Construction and application of lung-targeted lipid nanoparticles

Abstract

The invention relates to the technical field of lung-targeted drug delivery, and relates to a lung-targeted lipid molecule compound and a preparation method and application of lipid nanoparticles of the lung-targeted lipid molecule compound. The invention successfully develops a novel lipid nano-carrier drug delivery system through precise synthesis of lung-targeted lipid molecule compounds and preparation of lung-targeted lipid nano-particles based on the lung-targeted lipid molecule compounds. The system is mainly composed of innovatively synthesized lipoid molecules, auxiliary lipid, a stabilizer and the like, and a nano delivery platform which is stable in structure and high in targeting property is formed. Experiments prove that the system can efficiently entrap therapeutic drugs including fat-soluble and water-soluble macromolecules and micromolecules, especially mRNA, realizes accurate delivery of lung tissues, significantly improves the enrichment degree of the drugs in target organs, reduces the distribution of the drugs in non-targeted tissues, and improves the therapeutic effect of the drugs. The key factor of targeting lung tissues is a specific protein crown formed in a blood system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lung-targeted delivery of materials, in particular to lung-targeted delivery of compounds and materials, and a preparation method and application thereof. BACKGROUND

[0002] In the treatment of lung diseases, the targeting of drug delivery systems is a key factor affecting the therapeutic effect. In recent years, selective organ-targeted lipid nanoparticles (SORT LNPs) have shown significant advantages in the field of lung-targeted delivery due to their high drug encapsulation capacity and precise delivery characteristics. Through the design of specific lipid combinations, SORT LNPs can achieve high selectivity of drug accumulation in the lungs, effectively improve the bioavailability of drugs, and reduce the toxic side effects of drugs on non-target organs.

[0003] However, the SORT LNPs in the prior art face two major challenges: on the one hand, the types of SORT LNPs that can be used for lung targeting are limited, making it difficult to meet the treatment needs; on the other hand, the lung targeting mechanism of SORT LNPs has not been fully elucidated, especially in the in vivo environment, the protein corona formed on the surface of the nanocarrier has a decisive influence on its biological fate and targeting ability, but the systematic study on the composition of the protein corona on the surface of the lung-targeted SORT LNPs and its influence on the targeting efficiency is obviously insufficient, and there is a lack of in-depth exploration of understanding the structure-activity relationship from the molecular level, which seriously limits the further development of SORT LNPs in clinical applications. SUMMARY

[0004] Based on this, the present application discloses a lipid compound and a lipid nanoparticle composition containing the compound, which has the advantages of high encapsulation efficiency, high lung targeting, and low toxicity, especially low hepatotoxicity.

[0005] The application adopts the following technical solution, a lipid compound represented by formula (I):

[0006]

[0007] The dotted line represents a single bond or a double bond, when R1 is an alkyl group with 6 or 7 carbon atoms, the dotted line is a single bond, and when R1 is an alkyl group with 8 carbon atoms, the dotted line is a single bond or a double bond. R2 is one or more of an alkyl group, an alkene, or an aryl group. In the alkyl group, the alkene, or the aryl group, the carbon number is 1-12.

[0008] The present application provides a kind of lipid nanoparticle, it includes any one of the lipid compound described in claims 1-7. It is characterized in that the lipid nanoparticle includes neutral lipid, polyethylene glycol lipid, solid sterol lipid and one or more bioactive agents;Preferably, the neutral lipid is one of DSPC, DOPE, DPPC or POPC, the polyethylene glycol lipid is PEG2000DMG, the solid sterol lipid is cholesterol, and the bioactive agent is RNA, mRNA, siRNA, ASO (antisense oligonucleotide), DNA, tRNA, rRNA, miRNA, plasmid or snRNA.

[0009] The present application provides a pharmaceutical composition comprising the lipid compound or nanoparticle composition as described above, and a pharmaceutically acceptable carrier, drug.

[0010] The present application provides a method for producing a target polypeptide in a cell, the method comprising contacting the cell with the nanoparticle composition of any one of claims 3 or the pharmaceutical composition of claim 6 to deliver a therapeutic agent and / or prophylactic agent to the cell, wherein the therapeutic agent and / or prophylactic agent is mRNA encoding the polypeptide of interest and / or a pharmaceutically acceptable carrier, drug, whereby the mRNA is capable of being translated in the cell to produce the polypeptide of interest.

