Metal coordination lipid compound as well as composition and application thereof

Abstract

The invention relates to a metal coordination lipid compound, which is a compound shown in a chemical formula I, or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof.

Description

Technical Field

[0001] This invention application belongs to the field of gene therapy and new materials, and relates to a metal-coordinated lipid compound, its composition and application. Background Technology

[0002] The development of gene therapy is inseparable from nucleic acid delivery systems. In recent years, mRNA therapy has emerged as a rising star, showing great promise in areas such as vaccine development and protein replacement therapy. Messenger ribonucleic acid (mRNA) therapy overcomes the limitations of traditional nucleic acid therapies and demonstrates enormous potential in life science fields such as protein replacement therapy, gene editing, and vaccine development.

[0003] Following the approval of drugs such as BNT162b2 and mRNA-1273, lipid nanoparticles (LNPs) utilizing these technologies have become the most promising delivery system for mRNA drugs, ushering in a new era of mRNA therapy. However, the lack of efficient mRNA delivery vectors has become a key obstacle to the further development of mRNA therapy.

[0004] LNPs, as a relatively mature delivery system for mRNA drugs, are key carriers for promoting the implementation of mRNA therapy. A typical LNP consists of four components: ionizable lipids, phospholipids, cholesterol, and PEGylated lipids. Among them, ionizable lipids are the core component that determines delivery efficiency. Their structure includes amine groups, hydrophobic tail chains, and groups connecting the two. They encapsulate mRNA through charge interactions and mediate its entry into cells.

[0005] However, the development of existing ionizable lipids is mostly limited to the functional group modification of amine groups, hydrophobic tail chains or linker groups, which has limited improvement in delivery efficiency and is difficult to meet the needs of novel mRNA therapies for efficient delivery systems. Summary of the Invention

[0006] To address the shortcomings of existing technologies, there is an urgent need to develop breakthrough ionizable lipid modification strategies to significantly improve the mRNA delivery efficiency of LNPs. This invention provides a metal-coordinated lipid compound, its composition, and its applications. This invention specifically designs a metal-coordinated dimethylpyridinium amine element, which is modified onto an amine molecule to further obtain a metal-coordinated ionizable lipid, upon which LNPs are constructed. Through the synergistic effect of the specific lipid compound and the metal coordination, binding to phosphate-containing cell membranes and endosome membranes is enhanced, improving endocytosis, endosome escape, and mRNA release, unexpectedly and significantly increasing the mRNA delivery efficiency of LNPs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a compound represented by chemical formula (I), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: (I) M is a metal ion, L1-L2 are independent structures that are unrelated to each other, L1 represents Cm residue, L2 represents An residue (amine residue), R represents different hydrophobic molecule EP residue or Ac residue, and n represents the number of hydrophobic molecule residues, n is 0, 1, 2, 3, 4 or 5. The M is selected from Cu 2+ Ca 2+ Co 2+ Mg 2+ Mn 2+ Zn 2+ Fe 2+、 One or more of the following; preferably, M is selected from Ca. 2+ Co 2+ Mg 2+ Zn 2+ Fe 2+、 One or more of the following; more preferably, M is selected from Ca. 2+ Mg 2+ Zn 2+ One or more of the following; In some embodiments, the Cm residue is selected from one of the following structures: C1, C2, and C3: C1: C2: C3: ; Preferably, the Cm residue is selected from one of C1 and C2: C1: C2: ; In some embodiments, the amine residue is selected from one of the following structures: A1, A2, A3, A4, A5, A6, A7: A1: A2: A3: ; A4: A5: A6: ; A7: ; Preferably, the amine residue is selected from one of the following structures: A3, A5, A6, A7: A3: A5: A6: ; A7: ; In some embodiments, the hydrophobic EP residues are selected from one of the following structures: EP8, EP10, and EP12. EP8: ;EP10: ;EP12: ; Preferably, the hydrophobic EP residue is selected from one of the following structures: EP10, EP12: EP10: ;EP12: ; In some embodiments, the hydrophobic Ac residue is selected from one of the following structures: Ac8, Ac10, and Ac12: Ac8: Ac10: Ac12: ; Preferably, the hydrophobic Ac residue is selected from one of the following structures: Ac8, Ac12: Ac8: Ac12: ; “ "Indicates a connection site; In some embodiments, the metal-coordinated lipid compound is selected from C1-A7-EP10-ZnD, C1-A3-EP12-ZnD, and C2-A7-EP12-ZnD, and its structural formula is as follows: , , .

