Phosphonated ionizable lipids for selective delivery of nucleic acid drugs, and methods of making and using the same

Abstract

The application belongs to the technical field of biological medicine, and discloses a phosphonate ionizable lipid for selective delivery of nucleic acid drugs, and a preparation method and application thereof.The phosphonate ionizable lipid has a general structure of formula I, wherein Cn is a phosphonate-containing structure, Nn is a nitrogen-containing cyclic structure or a nitrogen-containing amine group, B is O or NH, and Ln is a connecting bond.The application further discloses a preparation method of the phosphonate ionizable lipid.The phosphonate ionizable lipid is used for preparing phosphonate ionizable lipid nanoparticles, and is used as a delivery carrier for selectively delivering nucleic acid drugs to hearts, lungs, spleens, livers and / or muscles.The ionizable lipid can effectively avoid the protein crown shielding effect existing in traditional targeted modification, can effectively and safely deliver nucleic acid drugs to extrahepatic organs, and is particularly suitable for delivering nucleic acid drugs to lungs and spleens.Further, the ionizable lipid has membrane stability and endosome escape functions.Further, the ionizable lipid has a general structure of formula I.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs, its preparation method, and its application. Background Technology

[0002] Currently, existing nucleic acid drug delivery carriers mainly accumulate in the liver after entering the body, making it difficult to effectively deliver nucleic acid drugs to other target organs besides the liver. To solve the problem of selective delivery of nucleic acid vaccines, existing strategies include: (1) modifying the surface of LNPs (lipid nanoparticles) with specific targeting molecules. However, after entering the bloodstream, the surface of LNPs will rapidly adsorb a large amount of plasma proteins, forming a protein crown, which often obscures the active sites of the targeting molecules, weakens their recognition with the target receptor, and leads to obvious off-target effects. (2) Introducing a fifth lipid component into traditional four-component LNPs, using specific lipids (e.g., adding anionic lipids to achieve spleen targeting, adding cationic lipids to achieve lung targeting) to change organ distribution, but this is often accompanied by obvious in vivo toxicity. (3) Relying on the random assembly of a large-scale lipid library and high-throughput in vivo screening to find the best lipids with organ selectivity in a limited lipid library, but the results of such screening are usually random and unpredictable. (4) By adjusting the types and ratios of phospholipids in LNPs, the membrane phase, protein adsorption behavior and in vivo distribution of the particles can be changed, thereby affecting organ-selective delivery. However, saturated phospholipids with high phase transition temperatures typically exhibit more compact molecular packing, which is beneficial for maintaining the structural integrity and cycling stability of LNPs, but may limit lipid rearrangement and endosome membrane fusion. Conversely, unsaturated phospholipids with low phase transition temperatures can improve membrane fluidity and promote endosome membrane perturbation, but may come at the cost of reduced particle stability. Therefore, relying solely on phospholipid composition regulation often makes it difficult to simultaneously achieve stability in the cycling stage and efficient membrane remodeling in the endosome stage.

[0003] One of the problems that needs to be solved is how to construct a phosphoesterified ionizable lipid that combines membrane stability and endosome escape capability, while reducing the interference of protein crowns on targeting function, thereby achieving efficient, safe and organ-selective mRNA delivery. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs, its preparation method, and its application. This invention overcomes the deficiencies of existing LNPs, such as significant liver accumulation during mRNA delivery, easy shielding of LNP surface targeting modifications by protein coronas, and the incompatibility between cyclic stability and endosome escape function caused by traditional multi-component physical mixing.

[0005] This invention covalently integrates a phosphonate head group and an ionizable amino group into the same lipid backbone, synergistically integrating cycling stability and endosome escape function at the molecular level. Simultaneously, this molecule can alter the surface electrochemical properties of LNPs to regulate the composition of the protein crown, thereby effectively circumventing the protein crown's shielding effect on targeting ability. Compared to ionizable lipids without phosphonate modification, the lipids of this invention exhibit significantly enhanced organ selectivity, overcoming the common extrahepatic delivery bottleneck of existing LNPs, and achieving efficient and safe mRNA targeted delivery to extrahepatic organs (especially the lungs and spleen).

[0006] The phosphoesterified ionizable lipids of the present invention are used to prepare lipid nanoparticles for selective delivery of nucleic acid drugs, particularly delivery carriers for selective delivery of nucleic acid drugs to the lungs and / or spleen.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A phosphonate-esterified ionizable lipid for selective delivery of nucleic acid drugs, with the general structural formula of Formula I:

[0009] Formula I: In the structure, N is nitrogen;

[0010] In formula I, Cn is , where n' is an integer from 0 to 20, preferably an integer from 0 to 5; * indicates a connection point;

[0011] Nn is * indicates a connection point;

[0012] B is either O or NH;

[0013] Ln acts as a linker bond, and its structure contains -(CH2). n1 -SS-(CH2) n2 -、-(CH2) n3 - where n1 = an integer from 0 to 6 (n1 is preferably 0, 1 or 2), n2 = an integer from 0 to 6 (n2 is preferably 0, 1 or 2), and n3 = an integer from 0 to 6;

[0014] T1 is m is an integer from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); * indicates a connection point;

[0015] T2 is m is an integer from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8); * indicates a connection point.

[0016] Alternatively, phosphoesterified ionizable lipids for selective delivery of nucleic acid drugs, with the structural formula II: Where C' is Where n' is an integer from 0 to 20, preferably an integer from 0 to 5; m' is an integer from 1 to 5, such as 2, 3, 4, etc.; * indicates a connection point;

[0017] T3 is or m1 is 1, 2, 3; R1 is C 10~18 Alkyl group, specifically an alkyl group with 11 carbon atoms or a branched alkyl group with 17 carbon atoms; m2 is 1, 2; R2 is C 14~17 Alkyl group, specifically a branched alkyl group with 15 carbon atoms; * indicates a junction;

[0018] T4 is or m1 is 1, 2, or 3; R1 is C 10~18 Alkyl group, specifically an alkyl group with 11 carbon atoms or a branched alkyl group with 17 carbon atoms; m2 is 1 or 2; R2 is C 14~17 Alkyl, specifically a branched alkyl group with 15 carbon atoms; * indicates a junction.

[0019] The phosphoesterified ionizable lipids for selective delivery of the nucleic acid drug include one or more of the following structures;

[0020] .

[0021] Alternatively, the phosphoesterified ionizable lipids for selective delivery of the nucleic acid drug may include one or more of the following structures:

[0022] (1) ,

[0023] (2) .