[0011] The present application provides a method for treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of claims 3 or the pharmaceutical composition of claim 6.

[0012] The present application provides a method for specifically delivering a therapeutic agent and / or prophylactic agent to an organ of a mammal, the method comprising administering to the mammal the nanoparticle composition of any one of claims 3 or the pharmaceutical composition of claim 6, the administration comprising contacting, feeding, injecting the mammal with the nanoparticle composition, whereby the therapeutic agent and / or prophylactic agent is delivered to the organ.

[0013] The present application provides a nanoparticle with protein crown regulation function, characterized by self-assembly of the compound of any one of claims 1 to 4 to form a protein crown in blood, body fluid or interstitial fluid. The preparation method of the protein crown is to mix the lipid nanoparticle with blood, body fluid or interstitial fluid, and then high-speed centrifugation to obtain the nanoparticle with protein crown regulation function. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Lung targeting mechanism for lipid nanoparticles.

[0015] Figure 2Luciferase protein expression after lipid nanoparticles made of different lipid compounds were delivered to A549 cells.

[0016] Figure 3 Fluorescence intensity of mice at organ sites after lipid nanoparticles made of different lipid compounds were injected into mice via tail vein, 12 hours later.

[0017] Figure 4 Flow chart of lipid nanoparticle incubation with plasma, PBS, lung / liver interstitial fluid, respectively.

[0018] Figure 5 Amount of surface adsorbed proteins.

[0019] Figure 6 Doltbot method to measure adsorption to specific proteins.

[0020] Figure 7 Isothermal titration calorimetry to determine binding affinity constant of lipid nanoparticles to albumin or IgG.

[0021] Figure 8 Molecular weight distribution profile of lipid nanoparticle adsorbed proteins.

[0022] Figure 9 Isoelectric point distribution profile of lipid nanoparticle adsorbed proteins.

[0023] Figure 10 Pictures of anti-tumor effect of lipid nanoparticle loaded with organic arsenical drugs.

[0024] Figure 11 Pictures of H&E staining of lipid nanoparticle loaded with organic arsenical drugs. DETAILED DESCRIPTION

[0025] TERMS

[0026] The term "alkyl" refers to an optionally substituted straight chain or branched chain saturated hydrocarbon comprising one or more carbon atoms.

[0027] The term "oleylamine" refers to an alkyl group as described herein, an unsaturated hydrocarbon chain containing 18 carbon atoms, having one carbon-carbon double bond at an intermediate position in the carbon chain (typically at C9-C10), with an amino (-NH2) functional group attached at the terminal end.

[0028] The term "RNA" refers to a ribonucleic acid that can be naturally occurring or non-naturally occurring. RNA includes, but is not limited to: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA, and a mixture of RNAs.

[0029] The term "nucleic acid" refers to a molecule comprising at least one phosphate backbone and at least one heteroatom. The heteroatom can be, but is not limited to, nitrogen, oxygen, and sulfur. The nucleic acid can be, but is not limited to, DNA, RNA, and a mixture of DNA and RNA.

[0029] The term "lipid component" refers to a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more cationic or ionizable lipids, pegylated lipids, structural lipids, or other lipids such as phospholipids.

[0030] The term "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system. The term "particle size" refers to the average diameter of a nanoparticle composition. The term "zeta potential" refers to the surface potential of lipids in a nanoparticle composition.

[0031] The term "encapsulation efficiency" refers to the ratio of the amount of therapeutic or prophylactic agent that becomes part of the nanoparticle composition to the initial total amount of therapeutic or prophylactic agent used to prepare the nanoparticle composition.

[0032] The term "delivery" refers to providing an entity to a target. For example, delivering a therapeutic or prophylactic agent to a subject can involve administering a nanoparticle composition comprising the therapeutic or prophylactic agent to the subject (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cell can involve contacting one or more cells with the nanoparticle composition.

[0033] The term "target cell" refers to one or a group of target cells. These cells can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal.

[0034] The term "expression" refers to the translation of mRNA into a polypeptide or protein and / or the polypeptide or protein.

[0035] The term "subject" refers to a target to which a treatment is intended to be applied, including but not limited to humans, other primates and other mammals, such as cows, pigs, horses, sheep, cats, dogs, mice or rats. Preferably, the subject can be a mammal, particularly a human.

