Ionizable lipids, lipid nanoparticles based on a biogenic creatine backbone, and nucleic acid delivery applications thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to ionizable lipids based on a creatine skeleton, lipid nanoparticles and nucleic acid delivery applications thereof. The ionizable lipids based on creatine have a chemical structure shown in general formula (I): wherein n is an integer of 1-6, R1 and R2 are each independently selected from alkyl with 6-20 carbon atoms, ether bond-containing alkyl with 6-20 carbon atoms, alkenyl with 6-20 carbon atoms, ether bond-containing alkenyl with 6-20 carbon atoms, alkynyl with 6-20 carbon atoms, and ether bond-containing alkynyl with 6-20 carbon atoms. The novel ionizable lipids based on the creatine mother nucleus are expected to improve the nucleic acid drug loading efficiency, cell uptake capacity and endosome escape efficiency while maintaining good safety, and provide a new technical path for constructing a safe and efficient lipid nanoparticle delivery system.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to ionizable lipids, lipid nanoparticles and their nucleic acid delivery applications based on the biogenic creatine backbone. Background Technology

[0002] Nucleic acid drugs are a class of exogenous DNA or RNA with therapeutic activity. They have advantages such as flexible design, high specificity, short development cycle, and low likelihood of drug resistance, and have been widely used in gene editing, protein replacement, and nucleic acid vaccines. However, their large molecular weight and negative charge make it difficult for them to penetrate cell membranes and enter cells. They are easily degraded by nucleases or cleared by the kidneys. Even if they enter cells, they are often unable to exert their effects due to lysosomal degradation, which limits their in vivo application.

[0003] Lipid nanoparticles (LNPs) are among the most promising nucleic acid delivery systems, capable of encapsulating and protecting various nucleic acids such as siRNA, mRNA, and microRNA, thereby improving their stability and cellular uptake efficiency in vivo. Currently approved clinical LNPs mostly utilize ionizable lipids such as DLin-MC3-DMA, SM-102, and ALC-0315, but these still suffer from high toxicity and insufficient delivery efficiency. Therefore, there is an urgent need to develop novel, low-toxicity, and highly efficient ionizable lipids and their LNP delivery systems to promote the clinical application of nucleic acid drugs.

[0004] Creatine is an endogenous nitrogenous organic acid widely found in body tissues and is a core component of the creatine / phosphocreatine energy buffer system, playing a crucial role in cellular energy storage, transport, and homeostasis. Its molecular structure contains functional groups such as guanidine, carboxyl, and N-methyl, exhibiting strong hydrophilicity, excellent biocompatibility, and a well-defined in vivo metabolic pathway, providing an ideal structural template for constructing ionizable lipids derived from natural small molecules. Incorporating the creatine backbone into the design of ionizable lipids offers several advantages. First, its nitrogenous basic groups (such as guanidine) endow lipids with ionizable properties under different pH conditions, enhancing their electrostatic binding and compressibility with negatively charged nucleic acids. Second, by covalently linking hydrophobic alkyl chains to derivatizable sites such as carboxyl groups, amphiphilic lipid molecules can be constructed, facilitating the formation of stable nanoparticles and achieving effective cell membrane fusion and endosome escape. However, how to precisely control the ionization behavior of creatine, optimize the self-assembly performance of lipids and the efficiency of nucleic acid encapsulation while preserving the good biocompatibility of creatine, and balance safety and delivery efficiency in complex physiological environments, remains a key technical challenge in the development of creatine-based ionizable lipids and high-performance nucleic acid delivery systems. Summary of the Invention

[0005] The purpose of this invention is to provide ionizable lipids, lipid nanoparticles, and their nucleic acid delivery applications based on a biogenic creatine backbone, thereby overcoming the shortcomings of existing technologies. Novel ionizable lipids based on a creatine core are expected to improve the loading efficiency, cellular uptake capacity, and endosome escape efficiency of nucleic acid drugs while maintaining good safety, providing a new technical path for constructing a safe and efficient lipid nanoparticle delivery system.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a creatine-based ionizable lipid is provided, having the chemical structure shown in general formula (I): ; Wherein, n is an integer from 1 to 6, and R1 and R2 are each independently selected from alkyl groups with 6 to 20 carbon atoms, alkyl groups with 6 to 20 carbon atoms containing ether bonds, alkenyl groups with 6 to 20 carbon atoms, alkenyl groups with 6 to 20 carbon atoms containing ether bonds, alkynyl groups with 6 to 20 carbon atoms, and alkynyl groups with 6 to 20 carbon atoms containing ether bonds.