[0008] Secondly, the present invention provides a method for preparing a metal-coordinated lipid compound: Step S100: Dissolve the compound corresponding to the Cm residue, dimethylpyridinium amine and / or an inorganic base in an organic solvent, react them, and process them after the reaction to obtain Cm-D; Step S200: Mix the compound corresponding to the An residue and the Cm-D obtained in step S100 in an organic solvent and react them. After the reaction is complete, process the mixture to obtain Cm-An-D. Step S300: Mix the compound corresponding to the EP residue or the compound corresponding to the Ac residue with the Cm-An-D obtained in step S200 in an organic solvent and react. After the reaction is completed, remove the solvent by rotary evaporation and purify to obtain Cm-An-EP-D or Cm-An-Ac-D. Step S400: The Cm-An-EP-D (or Cm-An-Ac-D) obtained in step S300 and the metal salt are mixed in an organic solvent and reacted. After the reaction is completed, the metal-coordinated Cm-An-EP-ZnD (or Cm-An-Ac-ZnD) final product is obtained by vacuum drying. Preferably, the compound corresponding to the Cm residue is selected from C1-1, C2-1, and C3-1, and its structural formula is as follows: , , ; Preferably, the compound corresponding to the An residue is selected from A1-1, A2-1, A3-1, A4-1, A5-1, A6-1, and A7-1, and its structural formula is as follows: , , , , , , ; Preferably, the compounds corresponding to the EP residues are selected from EP8-1, EP10-1, and EP12-1, and their structural formulas are as follows: , , ; Preferably, the compounds corresponding to the Ac residues are selected from Ac8-1, Ac10-1, and Ac12-1, and their structural formulas are as follows: , , ; Preferably, in step S100, the inorganic base is selected from at least one of anhydrous potassium carbonate, anhydrous sodium carbonate, and lithium hydroxide; Preferably, in step S100, the organic solvent is selected from at least one of dichloromethane, chloroform, ethanol, isopropanol, dimethyl sulfoxide, and dimethylformamide; preferably, dichloromethane, chloroform, or isopropanol. Preferably, in step S100, the reaction temperature is 15-80°C; more preferably, the reaction temperature is 25-75°C. Preferably, in step S100, the reaction time is 24-72 hours; more preferably, the reaction time is 36-60 hours. Preferably, in step S200, the organic solvent is selected from at least one of isopropanol, ethanol, dichloromethane, chloroform, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S200, the reaction temperature is 65-85°C; more preferably, the reaction temperature is 70-80°C. Preferably, in step S200, the reaction time is 4-20 hours; more preferably, the reaction time is 9-15 hours. Preferably, in step S300, the organic solvent is selected from at least one of isopropanol, ethanol, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S300, the reaction temperature is 65-85°C; more preferably, the reaction temperature is 70-80°C. Preferably, in step S300, the reaction time is 24-72 hours; more preferably, the reaction time is 36-60 hours. Preferably, in step S400, the metal salt is selected from one of Zn(NO3)2•6H2O, Cu(CF3SO3)2, Ca(CF3SO3)2, CoCl2•6H2O, MgCl2•6H2O, MnCl2•4H2O, and FeCl2•4H2O. Preferably, in step S400, the organic solvent is selected from at least one of isopropanol, methanol, ethanol, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S400, the reaction temperature is 15-60°C; more preferably, the reaction temperature is 20-25°C. Preferably, in step S400, the reaction time is 1-15 hours; more preferably, the reaction time is 2-6 hours.

[0009] Thirdly, the present invention provides a lipid nanoparticle comprising a metal-coordinated lipid compound as described in the first aspect or a metal-coordinated lipid compound prepared by the preparation method described in the second aspect. Furthermore, the lipid nanoparticles contain auxiliary lipids; Furthermore, the auxiliary lipid is selected from any one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids; Preferably, the phospholipid is selected from any one or a combination of dioleoylphosphatidylethanolamine (DOPE), distearate phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), myristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), and sphingomyelin (SM); and / or, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol, and ergosterol; and / or, the polymer-conjugated lipid is selected from lipids conjugated with polyethylene glycol, polylactic acid, polyamide, cationic polymers, polysarcosine, polylactic-co-glycolic acid copolymers, polyamino acids, polypeptides, and peptide-conjugated groups; and / or, the modifiable lipid is selected from lipids that can be modified by small molecule compounds, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules, metal ions, and combinations thereof. Preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) or distearate phosphatidylcholine (DSPC); and / or, the steroid is cholesterol and / or sitosterol; and / or, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC0159), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 1000 (DMG-PEG1000), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 5000 (DMG-PEG1000), etc. One or more of DMG-PEG5000, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and phospholipid-methoxy polyethylene glycol 2000 (DSPE-PEG2000); more preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE); and / or, the steroid is cholesterol; and / or, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid selected from 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000); Further, the molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:1-82; preferably, the molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:2-10; more preferably, the molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:3-10.

[0010] Fourthly, the present invention provides the application of the metal-coordinated lipid compound described in the first aspect, or the metal-coordinated lipid compound prepared by the preparation method described in the second aspect, or the lipid nanoparticles described in the third aspect, in the preparation of drug delivery carriers.

[0011] Preferably, the drug is a therapeutic agent or a preventive agent; Preferably, the therapeutic or preventative agent is a nucleic acid; Preferably, the nucleic acid is selected from: single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isomers, plasmid DNA, complementary DNA, antisense nucleic acid molecules, small interfering nucleic acids, small activating nucleic acids, asymmetric interfering nucleic acids, micronucleic acids, agomir, antagomir, Dicer enzyme substrate nucleic acids, hairpin nucleic acids, transfer RNA, messenger RNA, and circular RNA; more preferably, the nucleic acid is selected from: transfer RNA, messenger RNA, and circular RNA; most preferably, the nucleic acid is messenger RNA.

[0012] Fifthly, the present invention provides a pharmaceutical composition comprising lipid nanoparticles as described in the fourth aspect, and a therapeutic or preventative agent.

[0013] In a sixth aspect, the present invention provides a formulation comprising the pharmaceutical composition as described in the fifth aspect and pharmaceutically acceptable excipients.

[0014] Compared with the prior art, the beneficial effects of this invention application are as follows: (1) In this invention, lipid nanoparticles (LNPs) containing metal-coordinated lipid compounds encapsulate mRNA by means of the strong binding of phosphate groups and metal ligands on the basis of the original electrostatic interaction. At the same time, due to the competitive effect between metal ligands and phospholipid membranes (including cell membranes and endosomal membranes), the metal coordination and specific lipid compounds work together to enhance the endocytosis and endosomal escape capabilities of the formulation and effectively release mRNA in the cytoplasm.