[0036] lipid compounds

[0037] In one aspect, the present invention provides a lipid compound having a structure as shown in formula (I):

[0038] Dashed lines represent single or double bonds. When R1 is an alkyl group with 6 or 7 carbon atoms, the dashed line represents a single bond. When R1 is an alkyl group with 8 carbon atoms, the dashed line represents a single or double bond. R2 is one or more of an alkyl group, an alkene group, or an aryl group. The alkyl group, alkene group, or aryl group has 1 to 12 carbon atoms.

[0039] In a specific embodiment, the lipid compound of the present application comprises:

[0040]

[0041] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0042] The lipid compounds of the present invention (including lipid compounds of formula N14, N16, N18 and N18O) are ionizable cationic compounds, wherein the tertiary amine (H3C-N) portion can be protonated at less than physiological pH. This series of lipids is also a zwitterionic compound, having a protonable tertiary amine center and a long-chain alkyl group connected by an amide (C=O-NH). The compounds N14, N16 and N18 contain saturated alkyl chains of 14 carbons, 16 carbons and 18 carbons, respectively, and N180 contains an unsaturated alkyl chain, each of which contains a carbon-carbon double bond (C=C). Regardless of whether they are charged or not, such zwitterionic compounds are included within the scope of the present invention. The structural feature of the lipid compound is that the central tertiary amine is connected to two amide groups, each of which is further connected to a long-chain alkyl group, forming a symmetrical or nearly symmetrical molecular configuration, which gives it unique physicochemical properties and biological functions.

[0043] Nanoparticle compositions

[0044] The present invention relates to a lipid nanoparticle, which comprises the lipid compound of the present invention and may further comprise one or more other lipids.

[0045] In addition to the lipids of the present invention, such as the lipids of formula (I), the nanoparticle composition may also contain one or more cationic and / or ionizable lipids, including but not limited to 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDMA), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dilinoleoyl-N-[2-(4-(2-aminoethyl)piperazin-1-yl)ethyl]-N,N-dimethylamine (DLin KC2 DMA), 3β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC Chol), and the like.

[0046] phospholipids

[0047] The phospholipid is selected from one or more of the following: dipalmitoyl phosphatidyl choline (DPPC), dilauroylphosphatidyl choline (DLPC), dimyristoyl phosphatidyl choline (DMPC), dioleoylphosphatidyl choline (DOPC), distearoylphosphatidyl choline (DSPC), dioleoylphosphatidyl choline (DUPC), palmitoyloleoylphosphatidyl choline (POPC), 1, 2 dioleoyl SN glycerol 3 phosphoethanolamine (DOPE), 1, 2 diolyl sn glycerol 3 phosphate (1 glycerol) sodium salt (DOPG), or sphingomyelin.

[0048] PEG lipids

[0049] The lipid component of the nanoparticle composition can also include one or more PEG or PEG-modified lipids. PEG lipids can include, but are not limited to, polyethylene glycol- dioleoyl-sn-glycerol-3-phosphoester (PEG c DOMG), polyethylene glycol-dimyristoylglycerol (PEG DMG), polyethylene glycol-dilauroylphosphatidyl ethanolamine (PEG DLPE), polyethylene glycol-dimyristoylphosphatidyl ethanolamine (PEG DMPE), polyethylene glycol-dipalmitoylphosphatidyl choline (PEG DPPC), or polyethylene glycol-distearoylphosphatidyl ethanolamine (PEG DSPE) lipids.

[0050] Structural lipids

[0051] The lipid component of the nanoparticle composition can also include one or more structural lipids. Structural lipids can include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, soysterol, brassicasterol, tomatidine, tomatin, ursolic acid, alpha tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol.

[0052] Inventive effects

[0053] The present disclosure provides a series of structurally novel lipid compounds, and other lipid compounds assembled to prepare a lipid nanoparticle delivery system. These carriers have significant lung targeting ability, improve the lung targeting ability of non-targeted drug molecules or mRNA, improve the anti-tumor treatment efficiency of anti-tumor molecules and reduce their toxic side effects. Through high-throughput proteomics analysis and testing, the present disclosure systematically studies the evolution law of the protein corona formed by the nanoparticles in different body fluid environments, identifies the key protein components closely related to lung targeting, and accurately determines the interaction mechanism between the nanoparticles and the key plasma proteins by isothermal titration calorimetry, elucidates the protein corona formation mechanism and its structure-activity relationship for lung targeted delivery from the molecular level, and provides an innovative solution and theoretical basis for the precise treatment of lung diseases.

