Ionizable lipid compound with three-dimensional space structure as well as preparation method and application of ionizable lipid compound

Abstract

The invention relates to the technical field of nano delivery carriers, in particular to an ionizable lipid compound with a three-dimensional space structure and a preparation method and application thereof. The ionizable lipid compound provided by the invention comprises a three-dimensional space structure constructed by a polar head group, a connecting bond and a hydrophobic tail. Diversified ionizable lipid compounds with different three-dimensional space structures are constructed by changing various polar head groups, connecting bonds and hydrophobic tails contained in an ionizable lipid compound combinatorial library, and the ionizable lipid compounds have excellent mRNA (messenger ribonucleic acid) binding capacity. The invention also provides preparation and application of the ionizable lipid compound with the advantageous three-dimensional space structure. The novel ionizable lipid compound with an advantageous three-dimensional space structure has stronger binding capacity with mRNA, and lipid nanoparticles synthesized from the novel ionizable lipid compound have excellent physicochemical characteristics, high cell uptake rate, lysosome escape rate and cell and in-vivo transfection rate, and have the function of specifically targeting liver, spleen or lung.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-delivery carriers, and in particular to an ionizable lipid compound with an advantageous three-dimensional spatial structure and a preparation method and application thereof. Background Art

[0002] Benefiting from the successful development of two mRNA vaccines for COVID-19 (Moderna's mRNA-1273 and Pfizer / BioNTech's BNT162b2), mRNA-based therapies have gained great attention in the medical community. This has also prompted people to explore a variety of mRNA-based therapies, including vaccines, gene editing, protein replacement therapy, and cell therapy, and has promoted some therapies into the clinical exploration stage. However, the in vivo delivery of mRNA remains one of the important issues that need to be urgently addressed for clinical applications. Developing new delivery carriers to overcome various biological barriers, achieve precise and targeted delivery to specific organs, tissues or cells, and further improve the efficiency of lysosomal escape is an effective strategy to solve this problem. In the past few decades, lipid nanoparticles (LNPs) have become the main choice for mRNA delivery and have been widely used in clinical research. The key component of LNPs is an ionizable lipid molecule, which usually includes a three-part structure, a nitrogen-containing polar head group for mRNA binding, a hydrophobic tail for promoting self-assembly, and a connecting bond for linking the head and tail. The properties of ionizable lipids are crucial for improving the lysosomal escape rate of LNPs. Studies have shown that changes in polar head groups, linkers, and hydrophobic tails can greatly change the delivery efficiency of LNPs and their organ / tissue-specific targeting. However, there is limited research on how the spatial arrangement of the head and tail in these ionizable lipid compounds affects the delivery efficiency of mRNA and its targeted expression in specific organs or cells. There is an urgent need to understand the effects of ionizable lipid molecules and their three-dimensional spatial structures on their interactions with mRNA and their ability to specifically target specific organs, so as to improve the design of LNPs and enhance the efficacy of mRNA therapy. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a novel ionizable lipid compound with an advantageous three-dimensional spatial structure, and a preparation method and application thereof.

[0004] The present invention constructs diversified ionizable lipid compounds with different three-dimensional structures by changing the various polar head groups, connecting bonds, and hydrophobic tails contained in the ionizable lipid compound combinatorial library, and has excellent mRNA binding ability. The novel ionizable lipid compound with an advantageous three-dimensional spatial structure has a stronger mRNA binding ability, and the lipid nanoparticles synthesized therefrom have excellent physical and chemical characteristics, high cell uptake rate, lysosomal escape rate, cell and in vivo transfection rate, and the functional effect of specifically targeting the liver, spleen or lungs.

[0005] In the first aspect, the ionizable lipid compound with an advantageous three-dimensional spatial structure provided by the present invention comprises a three-dimensional spatial structure constructed by a polar head group, a connecting bond and a hydrophobic tail; the polar head group has an N number of 1 to 5, and the polar head group contains -CH3, -CH2CH3, -OH, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, , , , One or more of; the hydrophobic tail comprises one or more of a fully saturated tail chain, an unsaturated tail chain, a branched structure tail chain and a heteroatom-doped tail chain.

[0006] The ionizable lipid compound combinatorial library created by the present invention constructs diversified ionizable lipids with different three-dimensional spatial structures by changing the polar head group, degradable linker, and hydrophobic tail. The present invention also analyzes the three-dimensional spatial structure of the ionizable lipid molecules, and through high-throughput screening and molecular dynamics simulation, it is found that the excellent three-dimensional spatial structure can mediate the ionizable lipids to not only mediate the efficient binding of the ionizable lipids with mRNA and lysosomal escape, but also promote specific organ (liver, spleen or lung) targeting.

[0007] Preferably, the polar head group is any one of formulas H1-H14.

[0008] .

