Lipid nanoparticles based on a methadone derivative and uses thereof

Abstract

The application discloses a kind of lipid nanoparticles based on methyldopa derivative and application thereof, belong to medical technology field.The raw material composition of the lipid nanoparticles includes: methyldopa derivative, auxiliary lipid, cholesterol, the methyldopa derivative is prepared after condensation reaction by methyldopa and alcohol or amine containing fatty chain.The lipid nanoparticles based on methyldopa derivative provided in the application can effectively deliver nucleic acid drugs such as mRNA in animal body, with higher transfection efficiency.Meanwhile, under the degradation of in-vivo esterase, the head group of methyldopa derivative strong positive electric nature is also decomposed into electric neutral small molecule methyldopa, reducing the cytotoxicity caused by positive electric nature.The application provides lipid nanoparticles with good biological safety, which is conducive to improving the clinical application effect of mRNA and other nucleic acid drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a lipid nanoparticle based on a meldonium derivative and its application as a delivery carrier material in the preparation of mRNA vaccines and gene therapy products. Background Technology

[0002] mRNA drugs are an emerging technology that combines molecular biology with immunology. In vitro synthesized mRNA can encode proteins using the body's own expression system to prevent and treat diseases. Compared to traditional vaccines, mRNA vaccines offer the advantage of higher efficacy. Furthermore, mRNA can encode the entire protein structure and exhibit multiple antigenic epitopes, providing unique advantages in the design of preventative and therapeutic vaccines. In terms of production, compared to cultured DNA or proteins, mRNA manufacturing processes are more stable and easier to scale up.

[0003] Despite the numerous advantages of mRNA, many challenges remain in mRNA vaccine design. One major reason limiting its application is the lack of a safe and efficient delivery system. How to efficiently deliver mRNA into cells is one of the key issues that needs to be addressed in mRNA vaccine research.

[0004] To address the unstable, negatively charged nature of mRNA, which makes it difficult for cells to take up, scientists have developed a series of delivery systems, including lipid-based delivery systems (such as patent documents CN 112961065A and US20220378701A1), peptide-based delivery systems (such as patent document WO2021133931A1), and polymer-based delivery systems (such as patent documents EP3106177B1 and WO2022125713A1), among others. Among these, lipid-based lipid nanoparticles (LNPs) are among the most promising carrier materials due to their excellent biocompatibility and efficient delivery capabilities. Several drugs based on lipid nanoparticles (LNPs) have already been approved by the FDA.

[0005] Currently, the structure-activity relationships among the components of lipid nanoparticles (LNPs) are not fully understood. Lipid nanoparticles (LNPs) are typically composed of cationic lipids (ionizable lipids), phospholipids, PEG-modified lipids, and cholesterol. Among these, cationic lipids are the core and the essence, and the properties of each component can affect the overall formulation and biological characteristics of LNPs. Extensive systematic research is currently dedicated to designing ideal cationic lipids.

[0006] The overall structure of cationic lipids can be divided into three parts: (1) Head, the head group usually carries a positive charge and mainly participates in the processes of encapsulating nucleic acids, stabilizing LNPs, interacting with cell membranes, and promoting endosome escape. Clinically used ionizable lipids (DLin-MC3-DMA, SM-102, ALC-0315) contain tertiary amine heads and can undergo pH-dependent ionization; (2) Linker fragment, the linker fragment can connect the head and tail. Biodegradable linker fragments (such as esters, amides and thiols) are preferred, as they can usually be rapidly cleared in vivo, can be used in multiple doses and have reduced side effects; (3) Tail, the hydrophobic tail affects pKa, lipophilicity, fluidity and cohesion, thereby affecting the formation and efficacy of LNPs.

[0007] Since cationic lipids play a crucial role in mRNA delivery, screening for highly efficient and safe cationic lipids is of significant clinical importance. Mildonium, a commercially available cardioprotective drug, chemically named 3-(2,2,2-trimethylhydrazine)propionate dihydrate, has not yet been reported in studies on the development of cationic lipids through modification of mildonium. Summary of the Invention

[0008] The purpose of this invention is to provide a novel cationic lipid material with high biosafety for preparing lipid nanoparticles, which can be used as drug delivery carriers to achieve efficient mRNA delivery.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides lipid nanoparticles based on meldonium derivatives. The raw material composition of the lipid nanoparticles includes: meldonium derivatives, auxiliary lipids, and cholesterol. The raw materials can be self-assembled to form lipid nanoparticles. The meldonium derivative is prepared by a condensation reaction of meldonium with an alcohol or amine containing a fatty chain, that is, a cationic lipid obtained by reacting meldonium with an alcohol or amine containing a fatty chain through a condensation reaction.