[0054] Examples

[0055] This article uses the following abbreviations: A14: Tetradecylamine; A16: Hexadecylamine; A18: Octadecylamine; A18O: Oleylamine; AD14: Tetradecylacrylamide; AD16: Hexadecylacrylamide; AD18: Octadecylacrylamide; AD18O: Oleylacrylamide

[0056] Example 1: Synthesis of compounds

[0057] (a) Dissolve 2 mmol of long-chain fatty amine (A14-A18) in dichloromethane and add triethylamine as an acid-binding agent. Slowly add a dichloromethane solution of acryloyl chloride dropwise and stir for 3 hours. After completion of the reaction, purify by column chromatography to obtain the desired AD-alkylacrylamide product.

[0058] (b) AD-alkyl acrylamides (AD14-AD18) were mixed with N,N-bis(3-aminopropyl)methylamine and allowed to react for 7 days. During the reaction, the amino group in the diamine molecule acts as a nucleophile to attack the β-carbon of the α,β-unsaturated amide, forming a new carbon-nitrogen bond via a Michael addition mechanism.

[0059] Characterization of N14

[0060] 1 H NMR (400MHz, CDCl3) δ: 7.56-7.53 (t, J ＝ 8.0Hz, 2H), 3.24-3.19 (q, J=8.0Hz, 4H), 2.88-2.85 (t, J=8.0Hz, 4H), 2.67-2.64 (t, J=8.0Hz, 4H), 2.4 0-2.34(m, 8H), 2.20(s, 3H), 1.69-1.62(m, 4H), 1.50-1.45(m, 4H), 1.30-1.25(m, 32H), 0.90-0.86(t,J ＝ 8.0Hz,6H).

[0061] 13 C NMR (100MHz, CDCl3) δ: 172.48, 56.20, 48.12, 45.78, 42.20, 39.20, 35.64, 31 .94, 29.71, 29.68, 29.63, 29.61, 29.38, 29.36, 27.45, 27.08, 22.71, 14.15.

[0062] Characterization of N16

[0063] 1H NMR(400MHz,,CDCl3)δ:7.51-7.47(t,J=8.0Hz,2H),3.24-3.19(q,J=8.0Hz,4H),2.87-2.84(t,J=8.0Hz,4H),2.67-2.63(t,J=8.0Hz,4H),2.39-2.33(m,8H),2.19(s,3H),1.69-1.62(m,4H),1.52-1.45(m,4H),1.30-1.25(m,32H),0.90-0.86(t,J=8.0Hz,6H).

[0064] 13 C NMR(100MHz,CDCl3)δ:172.50,56.17,48.10,45.79,42.19,39.22,35.70,31.93,29.71,29.68,29.67,29.64,29.62,29.60,29.37,29.35,27.50,27.07,22.70,14.13.

[0065] N18的表征

[0066] 1 H NMR(400MHz,CDCl3)δ:7.53-7.49(t,J ＝ 8.0Hz,2H),3.24-3.19(q,J=8.0Hz,4H),2.87-2.84(t,J=8.0Hz,4H),2.67-2.63(t,J=8.0Hz,4H),2.40-2.35(m,8H),2.33(s,3H),1.67-1.64(m,4H),1.50-1.47(m,4H),1.30-1.25(m,48H),0.90-0.86(t,J=8.0Hz,6H).

[0067] 13 C NMR(100MHz,CDCl3)δ:172.49,56.16,48.10,45.78,42.19,39.23,35.69,31.93,29.71,29.69,29.67,29.64,29.62,29.60,29.37,29.35,27.47,27.07,24.07,14.13.

[0068] N18O的表征

[0069] 1 H NMR(400MHz,CDCl3)δ:7.56-7.53(t,J ＝8.0 Hz, 2H), 3.24-3.19 (q, J = 8.0 Hz, 4H), 2.88-2.85 (t, J = 8.0 Hz, 4H), 2.67-2.64 (t, J = 8.0 Hz, 4H), 2.40-2.34 (m, 8H), 2.20 (s, 3H), 1.69-1.64 (m, 4H), 1.50-1.45 (m, 4H), 1.30-1.25 (m, 32H), 0.90-0.86 (t, J = 8.0 Hz, 6H). ＝ 8.0 Hz, 4H), 2.40-2.34 (m, 8H), 2.20 (s, 3H), 1.69-1.64 (m, 4H), 1.50-1.45 (m, 4H), 1.30-1.25 (m, 32H), 0.90-0.86 (t, J = 8.0 Hz, 6H).