[0007] In a second aspect, a method for preparing creatine-based ionizable lipids is provided, comprising the step of obtaining the compound of formula (I) by an amidation reaction of the compound of formula (II) and the compound of formula (III); , ; Wherein, n, R1, and R2 are the same as n, R1, and R2 in claim 1.

[0008] Thirdly, a lipid nanoparticle is provided, comprising creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids.

[0009] Fourthly, it provides the application of lipid nanoparticles in nucleic acid drug delivery carriers.

[0010] Fifthly, a pharmaceutical composition is provided, comprising an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug and the carrier is lipid nanoparticles.

[0011] The beneficial effects of this invention are: This invention utilizes creatine-based ionizable lipids to prepare lipid nanoparticles. Creatine is a widely distributed endogenous nitrogenous organic acid in the body, playing a crucial role in energy metabolism and the phosphocreatine cycle, and possesses excellent biocompatibility and safety. Creatine-based ionizable lipids use the creatine structure as a biologically derived functional core, possessing nitrogen-containing polar groups, modifiable carboxyl sites, and tertiary amine nucleic acid compression groups, which facilitates the encapsulation of nucleic acid drugs, the self-assembly of lipid nanoparticles, and intracellular delivery. The lipid nanoparticles prepared in this way exhibit high transfection efficiency, good safety, excellent encapsulation effect, and strong drug delivery capability.

[0012] The present invention provides a simple synthetic route for ionizable lipids based on creatine, with mild reaction conditions and readily available raw materials. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 The image shows the TEM characterization of lipid nanoparticles in Example 4 of this invention. Figure 2 This is a graph showing the results of lipid nanoparticle size determination in Example 4 of the present invention; Figure 3 This is a graph showing the results of lipid nanoparticle encapsulation efficiency measurement in Example 4 of the present invention; Figure 4 This is a diagram showing the results of lipid nanoparticle transfection into RAW264.7 cells in Example 4 of this invention. Detailed Implementation

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] As mentioned above, In a first aspect, a creatine-based ionizable lipid is provided, having the chemical structure shown in general formula (I): ; Wherein, n is an integer from 1 to 6, and R1 and R2 are each independently selected from alkyl groups with 6 to 20 carbon atoms, alkyl groups with 6 to 20 carbon atoms containing ether bonds, alkenyl groups with 6 to 20 carbon atoms, alkenyl groups with 6 to 20 carbon atoms containing ether bonds, alkynyl groups with 6 to 20 carbon atoms, and alkynyl groups with 6 to 20 carbon atoms containing ether bonds.

[0017] In some other implementations, n is an integer from 2 to 5, preferably 2 or 3; R1 and R2 are each independently selected from straight-chain alkyl groups with 9 to 18 carbon atoms and straight-chain alkenyl groups with 9 to 18 carbon atoms; Preferably, R1 and R2 are each independently selected from straight-chain alkyl groups having 9 to 14 carbon atoms or straight-chain alkenyl groups having 9 to 14 carbon atoms.

[0018] In some other implementations, R1 and R2 are each independently selected from... , , , , .

[0019] In some other embodiments, the creatine-based ionizable lipids include the following compounds: JS-2-9; JS-2-14; JS-2-O; JS-3-9; JS-3-14; JS-3-O.

[0020] In a second aspect, a method for preparing creatine-based ionizable lipids is provided, comprising the step of obtaining the compound of formula (I) by an amidation reaction of the compound of formula (II) and the compound of formula (III); , ; Wherein, n, R1, and R2 are the same as n, R1, and R2 in claim 1.