[0015] (2) The novel metal-coordinated ionizable lipid is used for LNPs construction, which synergistically improves the efficiency of multiple links in the mRNA delivery process and significantly improves the mRNA delivery efficiency, with the highest delivery efficiency reaching 29 times that of the currently FDA-approved SM-102LNPs. Attached Figure Description

[0016] Figure 1 Chemical structure diagrams of sixteen DPA derivatives (Cm-An-D).

[0017] Figure 2 : Structural diagram of uncoordinated lipid compounds.

[0018] Figure 3 : The 1H NMR spectrum of the ionizable lipid C1-A7-EP10-D.

[0019] Figure 4 : The 1H NMR spectrum of the ionizable lipid C1-A3-EP12-D.

[0020] Figure 5: The 1H NMR spectrum of the ionizable lipid C2-A7-EP12-D.

[0021] Figure 6 Particle size and polydispersity index (PDI) plots of representative LNPs.

[0022] Figure 7 Zeta potential diagram of representative LNPs.

[0023] Figure 8 mRNA encapsulation efficiency of representative LNPs.

[0024] Figure 9 : Graph showing the effect of LNPs delivering mRNA in vitro.

[0025] Figure 10 : Cytotoxicity of LNPs on IGROV1 cells.

[0026] Figure 11 : Lipid fusion assay of LNPs.

[0027] Figure 12 Fluorescence images of IGROV1 cells treated with C1-A7-EP10-D LNPs and C1-A7-EP10-ZnD LNPs loaded with Cy5-mRNA, where scale bar: 5 μm.

[0028] Figure 13 mRNA release efficiency of representative LNPs.

[0029] Figure 14 : Percentage of mRNA released from LNPs after incubation with different antagonists, where NaCl, EDTA, Tween-20 and urea act as ion antagonists, metal chelators, hydrophobic interaction antagonists and hydrogen bond antagonists, respectively.

[0030] Figure 15 Figure: Screening results of in vivo FLuc mRNA delivery for different LNPs, where H: heart, Lu: lung, Li: liver, K: kidney, and S: spleen.

[0031] Figure 16 : Graph showing the improved delivery efficiency of representative zinc-coordinated LNPs after intravenous injection.

[0032] Figure 17 : Orthogonal screening results of C1-A7-EP10-ZnD LNP in vivo formulation; a) First round orthogonal screening diagram of C1-A7-EP10-ZnD LNPs, H: heart, Lu: lung, Li: liver, K: kidney, S: spleen; b) Effect of the four components on the in vivo delivery efficiency of mRNA.

[0033] Figure 18 The second round of orthogonal screening diagram for C1-A7-EP10-ZnD LNPs, where H: heart, Lu: lung, Li: liver, K: kidney, and S: spleen.

[0034] Figure 19 In vivo liver mRNA delivery efficiency diagram after intravenous injection of C1-A7-EP10-ZnD LNPs and SM-102 LNPs.

[0035] Figure 20 In vivo mRNA delivery efficiency diagram of C1-A7-EP10-ZnD LNPs and SM-102 LNPs after intramuscular injection.

[0036] Figure 21 In vivo mRNA delivery map of different metal-coordinated LNPs: C1-A7-EP10-D, H: heart, Lu: lung, Li: liver, K: kidney, S: spleen. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] 1 1H NMR spectroscopy determination: The NMR spectra were determined using a Varian INOVA 500 MHz NMR spectrometer at 25 °C with deuterated chloroform (CDCl3) as the solvent.

[0039] The particle size and zeta potential of LNPs were measured using dynamic light scattering (DLS) technology with the Zetasizer Nano-ZS from Malvern, UK.

[0040] The formula for calculating the lipid fusion rate (%) is: (F-Fmin) / (Fmax-Fmin)×100%.

[0041] Statistical analysis: Statistical analysis was performed using GraphPad Prism 9 software. Data are presented as mean ± standard deviation. When comparing data between two or more groups, two-tailed unpaired Student's t-tests or one-way ANOVA were used, respectively. When the p-value is less than 0.05 (…), a p-value of less than 0.05 is considered statistically significant. ), 0.01 ( ), 0.001 ( ) and 0.0001 ( When the difference is 0, it is considered to be statistically significant.

[0042] The SM-102 structure is as follows:

[0043] It is worth noting that the raw materials used in this invention application are all common commercially available products, and their sources are not specifically limited.

[0044] Example 1: Overall synthetic pathway of novel zinc-coordinated ionizable lipids Cm-An-EP-ZnD and Cm-An-Ac-ZnD Cm-1 represents different linking materials (C1-1, C2-1, C3-1), where Cm represents the corresponding residue; An-1 represents different small molecule amines (A1-1, A2-1, A3-1, A4-1, A5-1, A6-1, A7-1), where An represents the amine residue; EP-m represents hydrophobic molecules (EP8-1, EP10-1, EP12-1), Ac-m represents hydrophobic molecules (Ac8-1, Ac10-1, Ac12-1), where EP or Ac represents the corresponding residue; DPA is dimethylpyridinium, where D represents the corresponding residue.

[0045] Synthetic routes of Cm-An-EP-ZnD and Cm-An-Ac-ZnD (a); raw materials used (b)

[0046] Synthesis of C1-D: dichlorobenzyl, DPA, and anhydrous potassium carbonate were dissolved in dichloromethane and reacted at room temperature for 48 hours under nitrogen protection. After the reaction was complete, solid potassium carbonate was removed by filtration. After removing dichloromethane by rotary evaporation, the crude product was purified by silica gel column chromatography using dichloromethane:methanol (40:1, v / v) as the eluent. The purified product was then dried by rotary evaporation to obtain C1-D.