[0009] Preferably, the carbon chain length of the fully saturated tail chain is C6-C18; and / or the unsaturation degree of the unsaturated tail chain is 1 or 2; and / or the branch chain lengths of the branched structure tail chain are equal or unequal; and / or the heteroatom of the heteroatom-doped tail chain is O, S or SS.

[0010] More preferably, the hydrophobic tail is any one of formulas T1-T16.

[0011] .

[0012] Further preferably, the number of hydrophobic tails of the ionizable lipid compound is 1 to 4 (1T, 2T, 3T, 4T); and / or, the link is a degradable link, and the degradable link includes an ester bond ( ) or amide bond ( ).

[0013] The ionizable lipid compound provided by the present invention has polar head groups with different structures, degradable connecting bonds, and free combination of hydrophobic tails ( Figure 1 ) can construct ionizable lipids with diverse three-dimensional structures ( Figure 2 ). For example, the number of N in the polar head group (1, 2, 3, 4, 5), the number of hydrophilic and hydrophobic groups (-CH3, -CH2CH3, -OH, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, ), functional groups ( , , ). Connecting bonds such as ester bonds ( ) and amide bonds ( ). The hydrophobic tail includes a fully saturated tail chain (C6-C18 length), an unsaturated tail chain (unsaturation degree 1, 2), a branched structure tail chain, and a heteroatom doped tail chain (O, S, SS). And the number of hydrophobic tail chains (1T, 2T, 3T, 4T). The novel ionizable lipid compound with high transfection efficiency provided by the present invention has an advantageous three-dimensional spatial structure, which usually has a polar head group exposed to the outside, and a three-dimensional spatial structural feature with connecting bonds and hydrophobic tails facing the other side, which can improve the binding force to mRNA in the spatial structure, promote better cell uptake and lysosomal escape, and thus improve the cell or in vivo transfection efficiency.

[0014] Further preferably, the ionizable lipid compound is one of Lipid 1 to Lipid 116, and the structures of Lipid 1 to Lipid 116 are shown in the embodiments of the present invention (Table 1).

[0015] The present invention can construct a variety of ionizable lipids with different three-dimensional spatial structures by changing the polar head group, degradable linking bond, and hydrophobic tail. The ionizable lipid compound combination library contains 14 polar head groups, 2 degradable linking bonds (ester bonds and amide bonds), and 16 hydrophobic tails, which are freely combined into a new type of ionizable lipid compound. Through molecular dynamics simulation, the present invention converts the two-dimensional structure of the molecule into a three-dimensional spatial structure under free state conditions in an aqueous environment. The present invention connects the two-dimensional structure of the ionizable lipid compound molecule to the three-dimensional spatial structure to the delivery efficiency and organ targeting. It is also proposed that the three-dimensional spatial structure of the ionizable lipid compound can mediate the efficient delivery efficiency and organ targeting of the ionizable lipid. The lipid compound with an excellent three-dimensional spatial structure provided by the present invention has better cell uptake rate, lysosomal escape rate, and cell and in vivo transfection rate, and its lipid nanoparticles have the function of specifically targeting the liver, spleen or lungs.

[0016] It is further preferred that the polar head group in the three-dimensional structure is exposed to one side, and the connecting bond and the hydrophobic tail are facing the other side; preferably, the three-dimensional structure includes a cone, an inverted cone, a quasi-cone, a truncated cone, a wedge, a bullet, a trumpet, a spire, a spire, a pointed tip, a needle, and a funnel. The present invention constructs an ionizable lipid with a diverse three-dimensional structure by freely combining polar head groups, connecting bonds, and hydrophobic tails of different structures. Through molecular dynamics simulation, the present invention can convert the two-dimensional structure information of the molecule into three-dimensional structure information. The ionizable lipid compound has a three-dimensional structure formed by the polar head group being exposed, the connecting bond and the hydrophobic tail facing the other side. Advantageous three-dimensional spatial structures provided in the present invention include: conical shape, inverted conical shape, cone-like shape, circular truncated cone shape, wedge shape, bullet shape, horn shape, spire shape, steeple shape, tipped shape, needle shape, and funnel shape.

[0017] According to the present invention, lipid nanoparticles composed of novel ionizable lipid compounds and other commercial auxiliary lipids, such as DSPC / DOPE, cholesterol, DMG-PEG2000, etc., are used to deliver firefly luciferase mRNA (mFluc), and ionizable lipid compounds with high transfection efficiency are screened by in vitro cell transfection experiments. Ionizable lipids with advantageous three-dimensional spatial structures have better delivery efficiency, and their three-dimensional spatial structures have common characteristics. The present invention found that the three-dimensional spatial structural characteristics formed by the polar head group being exposed to the outside and the connecting bond and the hydrophobic tail facing the other side have a high transfection effect ( Figure 3 ). In the present invention, the ionizable lipids with advantageous three-dimensional spatial structures in the novel ionizable lipid compound combinatorial library have a more excellent ability to bind to mRNA, and the lipid nanoparticles (LNPs) synthesized therefrom deliver firefly luciferase mRNA (mFluc), and the LNPs have excellent physicochemical characteristics (including particle size (size), particle dispersibility index (PDI), encapsulation efficiency (EE%), etc.), higher cellular uptake rate and lysosomal escape rate, and high in vivo transfection efficiency. The novel ionizable lipid compound with advantageous three-dimensional spatial structure provided by the present invention, the synthesized lipid nanoparticles (LNPs) thereof have the ability to specifically adsorb proteins to form different protein coronas, and the protein coronas mediate specific organ targeting.