[0021] The compound shown in formula (I) was obtained by following the reaction route below; .

[0022] In some other embodiments, the amidation reaction is carried out by mixing the compound shown in formula (II) and the compound shown in formula (III) with triethylamine (TEA), then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to dissolve and mix them evenly, and reacting at 4~40 °C for 24-48 h; Preferably, the molar ratio of the compound shown in formula (II), the compound shown in formula (III), EDCI, HOBT and TEA is 1:(1.05~1.5):(1.05~1.5):(1.05-1.5):(2-5); More preferably, the molar ratio of the compound shown in formula (II), the compound shown in formula (III), EDCI, HOBT and TEA is 1: 1.2: 1.2: 1.2: 4.5.

[0023] In some other embodiments, purification by silica gel column chromatography is performed after the reaction. The mobile phase for silica gel column chromatography is a mixture of petroleum ether and ethyl acetate.

[0024] Thirdly, a lipid nanoparticle is provided, comprising creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids.

[0025] In some other embodiments, the molar content of the creatine-based ionizable lipids is 10-60%, preferably 15-45%, and more preferably 28-36%. The total molar content is the sum of the molar amounts of the creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids.

[0026] In other embodiments, the neutral lipid is selected from at least one of distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristoylphosphatidylcholine (DMPC), and egg yolk lecithin (EPC). Dioleoylphosphatidylethanolamine (DOPE) is preferred. The molar content of the neutral lipid is 10-50%, preferably 20-40%, and more preferably 30-35%.

[0027] In some other embodiments, the polyethylene glycol lipid is selected from dipalmitoylphosphatidylethanolamine-PEG (DPPE). The polyethylene glycol lipid is selected from at least one of the following: PEG, 1,2-distearyl-rac-glycero-3-methoxy-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dioleoylphosphatidylethanolamine-PEG (DOPE-PEG), 2-[(polyethylene glycol)-2000]-N,N-dicarboxyacetamide (ALC-0159), distearylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol-PEG (DMG-PEG), and dimethacrylate-PEG (DMA-PEG). Dimyristylglycerol-PEG (DMG-PEG) is preferred. The molar content of the polyethylene glycol lipid is 0.5-15%, preferably 0.5-5.0%, more preferably 2.0-3.0%.

[0028] In some other embodiments, the steroidal lipids are selected from at least one of cholesterol, alfalfa sterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholesterol, cocciferol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, sulfonic acid, deoxycholic acid, and lithocholic acid. Cholesterol is preferred. The molar content of the steroidal lipids is 20-60%, preferably 25-35%, and more preferably 30-35%.

[0029] In some other embodiments, the molar ratio of the creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids is (10~60): (10~50): (0.5~15): (20~60), preferably (15~45): (20~40): (0.5~5.0): (25~35), and more preferably (15~35): (30~35): (2.0~3.0): (20~35). Alternatively, the diameter of the lipid nanoparticles is 1 nm to 1000 nm. Lipid nanoparticles are in particle form. The diameter of lipid nanoparticles is in the range of 20 nm to 800 nm, or 50 nm to 500 nm, or 80 nm to 200 nm, or 1 nm to 100 nm, or 1 nm to 10 nm. When the particle size ranges from 1 nm to 1000 nm, it is commonly referred to as a nanoparticle.

[0030] Lipid nanoparticles can be prepared using any method known in the art. This includes, but is not limited to, mixing aqueous solutions of bioactive substances with organic solvent solutions of lipid compounds using conventional liposome extrusion, thin-film hydration, pipette mixing, vortex mixing, and microfluidic mixing methods.

[0031] In some other embodiments, an aqueous solvent is included, which is an acidic buffer. More specifically, the buffer comprises at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tris(hydroxymethyl)aminomethane-hydrochloric acid, potassium dihydrogen phosphate-sodium hydroxide, boric acid-borax, glycine-hydrochloric acid, phthalic acid-hydrochloric acid, potassium hydrogen phthalate, and sodium dihydrogen phosphate-citric acid, preferably sodium citrate.