[0024] When the structure of the phosphonate-esterified ionizable lipid for selective delivery of nucleic acid drugs is of Formula I, the preparation method of the phosphonate-esterified ionizable lipid for selective delivery of nucleic acid drugs includes the following steps:

[0025] 1) Reacting a phosphonate-containing compound with a compound containing p-nitrobenzene to obtain a phosphonate-modified compound;

[0026] The structure of phosphine ester-containing compounds is as follows: The structure of the compound containing p-nitrobenzene is

[0027] The structure of the phosphine-esterified compound is as follows:

[0028] In the structure, Cn, Nn, B, and Ln are defined as in Equation I;

[0029] 2) Remove the Boc protecting group from the phosphoesterified compound and then react it with an epoxide to obtain phosphoesterified ionizable lipids;

[0030] The structure of the epoxy alkane is as follows: , where m" is an integer from 2 to 7.

[0031] The reaction conditions in step 1) are 0~4℃ and carried out under a protective atmosphere; triethylamine is added during the reaction.

[0032] The reaction is carried out in an organic solvent, namely dichloromethane.

[0033] In step 2), the removal of the Boc protecting group refers to dissolving the phosphine-esterified modified compound in dichloromethane, adding a dioxane solution of hydrogen chloride, shaking, allowing it to stand, removing the supernatant, and obtaining an oily substance.

[0034] The reaction with epoxides involves dissolving the oily substance in ethanol and then adding the epoxide. The reaction conditions are 80-90℃ for 10-16 hours.

[0035] When the phosphonate ionizable lipid is of formula II, the method for preparing the phosphonate ionizable lipid for selective delivery of nucleic acid drugs includes the following steps: S1) reacting a phosphonate compound containing p-nitrobenzene with a compound containing hydroxyl groups to obtain the phosphonate ionizable lipid;

[0036] The structure of phosphonate compounds containing p-nitrobenzene is as follows: C' is as defined in Equation II;

[0037] The structure of compounds containing hydroxyl groups is as follows: , m', T3, T4 are as defined in Equation II.

[0038] The reaction was carried out using DCM as the reaction solvent and in the presence of TEA. The reaction temperature was room temperature. The reaction time was 6–15 hours.

[0039] The phosphoesterified ionizable lipids are used to prepare ionizable lipid nanoparticles for selective delivery of nucleic acid drugs, serving as carriers for selective delivery of nucleic acid drugs.

[0040] The ionizable lipid nanoparticles are obtained by dissolving ionizable lipids, auxiliary lipids, sterol lipids and PEG lipids in an organic solvent to obtain an oil phase; dissolving nucleic acid drugs in water or a buffer solution to obtain an aqueous phase; and mixing the oil phase and the aqueous phase under stirring conditions to obtain a nucleic acid drug delivery carrier.

[0041] The ionizable lipid is the above-mentioned phosphoesterified ionizable lipid.

[0042] The sterol lipids include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; preferably cholesterol.

[0043] The auxiliary lipids include one or more of phospholipids, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives; preferably phosphatidylethanolamine, specifically dioleoylphosphatidylethanolamine.

[0044] The PEG lipid is DMG-PEG2000.

[0045] The nucleic acid drugs mentioned are ribonucleic acid (RNA), messenger RNA (mRNA), ribosomal RNA (rRNA), circular RNA (circRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), microRNA, short hairpin RNA (shRNA), and deoxyribonucleic acid (DNA).

[0046] The molar ratio of the ionizable lipid: auxiliary lipid: cholesterol lipid: PEG lipid is (45~55): (5~15): (38~39): (1~2).

[0047] The molar percentages of ionizable lipids, cofactor lipids, cholesterol lipids, and PEG lipids in the total composition are as follows: ionizable lipids 45%-55%, cofactor lipids 5%-15%, cholesterol lipids 38%-39%, and PEG lipids 1%-2%. Total composition: ionizable lipids, cofactor lipids, cholesterol lipids, and PEG lipids.

[0048] The molar ratio (N / P ratio) of ionizable nitrogen atoms in the ionizable lipid to phosphorus atoms in the nucleic acid drug is 1:1 to 50:1.

[0049] The nanoparticles are used in the preparation of pharmaceutical formulations for the selective delivery of nucleic acid drugs to the heart, lungs, spleen, liver and / or muscles.

[0050] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0051] (1) The ionizable lipids of the present invention synergistically integrate membrane stability and endosome escape functions, fundamentally solving the structural contradiction that membrane stability and membrane perturbation functions are difficult to reconcile in traditional multi-component physical mixing systems. By covalently coupling the phosphonate head group and the ionizable amino group to the same lipid backbone, this single molecule can provide a stable lipid membrane structure at physiological pH and trigger efficient perturbation and escape in the acidic environment of the endosome, significantly improving the cytoplasmic delivery efficiency of mRNA.

[0052] (2) The ionizable lipids of the present invention can actively regulate the composition of the protein crown, effectively avoiding the protein crown shielding effect that is common in traditional targeted modification strategies. The phosphoesterified ionizable lipids provided by the present invention can change the surface electrochemical properties of LNPs and reshape the protein crown components, thereby maintaining the targeting ability (e.g., lungs, spleen) without the need for external targeting ligands, breaking through the dilemma of "targeted modification - protein crown shielding - loss of targeting", and giving LNPs true in vivo targeting ability to perform functions.

[0053] (3) The ionizable lipids of the present invention can selectively deliver nucleic acid drugs, breaking through the bottleneck of high accumulation in the liver during mRNA delivery of existing ionizable lipid LNPs. Compared with ionizable lipids without phosphoester modification, the ionizable lipids of the present invention can efficiently and safely deliver mRNA to extrahepatic organs, especially the lungs and spleen.

[0054] (4) By adjusting the structure of the phosphonate head group, ionizable amino group and lipid backbone, the present invention can rationally optimize the targeting needs of different organs, greatly enriching the types of nucleic acid drug carriers and effectively achieving selective delivery to non-hepatic organs. Attached Figure Description

[0055] Figure 1 The 1H NMR spectrum of the ionizable lipid C2-N4-L1-T16 in Example 1;

[0056] Figure 2 The 1H NMR spectrum of the ionizable lipid N4-L1-T16 in Comparative Example 1;

[0057] Figure 3 The 1H NMR spectrum of the ionizable lipid C2-N7-L1-T16 in Example 2;

[0058] Figure 4 The 1H NMR spectrum of the ionizable lipid N7-L1-T16 in Comparative Example 2;

[0059] Figure 5 In Example 4, phosphoesterified LNPs were used to siRNA under different formulations. luc Relative luminescence index delivered to B16 luc cells;

[0060] Figure 6 The bar chart shows the molar percentage of each lipid component in different formulations under the condition that the N / P ratio is fixed at 6 in the orthogonal experiment of Example 4.