[0018] In a second aspect, the present invention provides a method for preparing the ionizable lipid compound having an advantageous three-dimensional spatial structure, comprising the following steps.

[0019]

[0020] 1) Esterification reaction: The hydroxyl group of alcohol compound A1 is esterified in the presence of acryloyl chloride to obtain compound A2.

[0021] 2) Michael addition reaction: the compound A2 is mixed with a polar head group amine and subjected to Michael addition reaction to obtain an ionizable lipid compound A3 having a three-dimensional spatial structure.

[0022] Preferably, the method for preparing the ionizable lipid compound with a three-dimensional structure provided by the present invention comprises the following steps.

[0023]

[0024] 1) Amidation reaction: The amine group of the amine compound B1 is amidated in the presence of acryloyl chloride to obtain compound B2.

[0025] 2) Michael addition reaction: the compound B2 is mixed with a polar head group amine and subjected to Michael addition reaction to obtain an ionizable lipid compound B3 having a three-dimensional spatial structure.

[0026] The advantageous three-dimensional spatial structure ionizable lipid compounds of the present invention can be synthesized using existing methods in the art, for example, using one equivalent or more of an amine polar head group and two equivalents or more of a hydrophobic tail chain compound under appropriate conditions through a Michael addition reaction. The synthesis of the ionizable lipid compound is carried out in the absence of a solvent, and the synthesis can be carried out at a higher temperature in the range of 60-120°C. The obtained ionizable lipid compound can be optionally purified. For example, a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound, such as a product containing three hydrophobic tail chains (3T). The amine polar head group and the hydrophobic tail chain compound can be purchased commercially or prepared synthetically.

[0027] According to the present invention, examples of solvents used in esterification or amidation reactions include, but are not limited to, halogenated hydrocarbons (such as ultra-dry dichloromethane, ultra-dry dichloroethane, and ultra-dry chloroform), hydrocarbons (such as ultra-dry n-pentane, benzene, and toluene), nitriles (such as ultra-dry acetonitrile), and mixed solvents of two or more of these solvents. The Michael addition reaction may choose not to use a solvent. Amines may use N-methyl-2,2'-diaminodiethylamine, etc. The raw materials in the preparation method of the present invention may be commercially available or may be synthesized using conventional methods.

[0028] In a third aspect, the present invention provides an application of an ionizable lipid compound having a three-dimensional spatial structure in the preparation of a nucleic acid delivery system, wherein the delivery system is a lipid nanoparticle, wherein the lipid nanoparticle contains the ionizable lipid compound having a three-dimensional spatial structure or the ionizable lipid compound having a three-dimensional spatial structure obtained by the preparation method. The nucleic acid includes but is not limited to DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), endogenous microRNA (miRNA), messenger RNA (mRNA), RNA aptamer, small activating RNA (saRNA), etc.

[0029] In a fourth aspect, the present invention also provides a lipid nanoparticle, wherein the lipid nanoparticle contains the above-mentioned ionizable lipid compound with a three-dimensional spatial structure or the ionizable lipid compound with a three-dimensional spatial structure obtained by the above-mentioned preparation method; wherein the lipid nanoparticle also contains: 20-60 mol% of the ionizable lipid compound, 5-30 mol% of neutral lipid molecules, 30-60 mol% of cholesterol lipid molecules, and 0.5-5 mol% of PEGylated lipid molecules; more preferably, 35-48 mol% of the ionizable lipid compound, 10-15 mol% of neutral lipid molecules (DOPE), 35-50 mol% of cholesterol lipid molecules (cholesterol), and 1-2 mol% of PEGylated lipid molecules (DMG-PEG2000). Further preferably, the neutral lipid molecule is dioleoylphosphatidylethanolamine (DOPE), the cholesterol lipid molecule is preferably cholesterol, and the PEGylated lipid molecule is preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000). Preferably, the molar ratio of the novel ionizable lipid molecule with a dominant three-dimensional spatial structure, DOPE, cholesterol and DMG-PEG2000 is 45:15:38.5:1.5. Preferably, the molar ratio of the novel ionizable lipid molecule with a dominant three-dimensional spatial structure, DOPE, cholesterol and DMG-PEG2000 is 40:10:48.5:1.5. The present invention has discovered the potential biological mechanism of the three-dimensional structure of ionizable lipid compounds for promoting organ-targeted delivery. The most important thing is that the lipid nanoparticles composed of the dominant three-dimensional spatial structure ionizable lipids have a unique three-dimensional spatial structure that can mediate their adsorption of specific protein coronas in vivo, and then specifically target the liver and / or spleen and / or lungs through the action of the protein corona. The present invention provides a lipid compound and a delivery system for targeted delivery with excellent specific targeting effect, which can more efficiently deliver mRNA to target organs / cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 A schematic diagram of the polar head group, connecting bond, hydrophobic tail structure and function in a class of ionizable lipid combinatorial libraries provided by the present invention.