[0011] The molecular structure of meldonium has a trimethylhydrazine group at one end and a carboxyl group at the other. This invention utilizes the condensation of its carboxyl group with the hydroxyl group of an alcohol containing an aliphatic chain to form an ester bond, or with the amino group of an amine containing an aliphatic chain to form an amide bond, to construct meldonium derivatives. The hydrazine group forms a positively charged hydrophilic end, which can bind negatively charged drugs through electrostatic interactions.

[0012] Due to supramolecular interactions involving hydrophilicity and hydrophobicity, lipid materials undergo self-assembly, thereby producing the aforementioned lipid nanoparticles. These nanomaterials can serve as drug delivery carriers, encapsulating negatively charged drugs such as mRNA. Supramolecular and electrostatic forces are utilized to encapsulate negatively charged drugs within lipid materials, forming nanoparticles.

[0013] This invention demonstrates that lipid nanoparticles, formed by the self-assembly of meldonium derivatives and other lipid materials, exhibit high cell transfection efficiency as drug delivery carriers, effectively delivering drugs such as mRNA in vivo for translation into proteins. Upon entry into the body, the lipid nanoparticles undergo ester bond cleavage under the action of esterases, and the strongly positively charged head group of the meldonium derivative decomposes into a neutral small molecule, meldonium. This not only reduces the cytotoxicity caused by the positive charge but also facilitates the deconstruction of the loaded drug and the meldonium derivative, enabling its release into the cytoplasm for effective delivery.

[0014] Furthermore, the preparation method of the meldonium derivative includes: dissolving meldonium with an alcohol or amine containing an aliphatic chain in an organic solvent, and conducting a condensation reaction under the catalysis of a catalyst, and purifying the product to obtain the meldonium derivative.

[0015] Preferably, the alcohol containing a fatty chain can be, but is not limited to, any one of 2-n-octyl-1-dodecanool, 2-decyl-1-tetradecanool, 2-dodecylhexadec-1-ol, 2-hexyl-1-decanool, 8-hexadecanool, 7-tetradecanool, and castor oil. More preferably, the alcohol containing a fatty chain is 2-decyl-1-tetradecanool.

[0016] Preferably, the amine containing the fatty chain can be, but is not limited to, ditetradecylamine or didodecylamine.

[0017] Preferably, the molar ratio of meldonium to an alcohol or amine containing an aliphatic chain is 1:1-3.

[0018] Preferably, the organic solvent can be, but is not limited to, N,N-dimethylformamide.

[0019] Preferably, the catalyst can be, but is not limited to, an EDC / DMAP system.

[0020] Preferably, the condensation reaction conditions are: a temperature of 40-90°C and a reaction time of 6-72 hours. More preferably, the reaction is carried out by stirring and heating at 50°C for 12 hours.

[0021] After the reaction was completed, the solvent was removed by rotary evaporation, the initial product was separated, and the meldonium derivative was purified by silica gel chromatography. The eluent for silica gel chromatography was a mixture of n-hexane and ethyl acetate in a volume ratio of 1:1.

[0022] Furthermore, the lipid nanoparticles are formed by self-assembly of the aforementioned meldonium derivative with auxiliary lipids and cholesterol. The preparation methods for the lipid nanoparticles can include, but are not limited to, ethanol injection, thin-film method, and ultrasonic method. Specifically, when preparing drug-loaded lipid nanoparticles, the above methods are used to allow the lipid material and the drug to be loaded to self-assemble into nanoparticles in a buffer solution through supramolecular forces and electrostatic interactions.