[0047] Synthesis of C2-D / C3-D: 1,2,7,8-diepoxyoctane or 1,4-butanediol diacrylate and DPA were dissolved in isopropanol and reacted at 75°C for 48 hours. After removing the isopropanol by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (40:1, v / v) as the eluent. After rotary drying, C2-D / C3-D was obtained.

[0048] Synthesis of Cm-An-EP-ZnD / Cm-An-Ac-ZnD: An-1 and Cm-D were mixed in isopropanol. For C1-D, triethylamine was added as an acid-binding agent during the reaction, while for C2-D and C3-D, triethylamine was not required. After stirring at 75°C for 12 hours, the isopropanol was removed, and the crude product was obtained by extraction. The organic phase was dried with anhydrous sodium sulfate and then dried under vacuum to obtain Cm-An-D (see attached diagram). Figure 1 EP-m (epoxyoctane, epoxydecane, or epoxydodecane) or Ac-m (octyl acrylate, decyl acrylate, or dodecyl acrylate) and Cm-An-D were mixed in isopropanol and stirred at 75°C for 48 hours. The solvent was removed by rotary evaporation, and Cm-An-EP-D / Cm-An-Ac-D were obtained by silica gel column chromatography (see attached image). Figure 2 Cm-An-EP-D / Cm-An-Ac-D and zinc nitrate hexahydrate were mixed in isopropanol, stirred at room temperature for 6 hours, and then dried under vacuum to obtain the zinc-coordinated Cm-An-EP-ZnD / Cm-An-Ac-ZnD final product.

[0049] Example 2: Synthesis of zinc-coordinated ionizable lipid C1-A7-EP10-ZnD

[0050] Dichlorobenzyl (3.24 mmol), DPA (1.62 mmol), and anhydrous potassium carbonate (8.10 mmol) were added sequentially to a round-bottom flask, followed by the addition of dichloromethane (10 mL) under nitrogen protection. The reaction was carried out at room temperature for 48 hours. After removing the dichloromethane by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (40:1, v / v) as the eluent. The purified product was dried by rotary evaporation to obtain C1-D. A7-1 (tris(2-aminoethyl)amine, 6.35 mmol) and triethylamine (6.35 mmol) were mixed in isopropanol (10 mL), followed by the dropwise addition of C1-D (1.27 mmol). The mixture was stirred at 75 °C for 12 hours. The product was extracted with chloroform in saturated brine (saturated brine:chloroform = 1:3, v / v). The organic phase was dried over anhydrous sodium sulfate and then dried under vacuum to obtain C1-A7-D. C1-A7-D (1.63 mmol) and EP10-1 (decane oxide, 8.15 mmol) were mixed in isopropanol (10 mL) and reacted at 75 °C for 48 hours. After removing the isopropanol by vacuum drying, the product was purified by silica gel column chromatography using dichloromethane:methanol (30:1, v / v) as the eluent, and then dried by rotary evaporation to obtain C1-A7-EP10-D.

[0051] C1-A7-EP10-D 1 H NMR spectrum (with) Figure 3 ), 1H NMR(500 MHz, CDCl3, ppm): δ8.54–8.49(d,2H),7.70–7.64(m,2H),7.59(d,2H),7.37(d,2H),7.25(d,2H) ,7.17–7.12(m,2H),3.80–3.48(m,13H),2.86–1.99(m,22H),1.37–1.21(m,70H),0.87(t,15H). Mass calculated:1228.07,found[M+H] + (ESI-HRMS): 1229.07.

[0052] C1-A7-EP10-D (1.31 mmol) and zinc nitrate hexahydrate (1.31 mmol) were mixed in isopropanol (10 mL) and reacted at room temperature for 6 hours. The mixture was then dried by rotary evaporation to obtain C1-A7-EP10-ZnD.

[0053] Example 3: Synthesis of zinc-coordinated ionizable lipid C1-A3-EP12-ZnD

[0054] Dichlorobenzyl (3.24 mmol), DPA (1.62 mmol), and anhydrous potassium carbonate (8.10 mmol) were added sequentially to a round-bottom flask, followed by the addition of dichloromethane (10 mL) under nitrogen protection. The reaction was carried out at room temperature for 48 hours. After removing the dichloromethane by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (40:1, v / v) as the eluent. The purified product was dried by rotary evaporation to obtain C1-D. A3-1 (1,4-butanediamine, 7.80 mmol) and triethylamine (7.80 mmol) were mixed in isopropanol (10 mL), followed by the dropwise addition of C1-D (1.56 mmol). The mixture was stirred at 75 °C for 12 hours. The product was extracted with chloroform in saturated brine (saturated brine:chloroform = 1:3, v / v). The organic phase was dried over anhydrous sodium sulfate and then dried under vacuum to obtain C1-A3-D. C1-A3-D (2.12 mmol) and EP12-1 (epoxydodecane, 6.36 mmol) were mixed in isopropanol (10 mL) and reacted at 75 °C for 48 hours. After removing the isopropanol by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (30:1, v / v) as the eluent. The purified product was then dried by rotary evaporation to obtain C1-A3-EP12-D.

[0055] C1-A3-EP12-D 1 H NMR spectrum (with) Figure 4 ), 1H NMR(500 MHz, CDCl3, ppm): δ8.51(d,2H),7.70–7.64(m,2H),7.60(d,2H),7.36(d,2H),7.23(d,2H),7 .18–7.11(m,2H),3.83–3.60(m,11H),2.64–2.31(m,10H),1.44–1.24(m,58H),0.88(t,9H). Mass calculated:941.81,found [M+Na] + (ESI-HRMS): 964.82.

[0056] C1-A3-EP12-D (1.61 mmol) and zinc nitrate hexahydrate (1.61 mmol) were mixed in isopropanol (10 mL) and reacted at room temperature for 6 hours. The mixture was then dried by rotary evaporation to obtain C1-A3-EP12-ZnD.