[0032] Fourthly, it provides the application of lipid nanoparticles in nucleic acid drug delivery carriers.

[0033] Fifthly, a pharmaceutical composition is provided, comprising an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug and the carrier is lipid nanoparticles; The nucleic acid drugs include, but are not limited to, nucleic acid molecules such as siRNA, linear mRNA, tRNA, rRNA, cDNA, ASO, plasmid DNA, circular RNA, microRNA, long non-coding RNA, and their analogues; The mass ratio of creatine-based ionizable lipids to nucleic acid drugs in the lipid nanoparticles is (1~50):1, preferably (5~15):1, and more preferably 15:1.

[0034] The following detailed embodiments further illustrate the following: Example 1 Synthesis of ionizable lipid compound JS-2-9 The synthesis route is shown below: Specifically, the following steps are included: Step ①: Synthesis of intermediate 1c Compound 1a was dissolved in anhydrous acetonitrile, potassium carbonate and compound 1b were added, the mixture was stirred, heated in an oil bath to 80 °C until the reaction was almost complete, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 1c.

[0035] Step 2: Synthesis of intermediate 1d Compound 1c was dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added under ice bath conditions. The reaction mixture was then transferred to room temperature and stirred until substantially complete. The solvent was removed from the reaction solution by rotary evaporation under reduced pressure, and then 20 mL of dichloromethane was added to dissolve it. The reaction was quenched by slowly adding saturated sodium bicarbonate solution under ice bath conditions. The organic phase was further washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate 1d, which was used directly in the next reaction step.

[0036] Step 3: Synthesis of the final product SHS-2-9 Compounds 1d, 1e, and TEA were mixed and stirred for 30 min. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) were added. The mixture was stirred at room temperature for 48 h. After the reaction was found to be basically complete by thin-layer chromatography, the reaction solution was diluted with dichloromethane, washed with saturated sodium bicarbonate and saturated brine, and the organic phase was dried with anhydrous sodium sulfate. After filtration and concentration, the final product JS-2-9 was obtained by silica gel column chromatography.

[0037] 1 H NMR (400 MHz, CDCl3) δ 3.35–3.25 (m, 5H), 2.65–2.20 (m, 8H), 1.50–1.35 (m, 4H), 1.35–1.20 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0038] 13 C NMR (101 MHz, CDCl3) δ 172.4, 158.3, 56.2, 53.8, 52.0, 41.5, 36.1,31.9, 29.7, 29.5, 29.3, 27.4, 26.8, 22.7, 14.1.

[0039] Example 2 Synthesis of ionizable lipid compound JS-2-14 The synthesis route is shown below: Specifically, the following steps are included: Step ①: Synthesis of intermediate 2c Compound 2a was dissolved in anhydrous acetonitrile, potassium carbonate and compound 2b were added, the mixture was stirred, heated in an oil bath to 80 °C until the reaction was almost complete, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 2c.

[0040] Step 2: Synthesis of intermediate 2d Compound 2c was dissolved in dichloromethane, and TFA was added under ice bath conditions. The reaction mixture was then transferred to room temperature and stirred until substantially complete. The solvent was removed from the reaction solution by rotary evaporation under reduced pressure, and then 20 mL of dichloromethane was added to dissolve it. The reaction was quenched by slowly adding saturated sodium bicarbonate solution under ice bath conditions. The organic phase was further washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate 1d, which was used directly in the next reaction step.

[0041] Step 3: Synthesis of the final product JS-2-14 Compounds 2d, 2e, and TEA were mixed and stirred for 30 min. EDCI and HOBT were then added, and the mixture was stirred at room temperature for 48 h. After the reaction was detected by thin-layer chromatography to be basically complete, the reaction solution was diluted with dichloromethane, washed with saturated sodium bicarbonate and saturated brine, and the organic phase was dried with anhydrous sodium sulfate. After filtration and concentration, the final product JS-2-14 was obtained by silica gel column chromatography.