[0023] One method involves dissolving meldonium derivatives, auxiliary lipids, and cholesterol in an appropriate amount of ethanol. The ethanol solution containing the lipid material is then injected into a buffer solution containing the drug to be encapsulated, where they self-assemble to form nanoparticles. The ethanol is then removed by dialysis to obtain stable nanoparticles.

[0024] Preferably, the auxiliary lipids include phospholipids and polyethylene glycol-functionalized lipids; the phospholipids may be, but are not limited to, distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the polyethylene glycol-functionalized lipids may be, but are not limited to, dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0025] Preferably, the molar ratio of meldonium derivative, phospholipid, cholesterol, and polyethylene glycol functionalized lipid is 20-70:5-25:15-55:1-2. More preferably, the molar ratio of the above four components is 40-55:7-22:21-47:1-2. Even more preferably, the molar ratio of the above four components is 55:15:29:1.

[0026] The present invention also provides the application of the aforementioned meldonium derivative-based lipid nanoparticles as carriers in the preparation and delivery of nucleic acid drugs.

[0027] The lipid nanoparticles based on meldonium derivatives provided by this invention can effectively deliver nucleic acid drugs such as mRNA, and this nanomaterial has potential application value in nucleic acid drug development.

[0028] Specifically, the application includes: adding meldonium derivatives, auxiliary lipids, and cholesterol to an acidic buffer containing nucleic acids, and self-assembling to form lipid nanoparticles carrying nucleic acids, thereby preparing the nucleic acid delivery drug.

[0029] Preferably, meldonium derivatives, auxiliary lipids, and cholesterol are self-assembled in a buffer containing nucleic acids using an ethanol injection method to form lipid nanoparticles.

[0030] Furthermore, the delivered nucleic acid drug is an mRNA vaccine.

[0031] Preferably, the total mass ratio of lipid material to mRNA is 20-160:1. Since mRNA molecules are relatively large, insufficient lipid material makes it difficult to effectively encapsulate and protect the mRNA, thus reducing transfection efficiency. Excessive lipid material may reduce endosome escape efficiency, thereby decreasing transfection efficiency. Within a suitable mass ratio range, good encapsulation and transfection rates can be ensured. More preferably, the total mass ratio of lipid material to mRNA is 80:1.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) This invention utilizes meldonium to construct meldonium derivatives via a condensation reaction with alcohols or amines containing aliphatic chains, which are then used as cationic lipids in the preparation of lipid nanoparticles. The molecular structure of meldonium consists of a trimethylhydrazine group at one end and a carboxyl group at the other. A meldonium derivative is constructed by reacting aliphatic alcohols or amines with its carboxyl group in one step. The hydrazine group forms a positively charged hydrophilic end, which can bind negatively charged drugs through electrostatic interactions.

[0034] (2) The lipid nanoparticles based on meldonium derivatives provided by this invention can effectively deliver nucleic acid drugs such as mRNA in animals, exhibiting high transfection efficiency. Simultaneously, under the degradation of esterases in vivo, the strongly positively charged head group of the meldonium derivative decomposes into a neutral small molecule, meldonium, reducing the cytotoxicity caused by its positive charge. Compared to the commercially available ionizable lipid SM-102, the lipid nanoparticles prepared from the meldonium derivatives provided by this invention have better biocompatibility, which is beneficial for improving the clinical application efficacy of nucleic acid drugs such as mRNA. Attached Figure Description

[0035] Figure 1 The images show the transfection effects of the nanomedicines LNPs prepared in Examples 1-7 in mice.

[0036] Figure 2 for Figure 1 Quantitative analysis results of transfection effect.

[0037] Figure 3 The image shows the transfection effect of LNPs prepared under different ratios of the lipid material in Example 6 in mice.

[0038] Figure 4 for Figure 3 Quantitative analysis results of transfection effect.

[0039] Figure 5 The results are from a cell safety experiment of lipid nanoparticles, where LNP is a lipid nanoparticle based on SM-102 and mLNP-69 is a lipid nanoparticle based on formulation 9 of meldonium derivative.

[0040] Figure 6 The images show the results of in vivo safety experiments on lipid nanoparticles. PBS is the negative control, LNP is the lipid nanoparticle based on SM-102, and mLNP-69 is the lipid nanoparticle based on formulation 9 of meldonium derivatives. The same applies below.