[0057] Example 4: Synthesis of zinc-coordinated ionizable lipid C2-A7-EP12-ZnD

[0058] 1,2,7,8-diepoxyoctane (11.0 mmol) and DPA (2.20 mmol) were added to isopropanol (10 mL), and the reaction was carried out at 75 °C for 48 hours. After removing the isopropanol by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (40:1, v / v) as the eluent. After rotary evaporation and drying, C2-D was obtained. A7-1 (9.35 mmol) and C2-D (1.87 mmol) were mixed in isopropanol (10 mL), and stirred at 75 °C for 12 hours. The product after rotary evaporation was extracted with chloroform in saturated brine (saturated brine:chloroform = 1:3, v / v). The organic phase was dried over anhydrous sodium sulfate and rotary evaporated to obtain C2-A7-D. C2-A7-D (1.42 mmol) and EP12-1 (7.10 mmol) were mixed in isopropanol (10 mL), and the reaction was carried out at 75 °C for 48 hours. After removing isopropanol by rotary evaporation, the product was purified by silica gel column chromatography using dichloromethane:methanol (30:1, volume ratio) as eluent. C2-A7-EP12-D was obtained after rotary evaporation.

[0059] C2-A7-EP12-D 1 H NMR spectrum (with) Figure 5 ), 1H NMR(500 MHz, CDCl3, ppm): δ8.54(d,2H),7.63–7.56(m,2H),7.31(d,2H),7.16–7.11(m,2H),4 .01–3.82(m,4H),3.70(m,7H),2.87–2.25(m,26H),1.48–1.25(m,98H),0.88(t,15H). Mass calculated:1408.28, found [M+H] + (ESI-HRMS):1409.32.

[0060] C2-A7-EP12-D (1.17 mmol) and zinc nitrate hexahydrate (1.17 mmol) were mixed in isopropanol (10 mL) and reacted at room temperature for 6 hours. The mixture was then dried under vacuum to obtain C2-A7-EP12-ZnD.

[0061] Example 5: Lipid Synthesis with Other Metal Coordination C1-A7-EP10-D and equimolar amounts of Cu(CF3SO3)2 (or Ca(CF3SO3)2, CoCl2•6H2O, MgCl2•6H2O, MnCl2•4H2O, FeCl2•4H2O) were mixed in isopropanol and reacted at 60°C for 6 hours. The mixture was then dried by rotary evaporation to obtain C1-A7-EP10-MD, where M represents copper, calcium, cobalt, magnesium, manganese, iron, etc.

[0062] Example 6: Preparation and Characterization of Lipid Nanoparticles (LNPs) LNPs were prepared using an ethanol dilution method: ionizable lipids, dioleoylphosphatidylethanolamine (DOPE), cholesterol, and dimyristic-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2000 (DMG-PEG2000) were dissolved in ethanol. mRNA was diluted in sodium citrate buffer (10 mM, pH 4.0), maintaining a constant weight ratio of ionizable lipids to mRNA of 10 / 1. The two solutions were rapidly mixed at a 1 / 3 volume ratio of ethanol to water, and then incubated at room temperature for 15 minutes. LNPs were dialyzed for 2 hours before in vivo experiments.

[0063] When the molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 is 15 / 20 / 25 / 2, the particle size of the LNP formulation is approximately 100 nm, and zinc coordination has no significant effect on the particle size (see appendix). Figure 6 The zeta potential is near neutral, ranging from -5 to +5 mV (see attached image). Figure 7The encapsulation efficiency of mRNA was determined using the Quant-iT RiboGreen RNA Detection Kit. The encapsulation rate of coordinated LNPs exceeded 80%, which was higher than that of uncoordinated LNPs (see attached). Figure 8 ).

[0064] Example 7: In vitro mRNA delivery and cytotoxicity assay In in vitro screening experiments, human ovarian cancer IGROV1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) at 37°C and 5% carbon dioxide. IGROV1 cells were cultured at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in white 96-well plates, with three parallel wells per group. After 24 hours, lipid nanoparticles (LNPs) loaded with firefly luciferase (FLuc) mRNA were prepared and added to each well at a dose of 25 ng mRNA. Unless otherwise specified, the molar ratio of ionizable lipid / DOPE / cholesterol / DMG-PEG2000 used was 15 / 20 / 25 / 2. Luciferase expression and cytotoxicity were assessed using the Bio-Lumi™ II Firefly Luciferase Assay Kit and the Alamar Blue Assay Kit, respectively. Results showed that zinc-coordinated LNPs generally had higher in vitro delivery efficiency (see attached). Figure 9 Furthermore, all LNPs exhibited low in vitro toxicity (cell viability ≥80%) (see appendix). Figure 10 ).