[0042] 1 H NMR (400 MHz, CDCl3) δ 8.88–6.73 (br m, 3H), 3.70–2.95 (m, 5H), 2.70–2.35 (m, 8H), 1.95–1.85 (m, 2H), 1.50–1.20 (m, 48H), 0.90 (t, J = 6.8 Hz, 6H).

[0043] 13 C NMR (101 MHz, CDCl3) δ 205.8, 158.4, 56.6, 54.1, 52.8, 43.5, 36.6,31.9, 29.8–29.3, 27.5, 26.9, 23.2, 22.7, 14.1.

[0044] Example 3 Synthesis of ionizable lipid compound JS-3-O The synthesis route is shown below: Specifically, the following steps are included: Step ①: Synthesis of intermediate 3b Compound 3a and carbon tetrabromide were dissolved in dichloromethane. Triphenylphosphine, which is also soluble in dichloromethane, was added dropwise to the solution under an ice bath at 0 °C. After the addition was complete, the reaction was transferred to room temperature and stirred until the reaction was almost complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 3b.

[0045] Step 2: Synthesis of intermediate 3D Compound 3c was dissolved in anhydrous acetonitrile, potassium carbonate and compound 3b were added, the mixture was stirred, heated in an oil bath to 80 °C until the reaction was almost complete, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 3d.

[0046] Step 3: Synthesis of intermediate 3e Compound 3d was dissolved in dichloromethane, and TFA was added under ice bath conditions. The reaction mixture was then transferred to room temperature and stirred until substantially complete. The solvent was removed from the reaction solution by rotary evaporation under reduced pressure, and then 20 mL of dichloromethane was added to dissolve it. The reaction was quenched with saturated sodium bicarbonate solution under ice bath conditions. The organic phase was further washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate 3e, which was used directly in the next reaction step.

[0047] Step 4: Synthesis of the final product SHS-3-O Compounds 3e, 3f and TEA were mixed and stirred for 30 min. Then EDCI and HOBT were added and the mixture was stirred at room temperature for 48 h. After the reaction was basically complete as detected by thin-layer chromatography, the reaction solution was diluted with dichloromethane, washed with saturated sodium bicarbonate and saturated brine, and the organic phase was dried with anhydrous sodium sulfate. After filtration and concentration, the final product JS-3-O was obtained by silica gel column chromatography.

[0048] 1 H NMR (400 MHz, CDCl3) δ 8.90–6.70 (br m, 3H), 5.37–5.30 (m, 4H), 3.70–2.95 (m, 5H), 2.70–2.35 (m, 8H), 2.10–1.95 (m, 8H), 1.95–1.80 (m, 2H),1.55–1.20 (m, 48H), 0.90–0.85 (t, J = 6.8 Hz, 6H).

[0049] 13 C NMR (101 MHz, CDCl3) δ 205.8, 158.4, 130.0, 129.7, 56.6, 54.2,53.0, 43.6, 36.6, 31.9, 29.8–29.1, 27.3, 27.2, 26.9, 23.3, 22.7, 14.1.

[0050] Example 4 The lipid nanoparticles JS-2-9L, JS-2-14L, and JS-3-OL, based on the ionizable lipid compounds JS-2-9, JS-2-14, and JS-3-O, can be prepared by any of the following methods: Preparation method ①: pipette mixing method Ionizable lipid compounds JS-2-9, JS-2-14, and JS-3-O were each prepared into isopropanol solutions with a concentration of 10 mg / mL. These solutions were then reacted with DOPE (10 mg / mL), cholesterol (10 mg / mL), and DMG dissolved in ethanol. PEG (5 mg / mL) solutions were mixed in molar ratios of 30:30:35:2, 25:30:20:2.5, and 20:30:25:2, respectively, and diluted with isopropanol to a certain volume to obtain the organic phase. Then, the mRNA encoding EGFP (EGFP mRNA coding region sequence: Dissolve the ionizable lipid compound in citrate buffer to obtain an aqueous solution. Based on a mass ratio of 10:1 between the ionizable lipid compound and mRNA, take appropriate amounts of both aqueous and organic phase solutions. Rapidly inject the aqueous solution into the organic phase, pipette 70-80 times, and incubate at 0 °C for 10 min to obtain solutions of lipid nanoparticles JS-2-9L, JS-2-14L, and JS-3-OL.