[0061] Figure 7 Comparison of nanoparticle size and Zeta potential values ​​of phosphoesterified LNPs and non-phosphoesterified LNPs in Example 5;

[0062] Figure 8 In Example 6, a 0.1 mg / kg mRNA load was administered intravenously. luc SM102 Lung SORT LNPs, SM102 Spleen SORT LNPs, C2-N4-L1-T16-P6 LNPs, C2-N7-L1-T16-P0 LNPs, N4-L1-T16-P6 LNPs and N7-L1-T16-P0 LNPs were used for in vitro organ imaging and corresponding fluorescence quantification.

[0063] Figure 9 Different doses of siRNA were administered in Example 7. luc LNPs were injected intravenously into C57BL / 6j mice. Six hours later, blood was collected and the concentrations of IL-6, TNF-α and IL-1β in plasma were quantitatively detected by ELISA.

[0064] Figure 10 To track the stability and cell transfection efficiency of the SM102 Lung SORT, SM102 Spleen SORT, C2-N4-L1-T16-P6, C2-N7-L1-T16-P0, C2-N4-L1-T16-A2 and C2-N7-L1-T16-A5 LNPs formulations stored at 4°C for 21 days in Example 8; a: relative fluorescence, b: hydrodynamic diameter;

[0065] Figure 11 This is an example of the endosome escape ability test of phosphoesterified LNPs in Example 9;

[0066] Figure 12 This is a proteomic analysis of the protein canopy adsorbed on LNPs in Example 10. Detailed Implementation

[0067] The present invention will be described in further detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0068] Example 1

[0069] Synthesis of Compound 1:

[0070]

[0071] Compound C2 (50 mmol), triethylamine (60 mmol), and anhydrous dichloromethane (400 mL) were added sequentially to a dry reaction flask and stirred until dissolved. Acryloyl chloride (50 mmol) was slowly added dropwise at 0 °C under nitrogen protection, and the reaction was continued at 0 °C with stirring. Samples were taken every 0.5 h to monitor the reaction progress by TLC. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give a colorless oily product 1. Yield: 83%.

[0072] Synthesis of compound C2-N4:

[0073]

[0074] Compound 1 (10 mmol) and compound N4 ( mmol) were added sequentially to a dry reaction flask. The solvent was dissolved in 10 mmol of anhydrous ethanol (30 mL) by stirring. The reaction was continued at 75 °C for 12 h, and the reaction progress was monitored by TLC. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography to give a pale yellow oily product C2-N4. Yield: 77%.

[0075] Synthesis of compound 4:

[0076]

[0077] Compound 3 (50 mmol), p-nitrobenzene chloroformate (55 mmol), and anhydrous dichloromethane (200 mL) were added sequentially to a dry reaction flask and stirred until dissolved. Triethylamine (55 mmol) was slowly added dropwise at 0 °C under nitrogen protection, and the reaction was continued at 0 °C with stirring. Samples were taken every 0.5 h to monitor the reaction progress by TLC. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give a white solid product 4. Yield: 89%.

[0078] Synthesis of compound C2-N4-L1:

[0079]

[0080] Compound C2-N4 (10 mmol), compound 4 (10 mmol), and 100 mL of anhydrous dichloromethane were added sequentially to a dry reaction flask and stirred until dissolved. Triethylamine (10 mmol) was slowly added dropwise at 0 °C under nitrogen protection, and the reaction was continued at 0 °C with stirring. Samples were taken every 0.5 h to monitor the reaction progress by TLC. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give a pale yellow oily product C2-N4-L1. Yield: 81%.

[0081] Synthesis of phosphoesterified ionizable lipid C2-N4-L1-T16:

[0082]

[0083] In a dry reaction flask, compound C2-N4-L1 (2 mmol) was dissolved in dichloromethane (10 mL) and stirred until completely dissolved. A 4 M dioxane solution (5 mL) was added to the solution, shaken for 1 minute, and allowed to stand, resulting in the precipitation of an oily substance. The supernatant was decanted, and the remaining oily substance was dissolved in anhydrous ethanol (10 mL), followed by the addition of 1,2-epoxyhexadecane (4 mmol), and the reaction was carried out at 85 °C for 12 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the starting material C2-N4-L1 was completely consumed, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain a pale yellow oily product C2-N4-L1-T16, with a yield of 57%. The 1H NMR spectrum of C2-N4-L1-T16 is shown below. Figure 1 .

[0084] Comparative Example 1

[0085] Synthesis of compound N4-L1:

[0086]

[0087] Compound N4 (10 mmol), compound 4 (10 mmol), and 100 mL of anhydrous dichloromethane were added sequentially to a dry reaction flask and stirred until dissolved. Triethylamine (10 mmol) was slowly added dropwise at 0 °C under nitrogen protection, and the reaction was continued at 0 °C with stirring. Samples were taken every 0.5 h to monitor the reaction progress by TLC. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give a pale yellow oily product N4-L1. Yield: 88%.

[0088] Synthesis of nonphosphonate-esterified ionizable lipid N4-L1-T16:

[0089]

[0090] In a dry reaction flask, compound N4-L1 (2 mmol) was dissolved in dichloromethane (10 mL) and stirred until completely dissolved. A 4 M dioxane solution (5 mL) was added to this solution, shaken for 1 minute, and allowed to stand, resulting in the precipitation of an oily substance. The supernatant was decanted, and the remaining oily substance was dissolved in anhydrous ethanol (10 mL), followed by the addition of 1,2-epoxyhexadecane (4 mmol), and the reaction was carried out at 85 °C for 12 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the starting material N4-L1 was completely consumed, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to give a pale yellow oily product N4-L1-T16, with a yield of 66%. The 1H NMR spectrum of N4-L1-T16 is shown below. Figure 2 .

[0091] Example 2

[0092] Synthesis of phosphoesterified ionizable lipid C2-N7-L1-T16:

[0093]

[0094] The difference between this embodiment and Example 1 is that compound N7 is used. The method for synthesizing compound C2-N7-L1-T16 is the same as that for compound C2-N4-L1-T16, with a yield of 59%. The 1H NMR spectrum of C2-N7-L1-T16 is shown below. Figure 3 .

[0095] Comparative Example 2

[0096] Synthesis of compound N7-L1:

[0097]

[0098] The synthesis method of compound N7-L1 is the same as that of compound N4-L1, with a yield of 90%.