[0032] Figure 2A schematic diagram of the construction of ionizable lipids with diverse three-dimensional spatial structures provided by the present invention.

[0033] Figure 3 The invention provides a two-dimensional structure and a three-dimensional spatial structure of an ionizable lipid molecule.

[0034] Figure 4 This is a Lipid 6 H NMR spectrum provided in an embodiment of the present invention.

[0035] Figure 5 The Lipid 6 mass spectrum provided in the embodiments of the present invention.

[0036] Figure 6 This is a Lipid 8 H NMR spectrum provided in an embodiment of the present invention.

[0037] Figure 7 The Lipid 8 mass spectrum provided in the embodiments of the present invention.

[0038] Figure 8 This is a Lipid 54 H NMR spectrum provided in an embodiment of the present invention.

[0039] Fig. 9 The Lipid 54 mass spectrum provided in the embodiments of the present invention.

[0040] Fig.10 This is a Lipid 55 H NMR spectrum provided in an embodiment of the present invention.

[0041] Fig.11 The Lipid 55 mass spectrum provided in the embodiments of the present invention.

[0042] Fig.12 The molecular dynamics simulation process and results (molecular structure, 2D projection density map, 3D spatial structure) of some ionizable lipid molecules with high transfection efficiency provided in the embodiments of the present invention.

[0043] Fig.13 The 3D spatial structure of some ionizable lipid molecules (lipid 3, lipid 6, lipid 11, lipid 19, lipid 21, lipid 24, lipid 26, lipid 28) with high transfection efficiency provided by the embodiments of the present invention.

[0044] Fig.14 The 3D spatial structure of some ionizable lipid molecules (lipid 41, lipid 42, lipid 45, lipid 47, lipid 54, lipid 55, lipid 58) with high transfection efficiency provided by the embodiments of the present invention.

[0045] Fig.15 The molecular dynamics simulation of the interaction between lipid 8 and mRNA provided in the embodiment of the present invention.

[0046] Fig.16 The molecular dynamics simulation provided in the embodiment of the present invention is a process in which excess ionizable lipid molecules interact with mRNA to form an ionizable lipid molecule-mRNA load.

[0047] Fig.17 This is the confocal microscopy result of Cy5-mRNA-LNPs provided in the embodiment of the present invention.

[0048] Fig.18 Schematic diagram of relative luciferase activity of HEK293T cells 24 h after transfection with LNPs encapsulated with mFluc prepared with ionizable lipids having an advantageous three-dimensional spatial structure provided in an embodiment of the present invention.

[0049] Fig.19 The fluorescence expression in muscles and lymph nodes of mice provided in the embodiments of the present invention was shown 6 hours after intravenous injection of PBS, lipid 6, lipid 8, lipid 11, lipid 23, lipid 29, lipid 35, lipid 42, lipid 43, lipid 49, lipid 54, and lipid 55 LNP.

[0050] Fig. 20 The fluorescence expression of various organs of mice provided in the embodiments of the present invention was measured 6 hours after intravenous injection of PBS, lipid 6, lipid 8, lipid 11, lipid 23, lipid 29, lipid 35, lipid 42, lipid 43, lipid 49, lipid 54, and lipid 55 LNP.

[0051] Fig.21 The lipid 8 LNP, lipid 11 LNP, and lipid 80 LNP provided in the embodiments of the present invention mediate their specific organ targeting by specifically adsorbing proteins to form a protein corona. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] It should be understood that the following examples are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available products, or can be prepared by known methods. If the specific technology or conditions are not specified in the examples, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product instructions. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels.

[0054] Example 1 Advantageous three-dimensional spatial structure can be ionized lipid molecules, synthesis of lipid 6

[0055] Synthesis of Lipid 6: At room temperature, 200 mg of hydroxyethylethylenediamine solution and 2.4 g of (9Z,12Z)-9,12-diene octadecane acrylamide solution were mixed together, and then the mixture was heated to 120 °C and stirred overnight. When the reaction was completed by thin layer chromatography, 2.6 g of crude product was obtained, and then the target product was purified by silica gel column chromatography to obtain lipid 6. The H NMR spectrum of compound lipid 6 is shown in Figure 4 , mass spectrometry see Figure 5 .