[0051] Preparation method ②: Microfluidic mixing method The ionizable lipid compounds JS-2-9 / JS-2-14 / JS-3-O, DOPE, cholesterol, and DMG-PEG were prepared into an isopropanol solution at the same molar ratio as in preparation method ① to obtain the organic phase. The mRNA encoding EGFP was then dissolved in citrate buffer to obtain the aqueous phase. Based on a mass ratio of ionizable lipid compounds to mRNA of 10:1, appropriate amounts of the aqueous and organic phase solutions were taken. The organic and aqueous phases were respectively aspirated using syringes and connected to the organic phase inlet and aqueous phase inlet of the microfluidic mixing chip, respectively. The flow rate of the aqueous phase was set to 9 mL / min, the flow rate of the organic phase to 3 mL / min, the volumetric flow rate ratio of the aqueous phase to the organic phase to 3:1, and the total flow rate to 12 mL / min. The syringe pump was started to rapidly mix the two phases within the microfluidic chip. After the flow rate stabilized, the initial approximately 100–300 μL of eluent was discarded, and the eluent was collected to obtain a solution of lipid nanoparticles JS-2-9L, JS-2-14L, and JS-3-OL.

[0052] Performance testing 1. TEM morphology characterization of lipid nanoparticles The lipid nanoparticles were retrieved several times from a double-mesh square-hole copper mesh, dried at room temperature, and then photographed and observed under a transmission electron microscope. The results are as follows: Figure 1 As shown, the prepared lipid nanoparticles (JS-2-9L, JS-2-14L and JS-3-OL) have clear morphology, good dispersibility, and relatively neat and regular appearance, which proves that the prepared lipid nanoparticles are of good quality.

[0053] 2. Particle size determination of lipid nanoparticles Take 20 μL of the prepared lipid nanoparticle (JS-2-9L, JS-2-14L and JS-3-OL) solution, dilute it to 1 mL with 1×PBS solution, and determine the particle size of the lipid nanoparticle using a Malvern laser particle size analyzer.

[0054] Test results as follows Figure 2 As shown, the average hydrated particle size of lipid nanoparticles JS-2-9L is 115.3 nm, JS-2-14L is 126.2 nm, and JS-3-O is 113.4 nm, proving that the prepared lipid nanoparticles have uniform particle size and good quality.

[0055] 3. Determination of lipid nanoparticle encapsulation efficiency.

[0056] Prepare the standard curve solution according to the RiboGreen™ RNA Detection Kit instructions and obtain the relative fluorescence units using a fluorescence spectrophotometer. Dilute the lipid nanoparticles (JS-2-9L, JS-2-14L, and JS-3-OL) samples, and add 10 μL to each well of a cell culture plate, repeating for 6 wells. Add 40 μL of 2% Triton-100 to 3 wells containing the samples and treat for 5 min to rupture the lipid nanoparticles and release mRNA. Add 40 μL of 1×TE solution to the remaining 3 sample wells as a control, and add 50 μL of RiboGreen staining working solution to the remaining 6 sample wells. Use a Multimode Plate Reader (EnSight) system to read the fluorescence intensity, with the excitation light set to 480 nm and the emission light to 520 nm. After data processing, the RNA content in the solution before and after lipid nanoparticle demulsification can be obtained. Calculate the encapsulation efficiency using the following formula: Encapsulation efficiency (%) = (RNA content after demulsification - RNA content before demulsification) / RNA content after demulsification; Test results as follows Figure 3 As shown, the average encapsulation efficiency of the lipid nanoparticles JS-2-9L was 84.9%, JS-2-10L was 83.9%, and JS-3-OL was 83.0%, demonstrating that the prepared lipid nanoparticles can efficiently encapsulate mRNA.