[0065] Example 8: Measurement of membrane fusion by fluorescence resonance energy transfer (FRET) FRET probes NBD-PE (nitrobenzoxadiazole-phosphatidylethanolamine) and Rho-PE (rhodamine-phosphatidylethanolamine) were prepared in liposomes mimicking endosomes. Due to fluorescence resonance energy transfer to rhodamine, the fluorescence of NBD was weakened. When lipid fusion occurred, the distance between the two probes increased, leading to an enhanced NBD signal. Therefore, membrane fusion could be assessed by measuring fluorescence intensity. The mimicking endosomes were prepared as follows: DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) / DOPS (1,2-dioleoyl-sn-glycerol-3-phosphoserine) / DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine) / Rho-PE / NBD-PE (molar ratio 25 / 25 / 48 / 1 / 1) were mixed in chloroform, and the mixture was subjected to rotary evaporation and vacuum drying for 2 hours to obtain a lipid film. Next, buffer (pH 7.4) was added and the mixture was sonicated for 30 minutes to obtain a solution with a total lipid concentration of 1 mM. The total lipid concentration of the lipid nanoparticles (LNPs) was 1 mM, and the molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 was 5 / 20 / 25 / 2. 100 μL of buffer (pH 5.5), 1 μL of anionic endosome-mimicking liposomes, and 10 μL of LNPs were added to the wells of a black 96-well plate. After incubation at 37°C for 5 minutes or other times, the fluorescence intensity (F) was measured using a microplate reader at an excitation wavelength / emission wavelength of 465 / 520 nm. Anionic endosome-mimicking liposomes were used as the negative control (Fmin), and liposomes containing the FRET probe and Triton X-100 (1 wt.%) were used as the positive control (Fmax). LNPs containing zinc-coordinating lipids showed significantly higher lipid fusion membrane fusion results at different time points (5 min, 60 min, 180 min) than LNPs without zinc-coordinating lipids (see attached). Figure 11 ).

[0066] Example 9: Assay of endosome escape and cellular uptake Confocal imaging was used to determine endosome escape and cellular uptake of lipid nanoparticles (LNPs). IGROV1 cells were seeded at a density of 500,000 cells / well in confocal culture dishes. After 24 hours, 1.5 mL of fresh culture medium (containing 10% FBS) was added, and the cells were treated with LNPs containing 1 μg Cy5-mRNA. The LNPs were prepared from ionizable lipids / DOPE / cholesterol / DMG-PEG2000 at a molar ratio of 5:20:25:2. After 2 and 4 hours, the cells were washed three times with 1× phosphate-buffered saline (PBS), lysosomes were stained with Lysotracker Green DND 26 (1 / 5000 dilution) at 37°C for 2 hours, and the nuclei were stained with Hoechst 33342 (1 / 100 dilution) for 15 minutes. Cell imaging was performed using confocal microscopy. In the image, red indicates successful endosome entry into the cell, while the separation of red and green signals indicates successful endosome escape. The image demonstrates that zinc-coordinated LNPs can mediate effective endosome escape and cellular uptake. (See attached image) Figure 12 ).

[0067] Example 10: mRNA release assay mRNA release was determined by mixing LNPs with liposomes simulating anionosomes. The LNPs contained ionizable lipids / DOPE / cholesterol / DMG-PEG2000 in a molar ratio of 5 / 20 / 25 / 2 (total lipid concentration 1 mM). The lipid mixture of the endosomes was prepared in chloroform at a DOPS / DOPC / DOPE molar ratio of 25 / 25 / 50. Anionosome membranes were obtained by rotary evaporation, followed by vacuum drying for 2 hours to remove the solvent. The membranes were then hydrated by sonication in buffer (pH 5.5) to obtain a solution with a total lipid concentration of 10 mM. 100 μL of buffer (pH 5.5), 1 μL of LNPs, and 1 μL of liposomes simulating anionosomes were added to each well of a black 96-well plate and incubated at 37°C for 5 min or 30 min. mRNA release was detected using a microplate reader with the Quant-iT Ribogreen RNA assay kit. The results showed that the mRNA release rate of LNPs containing zinc-coordinating lipids was significantly increased, reaching 40%-60% at 30 min (see appendix). Figure 13 ).

[0068] Example 11: Interaction forces between mRNA and LNPs To determine the main interaction forces between mRNA and LNPs, the prepared LNPs were incubated in sodium chloride (NaCl, an electrostatic interaction antagonist), ethylenediaminetetraacetic acid (EDTA, a metal chelating agent), Tween-20 (a hydrophobic interaction antagonist), and urea (a hydrogen bond antagonist), with a final antagonist concentration of 100 mM for each. After 24 hours, the LNPs were dialyzed for 2 hours. The release of mRNA was detected using a microplate reader with the Quant-iT Ribogreen RNA assay kit. The release rates of naked mRNA and untreated LNPs were set as 100% and 0%, respectively. LNPs containing zinc-coordinating lipids encapsulate mRNA mainly through coordination and electrostatic interactions (see attached image). Figure 14 ).

[0069] Example 12: In vivo mRNA delivery Prepared LNPs were administered intravenously or intramuscularly to 6-8 week old C57BL / 6 mice at a luciferase (FLuc) mRNA dose of 0.25 mg / kg. Six hours later, mice were anesthetized with isoflurane and then intraperitoneally injected with 100 μL of D-luciferin substrate (30 mg / mL). Chemiluminescence in whole mice and isolated organs was measured using an IVIS Spectrum system (PerkinElmer). In the initial screening, the molar ratio of ionizable lipids / DOPE / cholesterol / DMG-PEG2000 was set at 15 / 20 / 25 / 2. Chemiluminescence signal and delivery efficiency were positively correlated, and LNPs with overall zinc coordination showed higher delivery efficiency than uncoordinated LNPs (see appendix). Figure 15 The in vivo delivery efficiency of the three representative zinc-coordinated lipids after intravenous injection was higher than that of the corresponding uncoordinated lipids (the luciferase activity expressed in the liver is as follows: C1-A3-EP12-D: 1.78×10). 6 p / sec / cm 2 / sr, C1-A3-EP12-ZnD: 6.54×10 6 p / sec / cm 2 / sr;C1-A7-EP10-D:4.89×10 6 p / sec / cm 2 / sr, C1-A7-EP10-ZnD: 3.50×10 7 p / sec / cm 2 / sr;C2-A7-EP12-D:3.11×10 6 p / sec / cm 2 / sr, C2-A7-EP12-ZnD: 1.87×10 7 p / sec / cm2 / sr) (attached) Figure 16 Subsequently, orthogonal experiments were used to optimize the C1-A7-EP10-ZnDLNPs formulation with different molar ratios. The results showed that when the molar ratio of ionizable lipid / DOPE / cholesterol / DMG-PEG2000 was set to 5 / 20 / 25 / 2, it exhibited higher luminescence intensity and a higher delivery efficiency, reaching 7.87 × 10⁻⁶. 7 p / sec / cm 2 / sr (attached) Figure 17 Appendix Figure 18 Using SM-102 LNPs as a positive control, the molar ratio of SM-102 / distearate phosphatidylcholine (DSPC) / cholesterol / DMG-PEG2000 was 50 / 10 / 38.5 / 1.5, and the nitrogen-to-phosphorus ratio (N / P) was 6. The optimized LNPs containing zinc-coordinating lipids showed significantly improved delivery efficiency after intravenous injection compared to SM-102 LNPs, with the luciferase signal reaching 29 times that of SM-102 LNPs (the SM-102 group signal was only 2.75 × 10⁻⁶). 6 p / sec / cm2 / sr (attached) Figure 19 The efficacy after intramuscular injection was also higher than that of SM-102 LNPs (C1-A7-EP10-ZnD: 8.45×10). 5 p / sec / cm 2 / sr, SM-102: 1.34×10 5 p / sec / cm2 / sr (attached) Figure 20 ).

