsive to amines and aldehydes in a weak acid environment, resulting in the release of benzaldehyde-amido polyethylene glycol 2000 in the long-chain part of Ben-PEG2000, while the short-chain part intermediate-2 remaining on the surface of the lipid nanoparticles exposes free primary amine functional groups, which can synergistically protonate with ionizable lipids in a weak acid environment, further fuse with the negatively charged endosomal membrane, disrupt the endosomal membrane structure, promote mRNA endosomal escape, and improve its transfection and expression.

[0098] (2) Compared with commercial lipid nanoparticles (such as MC3-LNP), the mRNA transfection efficiency of the lipid nanoparticles (Ben-LNP) prepared using the pH-responsive PEG lipid compound (Ben-PEG2000) is increased by 3-4 times. At the same time, its safety in vitro and in vivo has been greatly improved.

[0099] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0100] Example 1: Synthesis and Characterization of a pH-Responsive PEG Lipid Compound (Ben-PEG2000) Based on Benzimidoyl Bond

[0101] The synthesis route of a pH-responsive PEG lipid compound (Ben-PEG2000) based on benzimidoyl bond is as follows:

[0102]

[0103] The specific synthesis steps are as follows:

[0104] (1) Weigh 0.2 g of N-(tert-butoxycarbonylamino) polyethylene glycol 400-carboxylic acid (t-Boc-N-amino-PEG400-acid) (purchased from Xi'an Qianghua Biotechnology Co., Ltd., product number: K-MP-2262) and dissolve it in 5 ml of dichloromethane. Under ice bath, add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA), and 0.1 equivalent of 4-dimethylaminopyridine (DMAP), and activate it under ice bath for 1 h. Then add 1.1 equivalents of (purchased from Xi'an Qianghua Biotechnology Co., Ltd., product number: K-AM-11354), and stir and react at room temperature for 48 h.

[0105] (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.

[0106] (3)The above Intermediate-1 was dissolved in an appropriate amount of dichloromethane, and 15 equivalents of trifluoroacetic acid were added under ice bath conditions, followed by stirring at room temperature for 15 h.

[0107] (3)Under ice bath conditions, saturated sodium bicarbonate solution was added to the above system for neutralization, stirred for 15 min, diluted and extracted with dichloromethane, and rotary evaporated to obtain Intermediate-2.

[0108] (4)The above Intermediate-2 was dissolved in an appropriate amount of N,N-dimethylformamide, and 1.5 equivalents of benzaldehyde-amido polyethylene glycol-2000 (purchased from Xi'an Ruixi Biotechnology Co., Ltd., product number: R-BJQPE-2K) were added. Under nitrogen protection, the mixture was stirred at room temperature in the dark for 24 h. Then the system was dialyzed with a 2500 Da dialysis bag for 24 h and freeze-dried to obtain the target product Ben-PEG2000.

[0109] The prepared Ben-PEG2000 compound was subjected to structural characterization and pH responsiveness verification, as described below.

[0110] Structural Characterization of Ben-PEG2000 and Intermediates

[0111] The nuclear magnetic resonance hydrogen spectrum (1H NMR) and mass spectrometry (MS) were used to detect the chemical structure and molecular weight of the compound to verify whether the compound was successfully synthesized.

[0112] The NMR spectra of Intermediate-1 and Intermediate-2 are shown in Figure 1 a and b below. The chemical shift of 1.42 ppm belongs to the tert-butoxycarbonyl group (boc group, Figure 1 peak c in a), while in Figure 1 b, this peak was significantly weakened or disappeared, indicating that the reaction of removing the boc group from Intermediate-1 to Intermediate-2 was successful. In addition, Figure 1 c in is the mass spectrometry characterization of Intermediate-2, and the detected mass is the same as the target mass of 935.69, indicating that Intermediate-2 was successfully synthesized.

[0113] The 1H NMR spectrum of Ben-PEG2000 is shown in Figure 2 as shown. Among them, peak a belongs to the methyl hydrogen with a chemical shift of 0.88 ppm, and peaks b and c belong to the benzene ring hydrogen with chemical shifts of 7.86 and 8.08 ppm, respectively. The 1H NMR spectrum indicates the successful synthesis of Ben-PEG2000.

[0114] pH Responsiveness Verification of Ben-PEG2000

[0115] Take 0.03 g (0.01 mmol) of Ben-PEG2000 and dissolve it in 3 mL of citrate buffer solution (pH 5.0), MES buffer solution (pH 6.5), and PBS buffer solution (pH 7.4) respectively. Incubate at 37 °C and 100 rpm, and take 100 μL of samples at fixed points (0, 10 min, 20 min, 30 min, 60 min, 90 min, 120 min). Add 200 μL of borate buffer solution (pH 10), then add 100 μL of TNBS (0.1%), and incubate at room temperature for 1 h. Measure the absorbance value at 420 nm using an enzyme-linked immunosorbent assay reader. Use the intermediate-2 with the same amount of substance as the positive control group and the absence of Ben-PEG2000 as the blank control group.