[0041] Figure 7 for Figure 6 The results of inflammatory markers in the skin tissue of mice, from left to right, are the proportion of white blood cells, the proportion of neutrophils, and the number of neutrophils.

[0042] Figure 8 A physical image illustrating the anticancer effect of preparing tumor drugs using lipid nanoparticles.

[0043] Figure 9 This is a comparison chart of anti-cancer effects. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0046] The compounds and their abbreviations used in the examples are explained below:

[0047] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide, CAS No.: 1892-57-5; DMAP: 4-Dimethylaminopyridine, CAS No.: 1122-58-3;

[0048] DMF: N,N-dimethylformamide, CAS No.: 68-12-2;

[0049] Meldonium, CAS No.: 76144-81-5;

[0050] Didodecylamine, CAS No.: 3007-31-6;

[0051] N,N-bistetradecylamine, CAS No.: 17361-44-3;

[0052] 8-Hexadecyl alcohol, CAS No.: 19781-83-0;

[0053] 2-Hexyl-1-decyl alcohol, CAS No.: 2425-77-6;

[0054] 2-n-Octyl-1-dodecanool, CAS No.: 5333-42-6;

[0055] 2-Decyl-1-Tetradecaneol, CAS No.: 58670-89-6;

[0056] 2-Dodecylhexadec-1-ol, CAS No.: 72388-18-2;

[0057] D-Insect Luciferin Potassium Salt, CAS No.: 115144-35-9;

[0058] DSPC phospholipid, CAS No.: 816-94-4, has the following structural formula:

[0059]

[0060] DMG-PEG2000 lipid, CAS No.: 160743-62-4, structural formula as follows:

[0061]

[0062] Cholesterol, CAS No.: 57-88-5, structural formula as follows:

[0063]

[0064] Example 1

[0065] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0066] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and N,N-bisdodecylamine (403 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was obtained. The primary product was purified by silica gel chromatography (eluent: hexane: ethyl acetate = 1:1 (volume ratio)). After vacuum drying, the product THP-12A was obtained. The reaction process is as follows:

[0067]

[0068] (2) First, meldonium derivative THP-12A, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 400 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA (purchased from nearshore protein), stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0069] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0070] Example 2

[0071] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0072] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and N,N-bistetradecylamine (467 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was obtained. The primary product was purified by silica gel chromatography (eluent: hexane: ethyl acetate = 1:1 (volume ratio)) and dried under vacuum to obtain product THP-14A. The reaction process is as follows:

[0073]

[0074] (2) First, meldonium derivative THP-14A, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5, with a total mass of 400 micrograms. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0075] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0076] Example 3

[0077] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0078] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and 8-hexadecyl alcohol (276 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was obtained. The primary product was purified by silica gel chromatography (eluent: hexane: ethyl acetate = 1:1 (volume ratio)) and dried under vacuum to obtain the product THP-88. The reaction process is as follows:

[0079]

[0080] (2) First, meldonium derivative THP-88, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 400 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0081] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0082] Example 4

[0083] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0084] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and 2-hexyl-1-decyl alcohol (276 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was obtained. The primary product was purified by silica gel chromatography (eluent: n-hexane: ethyl acetate = 1:1 (volume ratio)). After vacuum drying, the product THP-710 was obtained. The reaction process is as follows:

[0085]

[0086] (2) First, meldonium derivative THP-710, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 400 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0087] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0088] Example 5

[0089] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0090] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and 2-n-octyl-1-dodecanool (340 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was separated. The primary product was purified by silica gel chromatography (eluent: n-hexane: ethyl acetate = 1:1 (volume ratio)). After vacuum drying, the product THP-812 was obtained. The reaction process is as follows:

[0091]

[0092] (2) First, meldonium derivative THP-812, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5, with a total mass of 400 micrograms. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0093] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0094] Example 6

[0095] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0096] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and 2-decyl-1-tetradecanool (404 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was obtained. The primary product was purified by silica gel chromatography (eluent: hexane: ethyl acetate = 1:1 (volume ratio)). After vacuum drying, the product THP-1014 was obtained. The reaction process is as follows:

[0097]

[0098] (2) First, meldonium derivative THP-1014, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 400 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0099] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0100] Example 7