[0070] Other divalent metals (Cu) 2+ Ca 2+ Co 2+ Mg 2+ Mn 2+ Fe 2+ Coordinated LNPs can still mediate high mRNA delivery efficiency, such as Ca. 2+ Mg 2+ The coordinated LNP delivered luciferase mRNA, and the enzyme activity signal reached 1.38 × 10⁻⁶. 7 p / sec / cm 2 / sr and 1.59×10 7 p / sec / cm 2 / sr (attached) Figure 21 Cobalt-coordinated LNPs can also deliver mRNA to the spleen, which holds promise for applications in immunotherapy.

[0071] In summary, this invention selects metal-coordinated lipid compounds, which exert an overall synergistic effect through specific lipid compounds and metal coordination. The specific lipid compounds with metal coordination interact with the phosphate groups of biological membranes, thereby improving endocytosis, endosome escape and mRNA release, unexpectedly and significantly improving the delivery efficiency of LNPs for nucleic acids.

[0072] Those skilled in the art will understand that the steps, measures, and schemes in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted; furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted; furthermore, the steps, measures, and schemes in the prior art that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above-described embodiments are merely illustrative of several implementation methods of this disclosure, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent for the embodiments of this disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this disclosure, and these all fall within the protection scope of the embodiments of this disclosure. Therefore, the protection scope of the embodiments of this disclosure should be determined by the appended claims. As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and detail can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

[0073] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A metal-coordinated lipid compound, characterized in that, Compounds represented by Formula I, or their stereoisomers, tautomers, or pharmaceutically acceptable salts thereof: （Ⅰ） in: M is a metal ion, L1-L2 are independent structures that are unrelated to each other, L1 represents Cm residue, L2 represents An residue (amine residue), R represents different hydrophobic molecule EP residue or Ac residue, and n represents the number of hydrophobic molecule residues, n is 0, 1, 2, 3, 4 or 5. The M is selected from Cu 2+ Ca 2+ Co 2+ Mg 2+ Mn 2+ Zn 2+ Fe 2+ One or more of the following; preferably, M is selected from Ca. 2+ Co 2+ Mg 2+ Zn 2+ Fe 2+ One or more of the following; more preferably, M is selected from Ca. 2+ Mg 2+ Zn 2+ One or more of the following; The Cm residues are selected from one of the following structures: C1, C2, and C3: C1: ;C2: ;C3: ; Preferably, the Cm residue is selected from one of C1 and C2: C1: C2: ; The amine residue is selected from one of the following structures: A1, A2, A3, A4, A5, A6, A7. A1: A2: A3: ; A4: A5: A6: ; A7： ； Preferably, the amine residue is selected from one of the following structures: A3, A5, A6, A7: A3: A5: A6: ; A7： ； The hydrophobic EP residue is selected from one of the following structures: EP8, EP10, and EP12. EP8: ;EP10: ;EP12: ; Preferably, the hydrophobic EP residue is selected from one of the following structures: EP10, EP12: EP10: ;EP12: ; The hydrophobic Ac residue is selected from one of the following structures: Ac8, Ac10, and Ac12. Ac8: ;Ac10: ;Ac12: ; Preferably, the hydrophobic Ac residue is selected from one of the following structures: Ac8, Ac12: Ac8: ;Ac12: ; " " " indicates a connection point.