[0116] As Figure 3 shown, in the environments of pH 5.5, 6.5, and 7.4, the hydrolysis rates of Ben-PEG2000 after 2 h are 85.62 ± 5.73%, 44.85 ± 4.46%, and 27.19 ± 3.14% respectively, and there are significant differences in the hydrolysis rates under each pH condition, indicating that Ben-PEG2000 has pH-responsive characteristics and hydrolyzes faster under acidic conditions than under neutral conditions.

[0117] Example 2: Optimization of the preparation process of nucleic acid lipid nanoparticles (Ben-LNP)

[0118] The present invention provides a novel nucleic acid lipid nanoparticle (Ben-LNP) and its preparation method, and the optimization process of its preparation process is as described in the following comparative examples.

[0119] Comparative Example 1: Influence of different raw material feeding ratios

[0120] Dissolve the PEG lipid compound (Ben-PEG2000), ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man prepared in Example 1 in anhydrous ethanol according to a molar ratio of 1.125 / 2.625 / 5.625:30:40:30:0.375 to form an organic phase. The specific groups are as follows:

[0121] Table 1 Raw material feeding ratios during the preparation of lipid nanoparticles

[0122]

[0123] Dilute the original mRNA solution to the target volume with DEPC water and mix it with the trisodium citrate solution to prepare an aqueous phase. The final concentration of trisodium citrate in the system is 50 mM. The volume ratio of the organic phase to the aqueous phase is 1:3, and the nitrogen-to-phosphorus ratio (N / P) is 3:1. Use a microfluidic system to quickly mix the organic phase and the aqueous phase to prepare lipid nanoparticles.

[0124] The obtained lipid nanoparticle solution was allowed to stand at room temperature for 20 min, and then dialyzed against PBS (1×) through a 14,000 MWCO dialysis bag for 2 h at 4 °C to obtain the Ben-LNP solution.

[0125] The Ben-LNP solution obtained in Comparative Example 1 was subjected to the following Experiments I-III respectively.

[0126] The results showed that in the lipid nanoparticle preparation process, under the condition that the feeding ratio (molar ratio) of Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man was 1.125:30:40:30:0.375, the grafting rate of Ben-PEG2000 and the mRNA transfection efficiency were significantly higher than those of other groups, and the transfection efficiencies of mCherry in DC2.4 and 293T cell lines were about 4 times and 3 times that of the commercial formulation MC3-LNP respectively; when the working concentration of Ben-LNP prepared in the 3 groups was below 1 μg / mL, the cell survival rate was as high as over 90%, indicating that Ben-LNP had good biosafety. Therefore, in the preparation process of lipid nanoparticles (Ben-LNP), it was preferred that the feeding ratio (molar ratio) of Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, and DMG-PEG2000-Man was 1.125:30:40:30:0.375.

[0127] (I) Evaluation of the grafting rate of Ben-PEG2000 in nucleic acid lipid nanoparticles (Ben-LNP) prepared under different Ben-PEG2000 feeding ratios

[0128] Take 200 μL of LNP grafted with different amounts of Ben-PEG2000, add 200 μL of borate buffer solution (0.1 M, pH 9.5), 100 μL of 10% (W / V) Triton X-100, allow to stand at room temperature for 20 min, then add 200 μL of TNBS (0.1%), incubate at room temperature for 1 h, and measure the absorbance value (A1) at 420 nm. Use the LNP prepared by replacing Ben-PEG2000 with the intermediate-2 in an equimolar ratio as the positive control group, and use PBS (0.01 M) to replace LNP 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. Calculate the final grafting rate of Ben-PEG2000 in LNP according to the following formula: Grafting rate (%) = 1 - (A1 - A0) / (A2 - A0).

[0129] The results showed that the grafting rate of the final Ben-PEG2000 in LNP was detected by the TNBS method (see Figure 4), the final grafting rates of Ben-PEG2000 with molar feeding ratios of 1.125, 2.625, and 5.625 were 44.53%, 11.07%, and 23.59%, respectively. Among them, the group 1 formula with a Ben-PEG2000 molar feeding ratio of 1.125 had the highest final grafting rate of Ben-PEG2000.

[0130] Table 2 Effect of Ben-PEG2000 feeding ratio on the grafting rate of lipid nanoparticles (Ben-LNP)

[0131]

[0132] (II) Cytotoxicity evaluation of nucleic acid lipid nanoparticles (Ben-LNP) prepared under different Ben-PEG2000 feeding ratios

[0133] 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 h. The results showed (see Figure 5 ): Ben-LNP prepared in groups 1-3 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.