[0101] This embodiment prepares lipid nanoparticles based on meldonium derivatives. The specific steps are as follows:

[0102] (1) Under the catalysis of EDC (314 mg, 1.64 mmol) and DMAP (33 mg, 0.27 mmol), meldonium (200 mg, 1.37 mmol) and 2-dodecylhexadecimalol (468 mg, 1.14 mmol) were dissolved in 5 mL of DMF. The mixture was stirred and heated at 50 °C for 12 h. The solvent was removed by rotary evaporation, and the primary product was separated. The primary product was purified by silica gel chromatography (eluent: hexane: ethyl acetate = 1:1 (volume ratio)). After vacuum drying, the product THP-1216 was obtained. The reaction process is as follows:

[0103]

[0104] (2) First, meldonium derivative THP-1216, DSPC, DMG-PEG2000 and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 400 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 5 μg luciferase mRNA (nearshore protein), stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0105] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0106] Test Example 1: Particle Size and Surface Potential Analysis of Lipid Nanoparticles

[0107] The lipid nanoparticles prepared in Examples 1-7 were tested using a Malvern particle size analyzer, and the results are as follows:

[0108] The average particle size of the LNP nanomaterial prepared in Example 1 is 255.3 nm, the distribution coefficient PDI = 0.479, and the zeta potential is -0.211 mV, indicating that the nanoparticles are electrically neutral.

[0109] The average particle size of the LNP nanomaterial prepared in Example 2 is 243.2 nm, the distribution coefficient PDI = 0.333, and the zeta potential is -0.227 mV, indicating that the nanoparticles are electrically neutral.

[0110] The average particle size of the LNP nanomaterial prepared in Example 3 is 310.0 nm, the distribution coefficient PDI = 0.168, and the zeta potential is -0.387 mV, indicating that the nanoparticles are electrically neutral.

[0111] The average particle size of the LNP nanomaterial prepared in Example 4 is 198.3 nm, the distribution coefficient PDI = 0.081, and the zeta potential is -0.408 mV, indicating that the nanoparticles are electrically neutral.

[0112] The average particle size of the LNP nanomaterial prepared in Example 5 is 221.6 nm, the distribution coefficient PDI = 0.138, and the zeta potential is 0.105 mV, indicating that the nanoparticles are electrically neutral.

[0113] The average particle size of the LNP nanomaterial prepared in Example 6 is 231.4 nm, the distribution coefficient PDI = 0.128, and the zeta potential is -0.054 mV, indicating that the nanoparticles are electrically neutral.

[0114] The average particle size of the LNP nanomaterial prepared in Example 7 is 241.7 nm, the distribution coefficient PDI = 0.164, and the zeta potential is 0.009 mV, indicating that the nanoparticles are electrically neutral.

[0115] Test Example 2: In vivo transfection experiment of lipid nanoparticles

[0116] 1. The PBS solution of dialyzed LNP was injected into mice subcutaneously. Six hours later, the luciferase substrate D-fluorescein potassium salt (10 mg / mL, 200 μL) was injected intraperitoneally. The fluorescence signal was observed using a small animal in vivo imaging system.

[0117] like Figure 1 As shown, we observed a significant fluorescent signal in the mice at 6 hours, indicating that the lipid nanoparticles were successfully taken up by the cells at the injection site and in the surrounding area. At the same time, the vector successfully delivered luciferase mRNA into the cytoplasm of the mice and successfully translated it into a large amount of protein.

[0118] The transfection effect of LNP nanomaterials prepared in each embodiment in mice was quantitatively analyzed using a small animal in vivo imaging system. For example... Figure 2 As shown, the tail chain has a significant impact on liposome performance. The liposomes in Example 6 have the best transfection effect and will be used in subsequent experiments.

[0119] It is evident that the aforementioned lipid nanoparticles can effectively deliver mRNA in animals, demonstrating potential clinical application value.

[0120] 2. Optimization of lipid nanoparticle formulation and in vivo transfection experiments

[0121] To improve the delivery efficiency of lipid nanoparticles, we designed nine different formulations, as shown in Table 1, to explore the optimal molar ratio to maximize the mRNA delivery efficiency of lipid nanoparticles.