[0099] Synthesis of nonphosphonate-esterified ionizable lipid N7-L1-T16:

[0100]

[0101] The synthesis method of compound N7-L1-T16 is the same as that of compound N4-L1-T16, with a yield of 69%. The 1H NMR spectrum of N7-L1-T16 is shown below. Figure 4 .

[0102] Example 3

[0103] Preparation of LNP formulations:

[0104] Phosphoesterified ionizable lipids (C2-N4-L1-T16 prepared in Example 1 or C2-N7-L1-T16 prepared in Example 2), the auxiliary lipid 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG2000 (purchased from Aladdin) were dissolved in anhydrous ethanol in the following two ratios, with the total lipid concentration controlled at 8 mM, to prepare ethanol-lipid phases: In the system containing C2-N4-L1-T16, the molar ratio of ionizable lipids, auxiliary lipids, cholesterol, and DMG-PEG2000 was 55:5:38:2; in the system containing C2-N7-L1-T16, the molar ratio of ionizable lipids, auxiliary lipids, cholesterol lipids, and DMG-PEG2000 was 50:10:38.5:1.5. Simultaneously, luciferase mRNA (mRNA...) was... luc The mRNA (mRNA molecular weight 665720 g / mol, purchased from Hefei Afana Biotechnology Co., Ltd.) was diluted with 10 mM sodium acetate buffer (pH 3.8) to form the aqueous phase. The aqueous phase and the ethanol lipid phase were mixed at a volume ratio of 3:1 under vortexing and allowed to stand at room temperature for 30 min to obtain the crude LNP suspension. Subsequently, the prepared crude LNP suspension was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4,000 × g for 15 min to remove most of the ethanol. Phosphate-buffered saline (PBS) was then added to adjust to the desired final volume to obtain C2-N4-L1-T16 LNPs and C2-N7-L1-T16 LNPs. In all formulations, the nitrogen-to-phosphorus molar ratio (N / P ratio) of ionizable lipids to mRNA is 8 (it is known that the molar amount of ionizable nitrogen atoms in 1 mol of C2-N4-L1-T16 is 2 mol, and the molar amount of ionizable nitrogen atoms in 1 mol of C2-N7-L1-T16 is 3 mol; the molar amount of phosphorus atoms in 1 mol of mRNA is 1958 mol). The preparation method of N4-L1-T16 LNPs is the same as that of C2-N4-L1-T16 LNPs, and the preparation method of N7-L1-T16 LNPs is the same as that of C2-N7-L1-T16 LNPs.

[0105] Example 4

[0106] Phosphoesterified LNPs for in vitro delivery of luciferase siRNA (siRNA) luc Performance evaluation and formulation screening of siRNA (molecular weight 13860 g / mol, purchased from Hefei Afana Biotechnology Co., Ltd.):

[0107] Logarithmic growth phase mouse melanoma B16-F10-luc cells were digested with trypsin, centrifuged, and resuspended in RPMI-1640 complete medium containing 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin. The culture was then prepared at approximately 1 × 10⁻⁶ cells / day. 4 siRNA was seeded at a density of 1 cell / well in white 96-well cell culture plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. The old culture medium was discarded, and siRNA-loaded cells were added to each well. luc Fresh complete culture medium (siRNA) for LNPs luc The final concentration was 10 nM (the molar amount of phosphorus atoms per mol of siRNA was 42 mol), and incubation was continued for 24 h. Subsequently, luciferase activity was measured using a multi-functional microplate reader. In vitro transfection details are shown below. Figure 5 .

[0108] Loaded siRNA luc Preparation of LNPs: To systematically optimize lipid composition, an orthogonal experimental design was used to investigate the molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000. The four-component lipid formulations (molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000) P0-P6 included: 50 / 10 / 38.5 / 1.5, 50 / 10 / 39 / 1, 50 / 10 / 38 / 2, 45 / 15 / 39 / 1, 45 / 15 / 38 / 2, 55 / 5 / 39 / 1, and 55 / 5 / 38 / 2. In the preparation methods of LNPs under each formulation, the nitrogen-phosphorus molar ratio (N / P ratio) of ionizable lipids to siRNA was 6, and other preparation steps and conditions were the same as in Example 3. Based on this, anionic lipid phosphatidylinositol (LAP) was further introduced to investigate five-component formulations (ionizable lipid / DOPE / cholesterol / DMG-PEG2000 / LAP) A1-A5, with molar ratios of 40 / 10 / 38.5 / 1.5 / 10, 45 / 10 / 38.5 / 1.5 / 5, 40 / 15 / 38.5 / 1.5 / 5, 45 / 5 / 38.5 / 1.5 / 10 and 50 / 5 / 38.5 / 1.5 / 5 (in the preparation methods of LNPs under each formulation, the nitrogen-phosphorus molar ratio (N / P ratio) of ionizable lipid to siRNA was 6, and other preparation steps and conditions were the same as in Example 3).

[0109] Figure 5 In Example 4, phosphoesterified LNPs were used to siRNA under different formulations. luc Relative luminescence index delivered to B16 luc cells.

[0110] Figure 6 The bar chart shows the molar percentage of each lipid component in different formulations under the condition that the N / P ratio is fixed at 6 in the orthogonal experiment of Example 4.

[0111] Figure 5 and Figure 6 In the formula, P0-P6 refer to the molar ratio of ionizable lipids / co-lipids / cholesterol lipids / PEG lipids, where P0 = 50 / 10 / 38.5 / 1.5; P1 = 50 / 10 / 39 / 1; P2 = 50 / 10 / 38 / 2; P3 = 45 / 15 / 39 / 1; P4 = 45 / 15 / 38 / 2; P5 = 55 / 5 / 39 / 1; P6 = 55 / 5 / 38 / 2, A1 -A5 refers to the molar ratio of ionizable lipids / co-lipids / cholesterol lipids / PEG lipids / LAP lipids in the formulation, where A1=40 / 10 / 38.5 / 1.5 / 10; A2=45 / 10 / 38.5 / 1.5 / 5; A3=40 / 15 / 38.5 / 1.5 / 5; A4=45 / 5 / 38.5 / 1.5 / 10; A5=50 / 5 / 38.5 / 1.5 / 5.

[0112] Figure 5 ionizable lipids Where Cn is When n' is 1, Cn is denoted as C2; when Ln is -SS-CH2-, Ln is denoted as L1; when B is NH;

[0113] Nn is (Referred to as N1 group) (Referred to as N2 group) (Referred to as N3 group) (Referred to as N4 group) (Referred to as N5 group) (Referred to as N6 group) (Referred to as the N7 group);

[0114] T1 / T2 is m is 2 (uniformly denoted as T10), 3 (uniformly denoted as T12), 4 (uniformly denoted as T14), 5 (uniformly denoted as T16), and 6 (uniformly denoted as T18).