[0070] 13 C NMR (100 MHz, CDC13) δ: 172.49, 130.00, 129.78, 58.40, 56.18, 48.11, 45.77, 42.20, 39.21, 35.63, 31.94, 29.78, 29.69, 29.63, 29.60, 29.53, 29.38, 29.34, 29.29, 27.43, 27.23, 27.08, 22.71, 18.45, 14.15.

[0071] Example 2: Preparation of Lipid Nanoparticles

[0072] Preparation of Lipid Solution: N-series compound: DSPC: Cholesterol: mPEG2000 DMG were dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5, the organic solvent was removed by rotary evaporation to form a uniform film, 15 mL PBS was added and hydrated at 40°C for 1 h, and then extruded through a membrane extruder (e.g. polycarbonate membrane with a pore size of 100 nm) to obtain lipid nanoparticles.

[0073] Preparation of mRNA solution: A certain amount of luciferase mRNA was dissolved in 20 mM citric acid buffer solution.

[0074] Preparation of Lipid Nanoparticles: The luciferase mRNA and lipid solution were mixed and stirred vigorously under ice bath conditions:

[0075] Solution replacement: The lipid nanoparticle solution was added to an ultrafiltration tube and centrifugal ultrafiltration was performed, and the phosphate buffer was replaced several times to obtain the finished product.

[0076] Example 3: Characterization of Lipid Nanoparticles

[0077] The particle size, polydispersity index (PDI) and zeta potential of the nanoparticle composition were determined using a Zetasizer Nano ZS.

[0078] Table 1. Physicochemical characterization of lipid nanoparticles prepared from lipid compounds synthesized in the examples of the present application.

[0079]

[0080] Example 4: In vitro cell expression detection

[0081] In vitro cell expression evaluation method: A549 cells were cultured in 96-well plates. After 24 hours of culture, the culture medium was replaced with cDMEM or serum-free DMEM, and FLuc mRNA lipid nanoparticles were added. After transfection treatment, fresh cDMEM was replaced for continued culture. Analytical reagents were added, and the luciferase (FLuc) expression level was detected using a multi-mode enzyme marker.

[0082] As a delivery carrier, the lipid nanoparticle significantly improved the transfection efficiency of luciferase Fluc mRNA and its protein expression level, and the results are shown in Figure 1 .

[0083] Example 5: In vivo protein expression study in mice

[0084] After the lipid nanoparticles were injected into the mouse body through the tail vein, the time course of protein expression could be evaluated by bioluminescence imaging. By comparing with the positive control, the amount of translated protein was compared to evaluate the targeted delivery performance of the lipid nanoparticles. Specifically, four kinds of lipid nanoparticles containing Fluc mRNA were injected into Balb / C mice through the tail vein, 6 hours, the mice were injected with D-luciferin substrate in the abdominal cavity, 15 minutes later, placed under the small animal living imaging instrument for whole body bioluminescence imaging, and the main organs were imaged ex vivo to detect the fluorescence intensity.

[0085] The strongest fluorescence signal was detected in the lung tissue of the lipid nanoparticles, and the fluorescence signal was weak in other tissues, indicating that the four kinds of lipid nanoparticles all showed significant lung targeting.

[0086] The results are shown in Figure 2 .

[0087] Example 6: Evolution of protein crown

[0088] By constructing a whole process simulation system for in vivo delivery of lipid nanoparticles: (1) Circulatory system module: the lipid nanoparticles were incubated in plasma for 1 hour to simulate the initial state of the stable protein crown formed by the nanocarrier in the blood circulation; (ii) Tissue penetration module: incubated in pH 7.4 PBS buffer for 60 minutes with continuous shaking to simulate the nanocarrier crossing the vascular endothelium; (iii) Targeting positioning module: lung interstitial fluid model and liver interstitial fluid model were selected for 1 hour continuous incubation to simulate the dynamic retention process of the nanocarrier in the organ microenvironment. This method systematically simulates the whole process of drug delivery of lipid nanoparticles in vivo, providing a reliable in vitro research model for evaluating the efficiency of nanomedicine delivery.