[0134] (III) Evaluation of transfection efficiency of nucleic acid lipid nanoparticles (Ben-LNP) prepared under different Ben-PEG2000 feeding ratios on DC2.3 and 293T cells

[0135] DC2.4 and 293T cells were diluted to 1×10 5 cells / mL and seeded in 24-well plates at 1 mL per well. The positive control group was the MC3-LNP group, and the experimental group was Ben-LNP. 400 ng of mCherry mRNA was transfected into each well. After 24 h of transfection, the culture medium was discarded, 500 μL of cell lysate was added to each well, and the cells were lysed at 1000 rpm for 20 min. The supernatant was aspirated, and the total protein content (BCA method) and the fluorescence intensity of mCherry (Ex = 590 nm, Em = 645 nm) were detected respectively. The cell transfection efficiency was detected by calculating the unit fluorescence intensity of mCherry.

[0136] The results are shown in Figure 6 : Figure 6As shown in a and b, taking the intracellular unit fluorescence intensity (RFU) as the standard, the unit fluorescence intensities transfected in DC2.4 cells in groups 1, 2, and 3 were 168481, 133295, and 94261 respectively; the unit fluorescence intensities transfected in 293T cells were 259837, 234245, and 169534 respectively, that is, the transfection efficiency of group 1 was higher. The Ben-LNP prepared by the formulation of group 1 was further compared with the commercial formulation MC3-LNP. Figure 6 As shown in c, the transfection efficiency of group 1 Ben-LNP for mCherry in DC2.4 and 293T cell lines was about 4 times and 3 times that of MC3-LNP respectively.

[0137] Comparative Example 2: Effect of different nitrogen-phosphorus ratios (N / P)

[0138] (1) Ben-PEG2000, ionizable lipid (BXA), cholesterol, DOPE, DMG-PEG2000-Man, and PEG lipid compound (Ben-PEG2000) prepared in Example 1 were dissolved in absolute ethanol according to (1.125:30:40:30:0.375 (molar ratio) to prepare an organic phase.

[0139] (2) The mRNA stock solution was diluted to the target volume with DEPC water and then mixed with the trisodium citrate solution to prepare an aqueous phase, and the final concentration of trisodium citrate in the system was 50 mM. The volume ratio of the organic phase to the aqueous phase was 1:3, and the nitrogen-phosphorus ratios (N / P) were 3:1, 4:1, and 5:1 respectively. A microfluidic system was used to rapidly mix the organic phase and the aqueous phase to prepare lipid nanoparticles.

[0140] Table 3 Different nitrogen-phosphorus ratios (N / P) in the preparation process of lipid nanoparticles

[0141]

[0142] (3) The obtained lipid nanoparticle solution was allowed to stand at room temperature for 20 min, and then dialyzed against PBS(1×) through a 14000 MWCO dialysis bag for 2 h at 4 °C to obtain a Ben-LNP solution.

[0143] Under different nitrogen-phosphorus ratios (N / P) obtained in Comparative Example 3, after the obtained nucleic acid liposome nanoparticles (Ben-LNP) were filtered and sterilized, the average particle size and PDI (polydispersity index) of Ben-LNP were characterized using a Malvern Zetasizer Nano ZS, and the encapsulation efficiency of mRNA was measured using a Ribogreen RNA quantification detection kit. The results are shown in Table 4:

[0144] Table 4 Characterization data of nucleic acid lipid nanoparticles prepared under different nitrogen-phosphorus ratios (N / P)

[0145]

[0146] The results showed that when the nitrogen-to-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 nitrogen-to-phosphorus ratio (N / P) of 3:1 was preferred.

[0147] Example 3: In vitro efficacy evaluation of nucleic acid-lipid nanoparticles (Ben-LNP)

[0148] (1) Prepare nucleic acid-lipid nanoparticles (Ben-LNP) encapsulating STING mRNA according to the optimized conditions in Example 2.

[0149] (2) Dilute the induced mouse bone marrow-derived dendritic cells (BMDC) to 5×10 5 cells / mL, seed them in a 24-well plate, and inoculate 1 mL per well. The negative control group was the PBS group, the positive control group was the MC3-LNP group, and the experimental group was Ben-LNP. After 24 h of drug stimulation, detect the contents of TNFα and IL6 in the supernatant of BMDC according to the elabscience ELISA instruction manual.

[0150] The experimental results showed (see Figure 7 ) that the average expression levels of the pro-inflammatory factors TNFα and IL6 of Ben-LNP encapsulating STING mRNA were as high as 649.04 pg / mL and 5591.49 pg / mL respectively, which were increased by 158.07% and 2245.88% respectively compared with the stimulation with the same concentration of MC3-LNP. Therefore, Ben-LNP can more effectively activate immune cells to produce TNFα and IL6, and then trigger a series of immune responses.