[0115] Figure 5 In the middle, C2-N1-L1-T10 represents Cn as , n' is 1; Nn is (denoted as N1 group); Ln is -SS-CH2-; B is NH; T1 / T2 is m is 2. In C2-N1-L1-T12~C2-N1-L1-T18, Cn, Ln, and B are the same as above, and m in T1 / T2 are 3, 4, 5, and 6 respectively.

[0116] C2-N3-L1-T16 indicates that Nn is... (denoted as N3 group), T1 / T2 is m is 5; Cn, Ln, and B are the same as above. In C2-N3-L1-T10~C2-N3-L1-T14, C2-N3-L1-T18, Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (uniformly denoted as T10), 3 (uniformly denoted as T12), 4 (uniformly denoted as T14), and 6 (uniformly denoted as T18) respectively.

[0117] For the N6 group in C2-N6-L1-T10~C2-N6-L1-T18, Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (unified as T10), 3 (unified as T12), 4 (unified as T14), and 6 (unified as T18).

[0118] The N2 group in C2-N2-L1-T10~C2-N2-L1-T18 is Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (uniformly denoted as T10), 3 (uniformly denoted as T12), 4 (uniformly denoted as T14), and 6 (uniformly denoted as T18) respectively.

[0119] The N4 group in C2-N4-L1-T10~C2-N4-L1-T18 is... Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (unified as T10), 3 (unified as T12), 4 (unified as T14), and 6 (unified as T18).

[0120] The N5 group in C2-N5-L1-T10~C2-N5-L1-T18 is... Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (unified as T10), 3 (unified as T12), 4 (unified as T14), and 6 (unified as T18).

[0121] The N7 group in C2-N7-L1-T10~C2-N7-L1-T18 is... Cn, Ln, and B are the same as above. In T1 / T2, m is 2 (unified as T10), 3 (unified as T12), 4 (unified as T14), and 6 (unified as T18).

[0122] The preparation methods for each lipid are the same as in Example 1.

[0123] SM102: .

[0124] from Figure 5 As can be seen, various phosphoesterified LNPs can effectively transfect siRNA in vitro. lucSome phosphoesterified LNPs exhibited superior transfection efficiency compared to SM102 (1-octylnonyl-8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) lipid nanoparticles. When the tail chain had 16 carbon atoms, phosphoesterified LNPs typically demonstrated higher transfection efficiency, highlighting their excellent siRNA transfection performance. Both excessively long and excessively short hydrophobic tail chains were detrimental to transfection; the C2-N3-L1-T16 / C2-N6-L1-T16 / C2-N4-L1-T16 series lipids perfectly conformed to this trend, with a compatibility close to 100%.

[0125] Example 5

[0126] LNP particle size and Zeta potential characterization:

[0127] The particle size and zeta potential of the prepared LNPs were measured using zetasizers for phosphoesterified and unphosphoesterified LNPs. The results showed that the zeta potential of the phosphoesterified LNPs was negatively charged or close to neutral, while the zeta potential of the unmodified LNPs was positive. This indicates that phosphoesterification can alter the surface electrochemical properties of LNPs. The test results are shown below. Figure 7 .

[0128] Figure 7 SM102-P0 refers to LNPs prepared from ionizable lipid SM102 under formulation P0 conditions, using the same preparation method as the LNPs in Example 3, and serves as a control group. C2-N4-L1-T16-P6 refers to LNPs prepared from ionizable lipid C2-N4-L1-T16 under formulation P6 conditions, using the same preparation method as the LNPs in Example 3; C2-N7-L1-T16-P0 refers to LNPs prepared from ionizable lipid C2-N7-L1-T16 under formulation P0 conditions, using the same preparation method as the LNPs in Example 3. N4-L1-T16-P6 refers to the LNP formulation obtained by preparing N4-L1-T16 under the P6 formulation conditions, and N7-L1-T16-P0 refers to the LNP formulation obtained by preparing N7-L1-T16 under the P0 formulation conditions. The preparation method is the same as the LNP preparation method in Example 3. P0 conditions: molar ratio of ionizable lipids / auxiliary lipids / cholesterol lipids / PEG lipids 50 / 10 / 38.5 / 1.5. P6 conditions: molar ratio of ionizable lipids / auxiliary lipids / cholesterol lipids / PEG lipids 55 / 5 / 38 / 2.

[0129] Figure 7Figure a shows the nanoparticle size distribution of SM102-P0 LNPs, representative phosphoesterified LNP formulations C2-N4-L1-T16-P6 and C2-N7-L1-T16-P0, and corresponding non-phosphoesterified LNP formulations N4-L1-T16-P6 and N7-L1-T16-P0. Figure 7 In the formula, b represents the Zeta potential value of the LNPs formulation (ionizable lipids C2-N1-L1-T14, C2-N1-L1-T16, C2-N3-L1-T10, C2-N3-L1-T12, C2-N2-L1-T18, C2-N5-L1-T12, C2-N4-L1-T16, C2-N7-L1-T16, N1-L1-T14, N1-L1-T16, N3-L1-T10, N3-L1-T12, N2-L1-T18, N5-L1-T12, N4-L1-T16, N7-L1-T16, each prepared by the same method as in Example 3) under P0 conditions. Figure 7 In the formula, c represents the Zeta potential value of the LNPs formulation (ionizable lipids C2-N1-L1-T14, C2-N1-L1-T16, C2-N3-L1-T10, C2-N3-L1-T12, C2-N2-L1-T18, C2-N5-L1-T12, C2-N4-L1-T16, C2-N7-L1-T16, N1-L1-T14, N1-L1-T16, N3-L1-T10, N3-L1-T12, N2-L1-T18, N5-L1-T12, N4-L1-T16, N7-L1-T16, the preparation method of each corresponding LNPs formulation is the same as in Example 3) under the P6 conditions.

[0130] Structure of ionizable lipids:

[0131] C2-N1-L1-T14: ;

[0132] C2-N1-L1-T16: ;

[0133] C2-N3-L1-T10: ;

[0134] C2-N3-L1-T12: ;

[0135] C2-N2-L1-T18: ;

[0136] C2-N5-L1-T12: ;

[0137] C2-N4-L1-T16: ;

[0138] C2-N7-L1-T16: ;

[0139] N1-L1-T14: ;

[0140] N1-L1-T16: ;

[0141] N3-L1-T10: ;

[0142] N3-L1-T12: ;

[0143] N2-L1-T18: ;

[0144] N5-L1-T12: ;

[0145] N4-L1-T16: ;

[0146] N7-L1-T16: .