[0056] Lipid 6: 1 H NMR (400 MHz, CDCl3) δ 6.80 (dt, J = 16.3, 5.4 Hz, 2H), 5.44– 5.25 (m, 12H), 3.56 (t, J = 5.0 Hz, 2H), 3.19 (td, J = 14.1, 7.0 Hz, 6H), 2.88– 2.71 (m, 8H), 2.68 (t, J = 6.1 Hz, 4H), 2.58 – 2.48 (m, 4H), 2.45 (d, J = 5.2Hz, 2H), 2.31 (dd, J = 12.8, 6.5 Hz, 6H), 2.05 (dd, J = 13.7, 6.8 Hz, 12H), 1.54– 1.44 (m, 6H), 1.38 – 1.25 (m, 48H), 0.89 (dd,J = 8.7, 5.0 Hz, 9H).

[0057] Example 2 Advantageous three-dimensional spatial structure can be ionized lipid molecules, synthesis of lipid 8

[0058] Synthesis of Lipid 8: At room temperature, 300 mg of N-methyl-2,2'-diaminodiethylamine solution and 2.1 g of (9Z,12Z)-9,12-diene octadecane acrylate solution were mixed together, and then the mixture was heated to 120 °C and stirred overnight. When the reaction was completed by thin layer chromatography, 2.4 g of crude product was obtained, and then the target product was purified by silica gel column chromatography to obtain lipid 8. The H NMR spectrum of compound lipid 8 is shown in Figure 6 , mass spectrometry see Figure 7 .

[0059] Lipid 8: 1 H NMR (400 MHz, CDCl3) δ 5.46 – 5.26 (m, 12H), 4.06 (dt, J =10.4, 6.8 Hz, 6H), 2.98 (t, J = 6.5 Hz, 2H), 2.84 – 2.71 (m, 12H), 2.60 (t, J= 16.7 Hz, 6H), 2.52 – 2.40 (m, 6H), 2.26 (s, 3H), 2.05 (q, J = 6.8 Hz, 12H), 1.61 (dd, J = 13.0, 6.5 Hz, 6H), 1.41 – 1.22 (m, 51H), 0.89 (dd, J = 8.8, 4.9Hz, 9H).

[0060] Example 3 Synthesis of lipid 54, an ionizable lipid molecule with an advantageous three-dimensional structure

[0061] Synthesis of Lipid 54: At room temperature, 200 mg of the solution and 1.9 g of (9Z,12Z)-9,12-diene octadecane acrylamide solution were mixed together, and then the mixture was heated to 120 °C and stirred overnight. When the reaction was completed by thin layer chromatography, 2.1 g of crude product was obtained, and then the target product was purified by silica gel column chromatography to obtain lipid 54. The H NMR spectrum of compound lipid 54 is shown in Figure 8 , mass spectrometry see Fig. 9 .

[0062] Lipid 54: 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 6.63 (s, 2H), 5.36 (qd, J = 10.9, 5.5 Hz, 12H), 3.21 (dt, J = 18.3, 6.7 Hz, 6H), 2.76 (q, J = 6.5 Hz,10H), 2.65 – 2.41 (m, 12H), 2.36 (t, J = 6.0 Hz, 2H), 2.31 (t, J = 6.2 Hz, 4H), 2.11 – 1.97 (m, 12H), 1.71 (s, 2H), 1.48 (d, J = 6.4 Hz, 6H), 1.38 – 1.26 (m,48H), 0.89 (t, J = 6.8 Hz, 9H).

[0063] Example 4: Synthesis of lipid 55, an ionizable lipid molecule with an advantageous three-dimensional structure

[0064] H11 head group synthesis: Synthesis of compound 1: Phthalic anhydride (11.0 g) was added to a solution of diethylenetriamine (3.2 g) dissolved in glacial acetic acid. The reaction mixture was refluxed for 2 hours, then the solvent was evaporated under reduced pressure, and 200 mL of hot ethanol was added thereto under stirring. Stirring was continued for 5 hours until the reaction was complete, and the precipitate was filtered, collected and dried to obtain compound 1.

[0065] Synthesis of compound 2: First, compound 1 (2.0 g) was dissolved in acetonitrile to prepare a solution. Potassium carbonate (3.8 g), potassium iodide (1.37 g) and bromopropanol (2.3 g) were added to the solution in sequence. The reaction mixture was stirred and refluxed overnight. After the reaction was complete, the reaction mixture was cooled and concentrated by rotary evaporation. Compound 2 was purified by C18 column (water / methanol).

[0066] Synthesis of Compound H11: Compound 2 (1.0 g) and hydrazine hydrate (1.3 g) were dissolved in a mixed solvent of chloroform and ethanol, and hydrazine hydrolysis of Compound 2 was performed. The mixture was stirred at room temperature for 24 hours, filtered, and the obtained transparent solution was evaporated under reduced pressure. Chloroform was added to the residue, and the mixture was stirred for another 24 hours and filtered again. The mixture was evaporated under reduced pressure to obtain the desired product Compound H11 as an oil.