[0151] Example 4: In vivo efficacy and safety evaluation of nucleic acid-lipid nanoparticles (Ben-LNP)

[0152] (1) Prepare nucleic acid-lipid nanoparticles (Ben-LNP) encapsulating STING mRNA according to the optimized conditions in Example 2.

[0153] (2) Construction of pancreatic cancer model: Shave the right hind limb and back of C57BL / 6 mice, and subcutaneously inject 2×10 6 Panc02 cells into each mouse. After 8 days, the subcutaneous pancreatic cancer model of mice was successfully constructed and could be used for subsequent experiments.

[0154] (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.

[0155] Table 5 Grouping of mice bearing pancreatic cancer

[0156]

[0157] (4) HE staining detection: At the end of treatment (day 20 after administration), the mice were euthanized, and their tumor tissues and major organs, including the heart, liver, spleen, lung, and kidney, 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 cryostat, and then the cell nuclei and cytoplasm were stained with hematoxylin and eosin (HE staining) respectively. Finally, the morphological changes of the tumor tissues and cells of each organ tissue were observed under a microscope.

[0158] Results:

[0159] (I) Body weight evaluation

[0160] According to Figure 9 : During the administration period, the body weights of the mice in all groups showed a steady growth trend, indicating good biosafety.

[0161] (II) Tumor size evaluation

[0162] According to Figure 8 : During the administration period, the tumor volumes 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 both were better than those of the PBS group, MC3 group, and PD-L1 group.

[0163] (III)HE staining in vivo safety evaluation

[0164] According to Figure 10, after 20 days of dosing, HE staining histological examination was performed on the tumor tissues of each group. The nucleus 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 the main organs (heart, liver, spleen, lung, kidney) showed that there were no obvious physiological morphological changes or tissue damage in the control group or the treatment group during the dosing period, and its acute pathological toxicity and adverse reactions were negligible. The treatment with Ben-LNP+PD-L1 had no obvious side effects on the mice and had good biosafety.

[0165] All documents mentioned in the present invention are cited in this application as references, as if each document was individually cited as a reference. 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 appended claims of this application.

Claims

1. A pH-responsive PEG lipid compound based on benzoylimine bond, whose chemical structure is shown in the following formula (I): （I） Among them, The PEG is PEG-2000.

2. The PEG lipid compound according to 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 pH-responsive PEG lipid compound based on benzoylimine bond as claimed in claim 1.

4. The lipid nanoparticle according to claim 3, wherein, The lipid nanoparticles further comprise an ionizable lipid, a phospholipid, a steroid and a PEG lipid.

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

375.

6. A preparation method of the pH-responsive PEG lipid compound based on benzoylimine bond as claimed in claim 1, comprising the following steps: (1) Weigh 0.2 g of N-(tert-butoxycarbonylamino) polyethylene glycol 400-carboxylic acid (t-Boc-N-amino-PEG400-acid) and dissolve it in 5 ml of dichloromethane. Under ice bath, add 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.5 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.1 equivalent of 4-dimethylaminopyridine (DMAP), and activate it under ice bath for 1 h. Then add 1.1 equivalents of , and stir at room temperature for 48 h; (2) Wash the system obtained in step (1) with water, extract with dichloromethane, and purify by column chromatography using a dichloromethane / methanol system to obtain intermediate-1; (3) Dissolve the above intermediate-1 in dichloromethane, add 15 equivalents of trifluoroacetic acid under ice bath conditions, and stir at room temperature for 15 h; (4) Add saturated sodium bicarbonate solution to neutralize the system in step (3) under ice bath conditions, stir for 15 min, dilute and extract with dichloromethane, and rotary evaporate to obtain intermediate-2; (5) Dissolve the above intermediate-2 in an appropriate amount of N,N-dimethylformamide, add 1.5 equivalents of benzaldehyde-amido polyethylene glycol-2000, stir at room temperature in the dark under nitrogen protection for 24 h; then dialyze the system with a 2500 Da dialysis bag for 24 h, and freeze-dry 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 benzoylimine bond as claimed in claim 1 in the preparation of lipid nanoparticles.

8. A preparation method of lipid nanoparticles, comprising the following steps: (S1) Mix the ionizable lipid, cholesterol, phospholipid, PEG lipid, and the pH-responsive PEG lipid compound based on benzoylimine bond as claimed in claim 1 in proportion and uniformly dissolve them in an organic solvent to prepare an organic phase; (S2) Provide an aqueous phase containing the drug to be loaded; (S3) Rapidly mix the organic phase and the aqueous phase to prepare lipid nanoparticles; (S4) Immediately dialyze the obtained lipid nanoparticles at 2 - 8 °C for 2 - 4 hours.

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

10. Use of the lipid nanoparticles as claimed in any one of claims 3 - 5 in the preparation of a pharmaceutical composition.

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