[0089] See the flow chart Figure 3 .

[0090] Protein concentration was detected by BCA Protein Assay Kit using a microplate reader.

[0091] See the results Figure 4 .

[0092] After the circulation system module (plasma incubation), the surface protein content of the lipid nanoparticles decreased in the tissue penetration module (PBS incubation), mainly due to the low protein concentration in PBS and the shedding of part of the surface proteins. In the targeted positioning module (lung / liver interstitial fluid incubation), the surface protein content increased again, which was attributed to the high protein concentration in the interstitial fluid and the high-affinity proteins not only competitively adsorbed but also served as a scaffold to promote the binding of more proteins.

[0093] Dot blot method: take the sample, cook for 5 min at 95℃. After activating the PVDF membrane with methanol for 30 s, wash it with TBST, and vertically spot at the pre-marked site. Block, incubate the primary antibody, incubate the secondary antibody, absorb the residual liquid with filter paper, add ECL developing solution, and use the chemiluminescence imaging system to collect the signal.

[0094] See the results Figure 5 .

[0095] Based on the protein adsorption experiment data, it can be found that the protein adsorption behavior on the material surface presents a significant modular response rule: in the circulation system module stage, the adsorption amount of albumin and IgG reaches the peak, which may be related to the high protein concentration in the blood circulation system promoting adsorption; after the tissue penetration module (PBS dynamic displacement) treatment, the surface protein load decreases, indicating that the dynamic displacement of physiological buffer can effectively remove non-specific adsorbed proteins; finally, after the targeted positioning module (lung / liver interstitial fluid incubation), the protein adsorption amount increases again, showing the active adsorption mechanism induced by the interstitial microenvironment.

[0096] Example 7: Study on the interaction mechanism between lipid nanoparticles and proteins

[0097] To explore the interaction between LNPs and BSA and IgG, isothermal titration calorimetry (ITC) was further studied. In the ITC experiment, BSA or IgG (as titrant) was added dropwise into the solution of lipid nanoparticles (as sample), and the heat change in the reaction process was monitored in real time, and the thermodynamic parameters such as binding constant (K a ), enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy change (ΔG) were calculated.

[0098] The binding process between L14 lipid nanoparticles and albumin showed biphasic characteristics: the initial stage was mainly driven by electrostatic force, and then shifted to a binding mode dominated by hydrophobic interaction. In contrast, the interaction mechanism between L16 lipid nanoparticles and albumin was mainly driven by electrostatic interaction. The binding process of L14 with immunoglobulin G (IgG) was dominated by electrostatic interaction. See Table 1 for results. Figure 6 .

[0099] The interaction of L18 and L18O lipid nanoparticles with albumin was hydrogen bond interaction, and the interaction of L16, L18 and L18O lipid nanoparticles with immunoglobulin G (IgG) was electrostatic interaction. See Table 2 for statistical structure.

[0100]

[0101] Example 8: Protein corona formed in different body fluid microenvironments is different.

[0102] By incubating lipid nanoparticles with plasma, lung interstitial fluid, and liver interstitial fluid, respectively (37°C, 1 hour), protein corona formed by lipid nanoparticles in different body fluid microenvironments was prepared. After incubation, the protein-LNP complex was separated by centrifugation and washed to remove unbound proteins.

[0103] The particle size and zeta potential of the protein-lipid nanoparticle complex were determined using a Zetasizer Nano ZS.

[0104] After incubation with tissue fluid, the particle size of each LNP increased significantly.

[0105] Results are shown in Table 3.

[0106]

[0107] The surface potential of LNPs changed significantly after forming a protein corona.

[0108] Results are shown in Table 4.

[0109]

[0110] The amount of protein adsorption was detected using BCA protein concentration assay.

[0111] LNPs exhibit differential protein corona formation characteristics in different biological environments.

[0112] Results are shown in Table 5.

[0113] Proteomics analysis:

[0114] Body fluid pretreatment: high-speed centrifugation was used to remove protein aggregates. 0.5 mL of lipid nanoparticles was taken, and 0.5 mL of body fluid (serum, pulmonary interstitial fluid, hepatic interstitial fluid) was added, and shaken at 37°C for 1 h. Gradient centrifugation was used to obtain purified nanoparticle-protein corona complexes. The protein concentration was determined.