[0067]

[0068] Synthesis of Lipid 55: 170 mg H11 and 1.2 g (9Z,12Z)-9,12-diene octadecyl acrylate solution were mixed together at room temperature, and then the mixture was heated to 120 °C and stirred overnight. When the reaction was completed by thin layer chromatography, 1.3 g of crude product was obtained, and then the target product was purified by silica gel column chromatography to obtain lipid 55. The H NMR spectrum of compound lipid 55 is shown in Fig.10 , mass spectrometry see Fig.11 .

[0069] Lipid 55: 1 H NMR (400 MHz, CDCl3) δ 5.44-5.27 (m, 12H), 4.05 (t, J =6.8 Hz, 6H), 3.74 (s, 2H), 2.84-2.72 (m, 12H), 2.54 (s, 6H), 2.46 (t, J = 7.0Hz, 6H), 2.05 (q, J = 6.8 Hz, 12H), 1.66 (dq, J = 41.6, 6.8 Hz, 8H), 1.44-1.20 (m, 53H), 0.89 (t, J = 6.8 Hz, 9H).

[0070] Furthermore, the present invention uses the same or optimized method to synthesize and characterize the ionizable lipid molecule library for subsequent high-throughput screening and molecular dynamics simulation of the ionizable molecule library. The structures of some ionizable lipid molecules are shown in the following table (Table 1).

[0071]

[0072] Example 5 High-throughput screening of ionizable lipid molecule library, molecular dynamics simulation of the three-dimensional spatial structure of ionizable lipids with high transfection efficiency, and establishment of an analysis method for the three-dimensional spatial structure of ionizable lipid molecules First, we screened for cell transfection using a class of ionizable lipid compound combinatorial libraries, which were constructed with polar head groups, connecting bonds, and hydrophobic tails and had diverse three-dimensional spatial structures. We performed molecular dynamics simulations on lipid molecules with high transfection effects, constructed a 2D projection density map of the molecular structure, and analyzed its 3D spatial structural characteristics. Fig.12 As shown, the molecular structural information of the ionizable lipid molecules is first converted into the language of molecular dynamics simulation, the most stable spatial structure of the molecules is dynamically simulated in an aqueous environment, and a 2D projection density map of the representative molecular spatial structure is made, and its 3D spatial structure characteristics are statistically analyzed.

[0073] The three-dimensional structure of lipid molecules simulated by molecular dynamics shows that highly transfected lipid molecules have such a structure, which is characterized by the polar head group being well exposed at one (top) end, and the connecting bonds and hydrophobic tails being regularly arranged toward the other end. It is also found that lipid molecules with three hydrophobic tail chain structures are more likely to form such a three-dimensional structure.

[0074] Depend on Fig.12 The results show that ionizable lipids with advantageous three-dimensional spatial structures have better delivery efficiency, and their three-dimensional spatial structures have common characteristics. It was found that the three-dimensional spatial structure characteristics formed by the polar head group being exposed, the connecting bond and the hydrophobic tail facing the other side have a high transfection effect. For example Fig.13 and Fig.14 , among which the advantageous three-dimensional spatial structures include but are not limited to the following descriptions: conical shape, inverted conical shape, cone-like shape, circular truncated cone shape, wedge shape, bullet shape, horn shape, spire shape, steeple shape, tipped shape, needle shape, funnel shape, etc.

[0075] Example 6 Ionizable lipid molecules such as Lipid 8 promote their binding to mRNA molecules The ionizable lipid Lipid 8 has an excellent three-dimensional spatial structure. This characteristic spatial structure exposes the ionizable head group at one end, which is beneficial to promote its binding with mRNA. To prove this, the present invention simulates the interaction between lipid 8 lipid molecules and mRNA under the condition of ethanol / water = 1 / 3 (v / v) by molecular dynamics simulation. Fig.15 As shown in the figure, during the simulation period of 0-20 ns, lipid 8 can quickly interact with mRNA and form a strong interaction at 8 ns. This may be because lipid 8 has a head group space structure that can expose the head, so that the ionizable positively charged amine group and the negatively charged phosphate group on the mRNA can be electrostatically adsorbed and bound or partially hydrogen bonded.

[0076] as follows Fig.16 As shown in the figure, by adding excess lipid 8 ionizable lipid molecules into the molecular simulation environment, it can be clearly seen that the advantageous three-dimensional spatial structure promotes lipid 8 to bind to mRNA, and at the end of the simulation (30 ns), it is shown that mRNA can bind more lipid 8 molecules compared to lipid molecules with ordinary structures. This makes lipid 8 show that its excellent three-dimensional spatial structure can promote its binding and loading with mRNA.