[0057] 4. Assay of the cell transfection capability of lipid nanoparticles Logarithmically growing or RAW264.7 cells were seeded into 48-well cell culture plates at a density of 10,000 cells per well. Lipid nanoparticles (JS-2-9L, JS-2-14L, and JS-3-OL) were placed on ice and diluted with 1×PBS to a mRNA concentration of 10 ng / μL. After RAW264.7 cells adhered to the wells, the diluted lipid nanoparticles were added, along with 150 ng of mRNA per well, with three replicates per group. An equal volume of PBS served as a negative control, and the commercially available mRNA transfection reagent LipoSmart mRNATransfection Reagent (Genetic Biotech) served as a positive control. The proportion of EGFP-positive cells was detected by flow cytometry 24 h after transfection.

[0058] Test results as follows Figure 4 As shown, the average transfection efficiency of the lipid nanoparticles JS-2-9L was 56.4%, JS-2-14L was 36.9%, and JS-3-OL was 46.7%, proving that the prepared lipid nanoparticles can efficiently transfect cells.

[0059] Example 5 Based on the determination of the encapsulation efficiency of other ionizable lipid nanoparticles, the encapsulation efficiency and average transfection efficiency of JS-3-9L, JS-3-14L, and JS-2-OL were measured using the method described in Example 4, as shown in Table 1.

[0060] Table 1 Encapsulation efficiency and average transfection efficiency of JS-3-9L, JS-3-14L, and JS-2-OL

[0061] Comparative Example 1 To further verify the necessity of lipid modification based on creatine structure in this invention, unmodified creatine was used as a control substance, and a creatine control preparation was prepared according to the lipid nanoparticle preparation method in Example 4.

[0062] Specifically, creatine was dissolved in citrate buffer to obtain an aqueous creatine solution; DOPE, cholesterol, and DMG-PEG were dissolved in ethanol or isopropanol, respectively, and mixed in the same proportion of auxiliary lipids as in Example 4 to obtain an organic phase. Then, the mRNA encoding EGFP was dissolved in citrate buffer containing creatine, and appropriate amounts of the aqueous and organic phase solutions were taken based on a creatine to mRNA mass ratio of 10:1.

[0063] When preparing the creatine control formulation using the pipette mixing method, the aqueous solution containing creatine and mRNA is rapidly injected into the organic phase containing DOPE, cholesterol, and DMG-PEG. The mixture is then pipetted 70–80 times and incubated at 0 °C for 10 min to obtain the creatine control formulation.

[0064] In the microfluidic mixing method, an aqueous solution containing creatine and mRNA, and an organic solution containing DOPE, cholesterol, and DMG-PEG were respectively aspirated using a syringe and connected to the aqueous and organic inlets of the microfluidic mixing chip. The flow rate of the aqueous phase was set to 9 mL / min, the flow rate of the organic phase to 3 mL / min, the volumetric flow rate ratio of the aqueous phase to the organic phase to be 3:1, and the total flow rate to be 12 mL / min. The syringe pump was started to rapidly mix the two phases within the microfluidic chip. After the flow rate stabilized, the initial approximately 100–300 μL of eluent was discarded, and the eluent was then collected to obtain the creatine control formulation.

[0065] The transfection efficacy of the creatine control formulation on RAW264.7 cells was then evaluated according to the method described in Example 4. Logarithmically growing RAW264.7 cells were seeded in 48-well cell culture plates at a density of 10,000 cells per well. After cell attachment, the creatine control formulation was added to each well to achieve a mRNA concentration of 150 ng, with three replicates per group. An equal volume of PBS was used as a negative control, and the commercially available mRNA transfection reagent LipoSmart mRNA Transfection Reagent was used as a positive control. The proportion of EGFP-positive cells was detected by flow cytometry 24 h after transfection.