2. The metal-coordinated lipid compound according to claim 1, characterized in that, The metal-coordinated lipid compound is selected from C1-A7-EP10-ZnD, C1-A3-EP12-ZnD, and C2-A7-EP12-ZnD, and its structural formula is as follows: 、 、 。 3. A method for preparing the metal-coordinated lipid compound as described in claim 1 or 2: Step S100: Dissolve the compound corresponding to the Cm residue, dimethylpyridinium amine and / or an inorganic base in an organic solvent, react them, and process them after the reaction to obtain Cm-D; Step S200: Mix the compound corresponding to the An residue and the Cm-D obtained in step S100 in an organic solvent and react them. After the reaction is complete, process the mixture to obtain Cm-An-D. Step S300: Mix the compound corresponding to the EP residue or the compound corresponding to the Ac residue with the Cm-An-D obtained in step S200 in an organic solvent and react. After the reaction is completed, remove the solvent by rotary evaporation and purify to obtain Cm-An-EP-D or Cm-An-Ac-D. Step S400: The product Cm-An-EP-D (or Cm-An-Ac-D) obtained in step S300 is mixed with a metal salt in an organic solvent and reacted. After the reaction is completed, the product is dried under vacuum to obtain the metal-coordinated Cm-An-EP-ZnD (or Cm-An-Ac-ZnD) final product. Preferably, the compound corresponding to the Cm residue is selected from C1-1, C2-1, and C3-1, and its structural formula is as follows: , , ; Preferably, the compound corresponding to the An residue is selected from A1-1, A2-1, A3-1, A4-1, A5-1, A6-1, and A7-1, and its structural formula is as follows: , , , , , , ; Preferably, the compounds corresponding to the EP residues are selected from EP8-1, EP10-1, and EP12-1, and their structural formulas are as follows: , , ; Preferably, the compounds corresponding to the Ac residues are selected from Ac8-1, Ac10-1, and Ac12-1, and their structural formulas are as follows: , , ; Preferably, in step S100, the inorganic base is selected from at least one of anhydrous potassium carbonate, anhydrous sodium carbonate, and lithium hydroxide; Preferably, in step S100, the organic solvent is selected from at least one of dichloromethane, chloroform, ethanol, isopropanol, dimethyl sulfoxide, and dimethylformamide; preferably, dichloromethane, chloroform, or isopropanol. Preferably, in step S100, the reaction temperature is 15-80°C; more preferably, the reaction temperature is 25-75°C. Preferably, in step S100, the reaction time is 24-72 hours; more preferably, the reaction time is 36-60 hours. Preferably, in step S200, the organic solvent is selected from at least one of isopropanol, ethanol, dichloromethane, chloroform, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S200, the reaction temperature is 65-85°C; more preferably, the reaction temperature is 70-80°C. Preferably, in step S200, the reaction time is 4-20 hours; more preferably, the reaction time is 9-15 hours. Preferably, in step S300, the organic solvent is selected from at least one of isopropanol, ethanol, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S300, the reaction temperature is 65-85°C; more preferably, the reaction temperature is 70-80°C. Preferably, in step S300, the reaction time is 24-72 hours; more preferably, the reaction time is 36-60 hours. Preferably, in step S400, the metal salt is selected from one of Zn(NO3)2•6H2O, Cu(CF3SO3)2, Ca(CF3SO3)2, CoCl2•6H2O, MgCl2•6H2O, MnCl2•4H2O, and FeCl2•4H2O. Preferably, in step S400, the organic solvent is selected from at least one of isopropanol, methanol, ethanol, dimethyl sulfoxide, and dimethylformamide; preferably, isopropanol or ethanol; more preferably, isopropanol. Preferably, in step S400, the reaction temperature is 15-60°C; more preferably, the reaction temperature is 20-25°C. Preferably, in step S400, the reaction time is 1-15 hours; more preferably, the reaction time is 2-6 hours.

4. A lipid nanoparticle comprising the metal-coordinated lipid compound as described in any one of claims 1-2 or the metal-coordinated lipid compound prepared by the preparation method described in claim 3.

5. The lipid nanoparticles as described in claim 4, characterized in that, The lipid nanoparticles further comprise auxiliary lipids selected from any one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids.

6. The lipid nanoparticles as described in claim 5, characterized in that, The phospholipid is selected from any one or a combination of dioleoylphosphatidylethanolamine (DOPE), distearate phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), myristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), and sphingomyelin (SM); and / or, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol, and ergosterol; and / or, the polymer-conjugated lipid is selected from lipids conjugated with polyethylene glycol, polylactic acid, polyamide, cationic polymers, polysarcosine, polylactic-co-glycolic acid copolymers, polyamino acids, polypeptides, and peptide clusters; and / or, the modifiable lipid is selected from lipids that can be modified by small molecule compounds, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules, metal ions, and combinations thereof. Preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) or distearate phosphatidylcholine (DSPC); and / or, the steroid is cholesterol and / or sitosterol; and / or, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid selected from 2-[(PEG)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 1000 (DMG-PEG1000), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 5000 (D... The steroid is selected from one or more of the following: MG-PEG5000, 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and phospholipid-methoxy polyethylene glycol 2000 (DSPE-PEG2000); more preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE); and / or, the steroid is cholesterol; and / or, the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid selected from 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000).

7. The lipid nanoparticles according to any one of claims 4-6, characterized in that, The molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:1-82; Preferably, the molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:2-10; More preferably, the molar ratio of the metal-coordinated lipid compound to the auxiliary lipid is 1:3-10.

8. The use of the metal-coordinated lipid compound as described in any one of claims 1-2, or the metal-coordinated lipid compound prepared by the preparation method as described in claim 3, or the lipid nanoparticles as described in any one of claims 4-7, in the preparation of drug delivery carriers; Preferably, the drug is a therapeutic agent or a preventive agent; Preferably, the therapeutic or preventative agent is a nucleic acid; Preferably, the nucleic acid is selected from: single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isomers, plasmid DNA, complementary DNA, antisense nucleic acid molecules, small interfering nucleic acids, small activating nucleic acids, asymmetric interfering nucleic acids, micronucleic acids, agomir, antagomir, Dicer enzyme substrate nucleic acids, hairpin nucleic acids, transfer RNA, messenger RNA, and circular RNA; more preferably, the nucleic acid is selected from: transfer RNA, messenger RNA, and circular RNA; most preferably, the nucleic acid is messenger RNA.

9. A pharmaceutical composition comprising lipid nanoparticles as described in any one of claims 4-7, and a therapeutic or preventative agent.

10. A formulation comprising the pharmaceutical composition of claim 9 and a pharmaceutically acceptable excipient.

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