[0077] Example 7 Lipid 8 and other ionizable lipid molecules encapsulate mRNA to prepare lipid nanoparticles with excellent physicochemical properties Ionizable lipid ALC-0315 or lipid 8, DOPE, cholesterol and DMG-PEG2000 were prepared in ethanol solution at a molar ratio of 45:15:38.5:1.5 as the organic phase, and firefly luciferase mRNA (mFluc) was dissolved in an aqueous solution of pH 4 as the aqueous phase. According to the volume ratio of the aqueous phase to the organic phase of 3:1, the nanoparticle suspension was prepared by microfluidics technology on a nanodrug manufacturing instrument (Shanghai Pengzan Bio, LNP S1). After preparation, the final lipid nanoparticle mFluc-LNP was obtained by ultrafiltration and concentration, and stored at 2-8 °C for use.

[0078] The particle size and Zeta potential of LNP were characterized using a Zetasizer Pro nanoparticle size potential analyzer (Malvern Panalytical). The encapsulation efficiency of mFluc-LNP was detected using the Ribogreen method using an F-280 fluorescence spectrophotometer. The test results of Example 7 are shown in Table 2.

[0079]

[0080] From the physicochemical data of lipid nanoparticles in Example 7, it can be seen that the particle size of the lipid nanoparticles mFluc-LNP prepared from ionizable lipid molecules with an advantageous three-dimensional spatial structure is mostly around 150 nm, with a narrow particle size distribution and a high encapsulation efficiency.

[0081] Example 8 Lipid 8 and other ionizable lipid molecules mFluc-LNP have excellent cellular uptake properties and lysosomal escape efficiency like Fig.17 As shown, Cy5-mRNA was encapsulated in LNPs (DOPE, cholesterol, DMG-PEG2000) prepared with lipid 8 as an ionizable lipid molecule, and confocal microscopy was used to capture the signal intensity of HEK293T cells taking up Cy5-mRNA-LNPs after co-incubation of Cy5-mRNA-LNPs with HEK293T cells for 2 h, 4 h, and 6 h. As can be seen from the figure, HEK293T cells can continue to take up lipid nanoparticles within 6 h, showing the excellent cellular uptake properties of lipid 8 LNP. The lysosomal escape experiment was conducted by using a confocal microscope to capture Cy5-mRNA-LNPs co-incubated with HEK293T cells for 6 h, lysosomal tracer (Lysotracker) staining endosomes / lysosomes and Hoechst33342 staining cell nuclei. Fig.17 Confocal microscopy results showed that Cy5-mRNA-LNPs had excellent HEK293T cell uptake characteristics and lysosomal escape rate, and it had very excellent lysosomal escape efficiency, releasing Cy5-mRNA into the cytoplasm.

[0082] Example 9 Lipid 8 and other ionizable lipid molecules mFluc-LNP have excellent in vitro cell transfection efficiency The transfection efficiency of the prepared mFluc-LNP on HEK293T cells was detected by the fluorescence reporter gene method using a multifunctional microplate reader (BioTek). The negative control was the cell culture medium without the addition of mFluc-LNP, and the positive control was the commercial ionizable lipid molecule ALC-0315-LNP. Fig.18 As shown, the LNP encapsulated mRNA prepared by the advantageous three-dimensional spatial structure ionizable lipid molecules has a very high cell transfection efficiency. It can be seen that compared with the commonly used commercial ALC-0315, the advantageous three-dimensional spatial structure ionizable lipid molecules have a higher cell transfection efficiency, and the advantage is very obvious. The phenomenon that such advantageous three-dimensional spatial structure ionizable lipid molecules have a higher cell transfection efficiency is also applicable to other lipid compounds with the same or similar spatial structure in the ionizable lipid molecule library of the present invention.

[0083] Example 10 Lipid 6 and other advantageous three-dimensional ionizable lipid nanoparticles have excellent transfection efficiency in animals According to the method described in Example 7, lipid nanoparticles (LNPs) were prepared using lipid 6 and other advantageous three-dimensional ionizable lipids and injected intramuscularly into 6-8 week old female / male C57BL / 6J mice. The mRNA dosage was 0.5 mg kg -1 PBS was used as a blank control. The fluorescence expression of the muscles and lymph nodes at the injection site of mice was shown in Figure 6. Fig.19 The results show that compared with lipid 8 and lipid 11, lipid 8 with ester bond ( ) has stronger in vivo muscle transfection ability. Compared with lipid 23 and lipid 29, lipid 29 with tail chain branching structure ( ) has stronger in vivo muscle transfection ability. Compared with lipid 8 and lipid 55, lipid 55 ( ) has stronger in vivo muscle transfection ability.

[0084] Example 11 Transfection and in vivo distribution experiment of lipid nanoparticles composed of ionizable lipid molecules with advantageous three-dimensional spatial structure in animals The lipid nanoparticles were injected into the tail vein of mice. According to the method described in Example 7, lipid nanoparticles were prepared using ionizable lipid molecules with advantageous three-dimensional spatial structures and injected into 6-8 week old female / male C57BL / 6J mice via the tail vein. The mRNA dosage was 0.5 mg kg -1 .