[0066] The test results showed that no effective EGFP-positive cell population was detected in the creatine control group, with an EGFP-positive cell ratio of 0.0%, which was basically consistent with the PBS negative control group. However, the JS-2-9L, JS-2-14L, and JS-3-OL prepared in this invention all showed significant EGFP-positive cell populations. These results indicate that unmodified creatine itself cannot replace the creatine-based ionizable lipids of this invention for mRNA delivery; only by further introducing a tertiary amine structure and a hydrophobic hydrocarbon chain into the creatine structure can the resulting ionizable lipids, together with auxiliary lipids, form lipid nanoparticles with effective nucleic acid delivery capabilities.

[0067] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A creatine-based ionizable lipid, characterized in that, It has the chemical structure shown in general formula (I): ； Wherein, n is an integer from 1 to 6, and R1 and R2 are each independently selected from alkyl groups with 6 to 20 carbon atoms, alkyl groups with 6 to 20 carbon atoms containing ether bonds, alkenyl groups with 6 to 20 carbon atoms, alkenyl groups with 6 to 20 carbon atoms containing ether bonds, alkynyl groups with 6 to 20 carbon atoms, and alkynyl groups with 6 to 20 carbon atoms containing ether bonds.

2. The creatine-based ionizable lipid as described in claim 1, characterized in that, n is an integer from 2 to 5, preferably 2 or 3; R1 and R2 are each independently selected from straight-chain alkyl groups with 9 to 18 carbon atoms and straight-chain alkenyl groups with 9 to 18 carbon atoms; Preferably, R1 and R2 are each independently selected from straight-chain alkyl groups having 9 to 14 carbon atoms or straight-chain alkenyl groups having 9 to 14 carbon atoms.

3. The creatine-based ionizable lipid as described in claim 1, characterized in that, R1 and R2 are each independently selected , , , , .

4. The creatine-based ionizable lipid as described in claim 1, characterized in that, Creatine-based ionizable lipids include the following compounds: JS-2-9; JS-2-14; JS-2-O; JS-3-9; JS-3-14; JS-3-O。 5. A method for preparing creatine-based ionizable lipids according to claim 1, characterized in that, The steps include obtaining the compound of formula (I) by amidation of the compound shown in formula (II) and the compound shown in formula (III); 、 ； Wherein, n, R1, and R2 are the same as n, R1, and R2 in claim 1.

6. The method for preparing creatine-based ionizable lipids as described in claim 5, characterized in that, The amidation reaction involves mixing the compound shown in formula (II) and the compound shown in formula (III) with triethylamine (TEA), then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to dissolve and mix them evenly, and reacting at 4~40 °C for 24-48 h. Preferably, the molar ratio of the compound shown in formula (II), the compound shown in formula (III), EDCI, HOBT and TEA is 1:(1.05~1.5):(1.05~1.5):(1.05-1.5):(2-5); More preferably, the molar ratio of the compound shown in formula (II), the compound shown in formula (III), EDCI, HOBT and TEA is 1: 1.2: 1.2: 1.2: 4.

5.

7. A lipid nanoparticle, characterized in that, Includes creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids as described in any one of claims 1-4.

8. The lipid nanoparticles as described in claim 7, characterized in that, The molar ratio of the creatine-based ionizable lipids, neutral lipids, polyethylene glycol lipids, and steroidal lipids is (10~60): (10~50): (0.5~15): (20~60), preferably (15~45): (20~40): (0.5~5.0): (25~35), and more preferably (15~35): (30~35): (2.0~3.0): (20~35). Alternatively, the diameter of the lipid nanoparticles may be 1 nm to 1000 nm.

9. The use of the lipid nanoparticles according to claim 7 or 8 in a nucleic acid drug delivery carrier.

10. A pharmaceutical composition, characterized in that, It includes an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug and the carrier is the lipid nanoparticles as described in claim 7 or 8; The nucleic acid drugs include, but are not limited to, nucleic acid molecules such as siRNA, linear mRNA, tRNA, rRNA, cDNA, ASO, plasmid DNA, circular RNA, microRNA, long non-coding RNA, and their analogues; The mass ratio of creatine-based ionizable lipids to nucleic acid drugs in the lipid nanoparticles is (1~50):1, preferably (5~15):1, and more preferably 15:1.

Images