[0115] Proteomic analysis showed that the protein corona formed by LNPs in the three biological environments had obvious composition specificity.

[0116] The molecular weight characteristics results are shown in Table 1. Figure 7 .

[0117] The isoelectric point characteristics results are shown in Table 2. Figure 8 .

[0118] The surface high-abundance protein results are shown in Table 6.

[0119]

[0120] Example 9 Anti-tumor application.

[0121] The anti-tumor drug PFZ2 independently developed by the research group was coated in lipid nanoparticles, and the anti-lung tumor ability of the drug-loaded nanoparticles was detected.

[0122] The results are shown in Table 7. Figure 9 The lipid nanoparticles themselves do not have anti-tumor activity, and the anti-tumor drug PFZ2 has certain anti-tumor activity. The PFZ2-loaded lipid nanoparticles have good anti-lung tumor effect, and the tumor inhibition rate reaches more than 90% compared with the control.

[0123] The lipid nanoparticles, the anti-tumor drug PFZ2, and the PFZ2-loaded lipid nanoparticles have good biocompatibility, and have no obvious toxic and side effects on organs and circulatory systems, and the results are shown in Table 8. Figure 10 .

[0124] The above description of the embodiments is to facilitate the understanding and application of the application by those of ordinary skill in the art. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to pay creative labor. Therefore, the present application is not limited to the embodiments herein, and any modifications, equivalent replacements, improvements, etc. made by those skilled in the art based on the disclosure of the present application without departing from the scope and spirit of the present application shall be included in the protection scope of the present application.

Claims

1. A compound, characterized in that The structural formula is shown in Formula 1: The dotted line represents a single bond or a double bond. When R1 is an alkyl group with 6 or 7 carbon atoms, the dotted line represents a single bond. When R1 is an alkyl group with 8 carbon atoms, the dotted line represents a single bond or a double bond. R2 is one or more of an alkyl group, an alkene group, or an aryl group.

2. A compound according to claim 1, characterized in that The alkyl, alkene or aryl group has 1 to 12 carbon atoms.

3. A nanoparticle composition comprising a lipid component, wherein the lipid component comprises the compound of any one of claims 1 or 2.

4. The lipid nanoparticle according to claim 3, characterized in that The lipid nanoparticles contain neutral lipids, polyethylene glycol lipids, sterol lipids and one or more bioactive agents; preferably, the neutral lipid is one of DSPC, DOPE, DPPC or POPC, the polyethylene glycol lipid is PEG2000 DMG, the sterol lipid is cholesterol, and the bioactive agent is RNA, mRNA, siRNA, ASO (antisense oligonucleotide), DNA, tRNA, rRNA, miRNA, plasmid or snRNA.

5. Use of the compound according to any one of claims 1 or 2 in the preparation of a lipid nanoparticle composition.

6. A pharmaceutical composition comprising the nanoparticle composition according to any one of claims 3 or 4 and a pharmaceutically acceptable carrier and a drug.

7. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition of any one of claim 3 or the pharmaceutical composition of claim 6 to deliver a therapeutic and / or prophylactic agent to the cell, wherein the therapeutic and / or prophylactic agent is mRNA and / or a pharmaceutically acceptable carrier, a drug, and the mRNA encodes the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

8. A method for treating a disease or disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of claim 3 or the pharmaceutical composition of claim 6.

9. A method for specifically delivering a therapeutic and / or prophylactic agent to an organ of a mammal, the method comprising administering the nanoparticle composition of any one of claim 3 or the pharmaceutical composition of claim 6 to the mammal, wherein the administration comprises contacting, feeding, or injecting the mammal with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the organ.

10. A nanoparticle with protein corona regulation function, characterized in that: It is obtained by self-assembly of the compound according to any one of claims 1 to 4 and can form a protein corona in blood, body fluids or interstitial fluids.

11. The method for preparing nanoparticles with protein corona regulation function according to claim 10, characterized in that: The method comprises the following steps: mixing the lipid nanoparticles according to claim 3 with blood, body fluids or interstitial fluid, and then subjecting the mixture to high-speed centrifugation to obtain nanoparticles with protein corona regulating function.

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