[0147] Example 6

[0148] LNPs mRNA was injected intravenously into C57BL / 6j mice. luc Delivery performance evaluation:

[0149] Five-week-old female C57BL / 6j mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were selected and injected via tail vein with mRNA-loaded mice at a dose of 0.1 mg / kg. lucThe LNP formulations are as follows: C2-N4-L1-T16 LNPs are formulated with a molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 of 55 / 5 / 38 / 2, and the preparation method is the same as in Example 3 (i.e., C2-N4-L1-T16-P6); C2-N7-L1-T16 LNPs are formulated with a molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 of 50 / 10 / 38.5 / 1.5, and the preparation method is the same as in Example 3 (i.e., C2-N7-L1-T16-P0); N4-L1-T16 LNPs are formulated with a molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 of 55 / 5 / 38 / 2, and the preparation method is the same as in Example 3 (i.e., N4-L1-T16-P6); N7-L1-T16... LNPs were prepared according to the same molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 as in Example 3 (i.e., N7-L1-T16-P0). Four hours later, 100 μL of a 10 mg / mL concentration of D-fluorescein potassium salt substrate was injected intraperitoneally into mice. Five minutes later, the major organs and tissues (brain, heart, lung, liver, spleen, and kidney) of the mice were removed and observed and photographed using an imaging system to record the chemiluminescence intensity of the isolated organs and tissues. The in vivo delivery efficiency and distribution results are shown in [the table below]. Figure 8 .

[0150] SM102 Lung SORT LNPs (SM102 / DOPE / cholesterol / DMG-PEG2000 / DOTAP molar ratio = 25:5:19.25:0.75:50, DOTAP being 1,2-dioleoyl-3-trimethylammonium propane chloride) and SM102 Spleen SORTLNPs (SM102 / DOPE / cholesterol / DMG-PEG2000 / 18PA molar ratio = 35:7:27:1.1:30, 18PA being 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt) were used as control groups for lung selectivity and spleen selectivity, respectively.

[0151] Figure 8Image a shows the in vitro major tissue imaging of each LNP formulation 4 hours after intravenous injection into mice, including the brain, heart, lung, liver, spleen, and kidney. Among them, SM102 Lung SORT is a known lung-targeting LNP formulation; SM102 Spleen SORT is a known spleen-targeting LNP formulation; C2-N4-L1-T16-P6 is a phosphonate-modified lung-targeting LNP formulation; N4-L1-T16-P6 is a non-phosphonate-modified LNP formulation; C2-N7-L1-T16-P0 is a phosphonate-modified spleen-targeting LNP formulation; and N7-L1-T16-P0 is a non-phosphonate-modified LNP formulation. Figure 8 b represents the statistical analysis of the average fluorescence values ​​of various organs after treatment with SM102 Lung SORT, C2-N4-L1-T16-P6, and N4-L1-T16-P6. Figure 8 c represents the statistical analysis of the average fluorescence values ​​of various organs after treatment with SM102 Spleen SORT, C2-N7-L1-T16-P0, and N7-L1-T16-P0.

[0152] The results show that the phosphoesterified ionizable lipids C2-N4-L1-T16 and C2-N7-L1-T16 described in this invention can effectively ionize mRNA. luc Selective delivery to target organs other than the liver and efficient gene editing were achieved, with multiple phosphoesterified ionizable lipids demonstrating superior delivery efficiency and selectivity compared to the control group. In contrast, unmodified ionizable lipids failed to deliver mRNA. luc Delivery to target organs other than the liver.

[0153] Example 7

[0154] Immunogenicity and toxicity testing of phosphoesterified LNP formulations:

[0155] To evaluate the in vivo immunogenicity of phosphonate-esterified LNPs, 5-week-old wild-type C57BL / 6J mice (half male and half female) were used. siRNA-loaded mice were prepared according to the method described in Example 3. lucThe LNP formulations were administered via tail vein injection at a volume of 100 μL per mouse. The LNP formulations included C2-N4-L1-T16-P6, C2-N7-L1-T16-P0, N4-L1-T16-P6, and N7-L1-T16-P0, with different siRNA doses (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg siRNA / kg mouse) to investigate dose-immunogenicity. SM102-P0 LNPs, SM102 Lung SORT LNPs, and SM102 Spleen SORT LNPs were used as controls. Six hours after administration, whole blood samples were collected, and the mice were subsequently euthanized. Blood samples were centrifuged at 4 ℃, 1,500 × g for 10 min to separate plasma. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the plasma were detected using the corresponding enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Enzyme Immunosorbent Assay Co., Ltd., China) according to the manufacturer's instructions. The test results are as follows: Figure 9 .

[0156] C2-N4-L1-T16-P6 refers to LNPs preparations obtained by using ionizable lipid C2-N4-L1-T16 under formulation P6 conditions; C2-N7-L1-T16-P0 refers to LNPs preparations obtained by using ionizable lipid C2-N7-L1-T16 under formulation P0 conditions. N4-L1-T16-P6 refers to LNPs preparations obtained by using N4-L1-T16 under formulation P6 conditions, and N7-L1-T16-P0 refers to LNPs preparations obtained by using N7-L1-T16 under formulation P0 conditions. P0 conditions: Molar ratio of ionizable lipid / auxiliary lipid / cholesterol lipid / PEG lipid 50 / 10 / 38.5 / 1.5. P6 conditions: Molar ratio of ionizable lipid / auxiliary lipid / cholesterol lipid / PEG lipid 55 / 5 / 38 / 2. siRNA is used in the preparation of LNPs formulations. luc Replace mRNA luc The molecular weight of siRNA was 13860 g / mol, N / P=6, and other conditions were the same as those for the preparation of LNPs in Example 3.

[0157] SM102-P0 LNPs refer to LNPs prepared from ionizable lipid SM102 under P0 formulation conditions. siRNA luc Replace mRNA luc N / P=6, and the preparation method is the same as that for LNPs in Example 3.

[0158] SM102 Lung SORT LNPs: SM102 / DOPE / cholesterol / DMG-PEG2000 / DOTAP molar ratio = 25:5:19.25:0.75:50; SM102 Spleen SORT LNPs: SM102 / DOPE / cholesterol / DMG-PEG2000 / 18PA molar ratio = 35:7:27:1.1:30. siRNA luc Replace mRNA luc N / P=6.

[0159] The results showed that the levels of inflammatory cytokines induced by phosphoesterified LNPs at all doses were lower than those of the control groups and the unphosphoesterified LNPs components, indicating that the LNPs preparations made from the phosphoesterified ionizable lipids of the present invention have significantly reduced immunogenicity and thus have higher safety for in vivo application.