[0085] Mice were injected with blank control PBS, lipid 6 LNP, lipid 8 LNP, lipid 11 LNP, lipid 23 LNP, lipid 29 LNP, lipid 35 LNP, lipid 42 LNP, lipid 43 LNP, lipid 49 LNP, lipid 54 LNP, lipid 55 LNP, and lipid 80 LNP through the tail vein. The fluorescence expression of each organ 6 hours later is shown in Fig. 20. The results showed that most LNPs were highly efficient in transfecting the spleen, and several cases could efficiently and specifically target a single organ. After intravenous injection (IV) of lipid 8 LNP lipid nanoparticles, the fluorescence expression in various organs of mice was mainly distributed in the spleen (about 92%), the liver (about 5%), the lung (about 3%), the heart (0%), and the kidney (0%), which showed that it could specifically target the spleen. After intravenous injection (IV) of lipid 11 LNP lipid nanoparticles, the fluorescence expression in various organs of mice was mainly distributed in the lung (about 93%), the liver (about 5%), the spleen (about 2%), the heart (0%), and the kidney (0%), which showed that it could specifically target the lung. After intravenous injection (IV) of lipid 80 LNP lipid nanoparticles, the fluorescence expression in various organs of mice was mainly distributed in the liver (about 95%), the spleen (about 4%), the lung (about 1%), the heart (0%), and the kidney (0%), which showed that it could specifically target the liver. No fluorescence expression was detected in various organs of mice in the PBS blank control group. It can be seen that lipid molecules with advantageous three-dimensional spatial structures can efficiently and specifically adsorb proteins with different characteristics to form specific protein coronas, and then mediate organ-specific expression of mRNA through protein coronas. The difference in the number of connecting bonds (ester bonds or amide bonds) and hydrophobic tails affects the specific adsorption and binding of lipid molecules with advantageous three-dimensional spatial structures to different proteins ( Fig.21 ), better achieving specific targeting of different organs.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ionizable lipid compound having a three-dimensional structure, characterized in that: The ionizable lipid compound comprises a three-dimensional structure constructed by a polar head group, a connecting bond and a hydrophobic tail; the polar head group has an N number of 1 to 5, and the polar head group comprises -CH3, -CH2CH3, -OH, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, , , , One or more of; the hydrophobic tail comprises one or more of a fully saturated tail chain, an unsaturated tail chain, a branched structure tail chain and a heteroatom-doped tail chain.

2. The ionizable lipid compound according to claim 1, characterized in that The polar head group is any one of formula H1-H14: 。 3. The ionizable lipid compound according to claim 1, characterized in that The carbon chain length of the fully saturated tail chain is C6-C18; and / or the unsaturation degree of the unsaturated tail chain is 1 or 2; and / or the branch chain lengths of the branched structure tail chain are equal or unequal; and / or the heteroatom of the heteroatom-doped tail chain is O, S or SS.

4. The ionizable lipid compound according to claim 3, characterized in that The hydrophobic tail is any one of formulas T1-T16: 。 5. The ionizable lipid compound according to claim 4, characterized in that The number of hydrophobic tails of the ionizable lipid compound is 1 to 4; and / or the connecting bond is a degradable connecting bond, and the degradable connecting bond includes an ester bond or an amide bond.

6. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is one of Lipid 1~Lipid 116.

7. The ionizable lipid compound according to any one of claims 1 to 6, characterized in that In the three-dimensional spatial structure, the polar head group is exposed to one side, and the connecting bond and the hydrophobic tail are facing the other side; the three-dimensional spatial structure includes cone, reverse cone, quasi-cone, truncated cone, wedge, bullet, trumpet, spire, spire, pointed tip, needle, and funnel.

8. The method for preparing the ionizable lipid compound having a three-dimensional structure according to any one of claims 1 to 7, characterized in that: The following steps are involved: ； 1) Esterification reaction: esterify the hydroxyl group of alcohol compound A1 in the presence of acryloyl chloride to obtain compound A2; 2) Michael addition reaction: mixing the compound A2 with a polar head group amine and performing Michael addition reaction to obtain an ionizable lipid compound A3 having a three-dimensional spatial structure; or: ； 1) Esterification reaction: the amine group of the amine compound B1 is amidated in the presence of acryloyl chloride to obtain compound B2; 2) Michael addition reaction: the compound B2 is mixed with a polar head group amine and subjected to Michael addition reaction to obtain an ionizable lipid compound B3 having a three-dimensional spatial structure.

9. Use of an ionizable lipid compound having a three-dimensional spatial structure in the preparation of a nucleic acid delivery system, characterized in that: The delivery system is a lipid nanoparticle, and the lipid nanoparticle contains the ionizable lipid compound with a three-dimensional structure according to any one of claims 1 to 7 or the ionizable lipid compound with a three-dimensional structure obtained by the preparation method according to claim 8.

10. A lipid nanoparticle, characterized in that: The lipid nanoparticles contain the ionizable lipid compound with a three-dimensional structure described in any one of claims 1 to 7 or the ionizable lipid compound with a three-dimensional structure obtained by the preparation method described in claim 8.

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