[0160] Example 8

[0161] Storage stability testing of phosphoesterified LNP formulations:

[0162] To evaluate the storage stability of phosphonate-esterified LNPs formulations, SM102 LungSORT, SM102 Spleen SORT, C2-N4-L1-T16-P6, C2-N7-L1-T16-P0, C2-N4-L1-T16-A2, and C2-N7-L1-T16-A5 LNPs formulations were prepared according to the method in Example 3. The samples were stored at 4°C for 21 days, and in vitro transfection efficiency and particle size were measured every 7 days. B16 luc cells were used for in vitro transfection. The test results are as follows: Figure 10 .

[0163] LNP formulations of SM102 Lung SORT, SM102 Spleen SORT, C2-N4-L1-T16-P6, and C2-N7-L1-T16-P0 are described in Example 7.

[0164] C2-N4-L1-T16-A2 refers to an LNP formulation prepared from ionizable lipid C2-N4-L1-T16 in an A2 formulation (the molar ratio of ionizable lipid / helper lipid / cholesterol lipid / PEG lipid / LAP lipid is 45 / 10 / 38.5 / 1.5 / 5). siRNA luc Replace mRNA luc N / P=6, and the preparation method is the same as that for LNPs in Example 3.

[0165] C2-N7-L1-T16-A5 LNPs formulations refer to LNPs formulations prepared from ionizable lipid C2-N7-L1-T16 in an A5 formulation (the molar ratio of ionizable lipid / helper lipid / cholesterol lipid / PEG lipid / LAP lipid is 50 / 5 / 38.5 / 1.5 / 5). siRNA luc Replace mRNA luc N / P=6, and the preparation method is the same as that for LNPs in Example 3.

[0166] The results showed that LNPs containing anionic lipids (LAP) began to disintegrate on day 7, and after day 14, the transfection efficiency decreased by more than 50%, with the particle size increasing to over 200 nm. In contrast, phosphoesterified LNPs without LAP did not show a significant decrease in transfection efficiency on day 14, and the particle size remained stable. This indicates that the phosphoesterified LNP formulation of this invention possesses better storage stability and maintains stable transfection efficiency.

[0167] Example 9

[0168] In vivo escape ability test of phosphoesterified LNP formulations:

[0169] To evaluate the endosome escape ability of phosphonate-esterified LNPs, B16 cells in exponential growth stages were cultured in confocal microscopy dishes. When the cell density reached 30%, LNPs loaded with Cy5-siRNA (a Cy5 fluorescently labeled siRNA), namely C2-N4-L1-T16-P6, C2-N4-L1-T16-A2, N4-L1-T16-P6, C2-N7-L1-T16-P0, C2-N7-L1-T16-A5, and N7-L1-T16-P0, were added according to the method in Example 3. The cells were further cultured in a 37°C, 5% CO2 incubator. Time-lapse imaging (2-8 hours) using a laser confocal microscope was performed to observe the endosome escape process. Lysosomes were labeled with LysoTracker Green, and cell nuclei were labeled with Hoechst 33342. The test results are as follows: Figure 11 .

[0170] C2-N4-L1-T16-P6 refers to LNPs prepared from ionizable lipid C2-N4-L1-T16 under formulation P6 conditions; C2-N7-L1-T16-P0 refers to LNPs prepared from ionizable lipid C2-N7-L1-T16 under formulation P0 conditions. N4-L1-T16-P6 refers to LNPs prepared from N4-L1-T16 under formulation P6 conditions, and N7-L1-T16-P0 refers to LNPs prepared from N7-L1-T16 under formulation P0 conditions. P0 conditions: molar ratio of ionizable lipid / auxiliary lipid / cholesterol lipid / PEG lipid 50 / 10 / 38.5 / 1.5. P6 conditions: molar ratio of ionizable lipid / auxiliary lipid / cholesterol lipid / PEG lipid 55 / 5 / 38 / 2. In LNPs formulation preparation, siRNA replaces mRNA. luc The molecular weight of siRNA was 13860 g / mol, N / P=6, and other conditions were the same as those for the preparation of LNPs in Example 3.

[0171] C2-N4-L1-T16-A2 refers to an LNP formulation prepared from the ionizable lipid C2-N4-L1-T16 in an A2 formulation (the molar ratio of ionizable lipid / helper lipid / cholesterol lipid / PEG lipid / LAP lipid is 45 / 10 / 38.5 / 1.5 / 5), with siRNA replacing mRNA. luc N / P=6, and the preparation method is the same as that for LNPs in Example 3.

[0172] C2-N7-L1-T16-A5 LNPs formulations refer to LNPs formulations prepared using the ionizable lipid C2-N7-L1-T16 in an A5 formulation (the molar ratio of ionizable lipid / helper lipid / cholesterol lipid / PEG lipid / LAP lipid is 50 / 5 / 38.5 / 1.5 / 5), with siRNA replacing mRNA. luc N / P=6, and the preparation method is the same as that for LNPs in Example 3.

[0173] The results showed that the phosphonate-modified C2-N4-L1-T16-P6 and C2-N7-L1-T16-P0 LNPs exhibited better endogenous escape ability than the unmodified N4-L1-T16-P6 and N7-L1-T16-P0 LNPs. This indicates that the phosphonate-modified LNPs of this invention possess good endogenous escape ability while retaining stability.

[0174] Example 10:

[0175] Proteomic analysis of protein canopy adsorbed on LNPs:

[0176] The three LNPs (SM102 Spleen SORT, N7-L1-T16-P0, and C2-N7-L1-T16-P0) exhibit completely different distribution characteristics in vivo, which may be attributed to differences in the composition of the protein canopy formed on their surfaces. To verify this hypothesis, the differences in the adsorption of phosphoesterified LNPs to serum proteins were assessed. Mouse serum was placed in 1.5 mL centrifuge tubes, and the mRNA-loaded SM102 Spleen SORT, N7-L1-T16-P0, and C2-N7-L1-T16-P0 LNPs were co-incubated with the mouse serum. After 30 minutes, the protein precipitate was collected by centrifugation at 13000 g for 15 minutes at 4°C, followed by washing once with 1×PBS. The obtained protein precipitate was stored on dry ice for proteomics analysis. The proteomics detection was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The test results are as follows: Figure 12 . Figure 12 In the middle a: N7-L1-T16-P0 (left) and SM102 Spleen SORT (right); Figure 12 Middle b: N7-L1-T16-P0 (left) and C2-N7-L1-T16-P0 (right); Figure 12 c: SM102 Spleen SORT (left) and C2-N7-L1-T16-P0 (right).

[0177] SM102 Spleen SORT: SM102 / DOPE / cholesterol / DMG-PEG2000 / 18PA molar ratio = 35:7:27:1.1:30, prepared by the same method as LNPs in Example 3.

[0178] C2-N7-L1-T16-P0 refers to LNP formulations obtained by preparing ionizable lipid C2-N7-L1-T16 under P0 formulation conditions. N7-L1-T16-P0 refers to LNP formulations obtained by preparing N7-L1-T16 under P0 formulation conditions. P0 conditions: molar ratio of ionizable lipid / auxiliary lipid / cholesterol lipid / PEG lipid 50 / 10 / 38.5 / 1.5. The preparation method is the same as the LNP preparation method in Example 3.

[0179] The results showed that, regarding key apolipoproteins, the abundance of ApoE was significantly increased in the N7-L1-T16-P0 group compared to the C2-N7-L1-T16-P0 group, while the ApoE level in the SM102 Spleen SORT group was comparable to that in the C2-N7-L1-T16-P0 group. This indicates that unmodified phosphoester LNPs have a stronger tendency to adsorb apolipoproteins, which may be one of the important reasons why they are difficult to selectively deliver nucleic acid drugs to extrahepatic tissues.

[0180] This invention can phosphoesterify SM102 and ALC-0315, and the reaction equation for the preparation is as follows:

[0181] .

[0182] .

[0183] This invention provides a phosphonate-based ionizable lipid with a phosphonate group and an ionizable amino group at its head, a degradable linker in the middle, and a hydrophobic tail chain of controllable length on the periphery. The preparation process of this ionizable lipid requires minimal equipment and is highly operable. Through in vitro and in vivo screening, formulation optimization, and safety assessment, the phosphonate-based ionizable lipid of this invention significantly improves its effectiveness and safety for nucleic acid delivery. Furthermore, selective delivery to different sites can be achieved by adjusting different structural groups and formulations.

Claims

1. A phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs, characterized in that: Its general structural formula is Formula I: Formula I: ; In formula I, N represents nitrogen; Cn represents... , where n' is an integer from 0 to 20; * indicates a connection point; Nn is ; B is either O or NH; Ln acts as a connecting bond, with a structure of -(CH2). n1 -SS-(CH2) n2 - where n1 = an integer from 0 to 6, and n2 = an integer from 0 to 6; T1 is m is an integer from 1 to 8; T2 is m is an integer from 1 to 8.

2. The phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs according to claim 1, characterized in that: In Equation I, n' in the Cn structure is 0, 1, 2, 3, 4, or 5; the Ln structure acts as a connecting bond, and its structure is -(CH2). n1 -SS-(CH2) n2 - where n1 is 0, 1, or 2, and n2 is 0, 1, or 2.

3. The phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs according to claim 1, characterized in that: Includes one or more of the following structures; 。 4. The method for preparing phosphoesterified ionizable lipids for selective delivery of nucleic acid drugs according to any one of claims 1 to 2, characterized in that: Includes the following steps: 1) Reacting a phosphonate-containing compound with a compound containing p-nitrobenzene to obtain a phosphonate-modified compound; The structure of phosphine ester-containing compounds is as follows: The structure of the compound containing p-nitrobenzene is The structure of the phosphine-esterified compound is as follows: ； In the structure, Cn, Nn, B, and Ln are defined in Equation I as described in claim 1 or 2; 2) Remove the Boc protecting group from the phosphoesterified compound and then react it with an epoxide to obtain phosphoesterified ionizable lipids; The structure of the epoxy alkane is as follows: , where m" is an integer from 2 to 7.

5. The method for preparing phosphoesterified ionizable lipids for selective delivery of nucleic acid drugs according to claim 4, characterized in that: The reaction conditions in step 1) are 0~4℃ and carried out under a protective atmosphere; triethylamine is added during the reaction. The reaction is carried out in an organic solvent, namely dichloromethane; In step 2), the removal of the Boc protecting group refers to dissolving the phosphine-esterified modified compound in dichloromethane, adding a dioxane solution of hydrogen chloride, shaking, allowing it to stand, removing the supernatant, and obtaining an oily substance. The reaction with epoxides refers to dissolving the oily substance in ethanol and then adding epoxides to react; the reaction conditions are 80~90℃ for 10~16h.

6. The application of the phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs according to any one of claims 1 to 3, characterized in that: The phosphoesterified ionizable lipids for selective delivery of nucleic acid drugs are used to prepare phosphoesterified ionizable lipid nanoparticles, which serve as delivery carriers for selective delivery of nucleic acid drugs to the heart, lungs, spleen, liver, and / or muscles.

7. A phosphine-esterified ionizable lipid nanoparticle, characterized in that: The following method was used to prepare the nanoparticles: ionizable lipids, auxiliary lipids, sterol lipids, and PEG lipids were dissolved in an organic solvent to obtain an oil phase solution; nucleic acid drugs were dissolved in water or a buffer solution to obtain an aqueous phase; and the oil and aqueous phases were mixed under stirring to obtain phosphoesterified ionizable lipid nanoparticles, which are selective delivery carriers for nucleic acid drugs. The ionizable lipid is a phosphoesterified ionizable lipid for selective delivery of nucleic acid drugs as defined in any one of claims 1 to 3; The sterol lipids include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; The auxiliary lipids include one or more of phospholipids, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols and their derivatives; The PEG lipid is DMG-PEG2000; The nucleic acid drugs are mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, microRNA, shRNA, and DNA; The molar ratio of ionizable lipid: auxiliary lipid: cholesterol lipid: PEG lipid is (45~55):(5~15):(38~39):(1~2); The molar ratio (N / P ratio) of ionizable nitrogen atoms in the ionizable lipid to phosphorus atoms in the nucleic acid drug is 1:1 to 50:

1.

8. The phosphoesterified ionizable lipid nanoparticles according to claim 7, characterized in that: The sterol lipid is cholesterol; the auxiliary lipid is dioleoylphosphatidylethanolamine; The molar percentages of ionizable lipids, cofactor lipids, cholesterol lipids, and PEG lipids in the total composition are as follows: ionizable lipids 45%-55%, cofactor lipids 5%-15%, cholesterol lipids 38%-39%, and polyethylene glycol lipids 1%-2%; total composition: ionizable lipids, cofactor lipids, cholesterol lipids, and PEG lipids.

9. The application of the phosphoesterified ionizable lipid nanoparticles according to claim 7, characterized in that: The phosphoesterified ionizable lipid nanoparticles are used to prepare drug formulations for selective delivery of nucleic acid drugs to the heart, lungs, spleen, liver and / or muscles.

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