[0122] Table 1

[0123]

[0124]

[0125] Meldonium derivative THP-1014, DSPC, cholesterol, and DMG-PEG2000 were dissolved in 20 μL of ethanol at the molar ratio shown in Table 1, with a total mass of 240 μg. Under vortex conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg luciferase mRNA, stirred vigorously for 20 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0126] The dialyzed LNP PBS solution was injected subcutaneously into mice. Six hours later, the luciferase substrate D-fluorescein potassium salt (10 mg / mL, 200 μL) was injected intraperitoneally, and the fluorescence signal was observed using a small animal in vivo imaging system.

[0127] like Figure 3 As shown, we observed a significant fluorescence signal in mice at 6 hours, with clear differences between different groups. This indicates that adjusting the ratio of the four components can significantly affect and improve the delivery efficiency of lipid nanoparticles for mRNA.

[0128] We used a small animal in vivo imaging system to quantitatively analyze the in vivo transfection effect of this formulation in mice, such as... Figure 4 As shown, adjusting the ratio and optimizing the formulation can significantly improve the delivery efficiency of lipid nanoparticles. Subsequent experiments will be conducted using formulation number 9.

[0129] Test Example 3: Safety Experiment of Lipid Nanoparticles

[0130] 1. Detection of the biosafety of lipid nanoparticles at the cellular level

[0131] Empty lipid nanoparticles were prepared using meldonium derivative THP-1014, DSPC, cholesterol, and DMG-PEG2000 according to formulation No. 9, with total lipid material weights of 40 μg, 200 μg, 400 μg, and 4000 μg, respectively (based on a liposome to mRNA mass ratio of 80, corresponding to 0.5 μg, 2.5 μg, 5 μg, and 50 μg of mRNA that can be encapsulated, respectively). A control group was also included, in which commercially available SM-102 was used to replace the empty LNPs prepared using THP-1014.

[0132] Mouse kidney cells were seeded into colorless, clear 96-well plates. The cells adhered and grew, reaching a density of 5 × 10⁶ cells / well. 4 / well. Then add the prepared empty LNPs, corresponding to deliverable mRNA masses of 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, and 50 μg / mL. After incubation for 24 h, change the culture medium and evaluate cytotoxicity using a CCK8 cytotoxicity assay.

[0133] The results are as follows Figure 5 As shown, at high concentrations, cationic liposomes based on meldonium derivatives have better biocompatibility than commercially available SM-102-based cationic liposomes, which is beneficial for broadening the application scenarios of cationic lipids.

[0134] 2. In vivo safety experiments of lipid nanoparticles

[0135] On days 1 and 4, empty lipid nanoparticles (with a liposome to mRNA mass ratio of 80, corresponding to a mRNA loading capacity of 25 μg) prepared according to formula No. 9 and totaled 2 mg of lipid material were injected subcutaneously into the abdomen of mice (n=5). On day 7, the mice were sacrificed, and 1 cm × 1 cm skin samples were dissected from the injection site for flow cytometry analysis to determine the content of leukocytes and neutrophils in the tissue.

[0136] like Figure 6 As shown, it is visible to the naked eye that after injection of SM-102-based cationic liposomes, mice developed obvious skin hyperplasia and hardening on their abdomens, and some mice also developed small vesicles. In contrast, mice injected with meldonium derivative liposomes had skin conditions that were closer to those of mice in the PBS group under natural conditions.

[0137] like Figure 7 As shown, after injection of SM-102-based cationic liposomes, mice had a higher proportion of leukocytes and neutrophils in their skin tissue, as well as a greater number of neutrophils, suggesting a strong inflammatory response. In contrast, cationic liposomes based on meldonium derivatives showed better biocompatibility.

[0138] Application Example 1: Application of lipid nanoparticles in tumor vaccines

[0139] On day 1, mice were injected with 1 million B16-OVA tumor cells via subcutaneous injection. On days 5, 8, and 11, lipid nanoparticles containing 5 μg of OVA mRNA, prepared according to formula No. 9 above, were injected into the mice via subcutaneous injection.

[0140] like Figure 8 , 9 As shown, cationic lipid nanoparticles based on meldonium derivatives have the same antitumor effect as cationic lipid nanoparticles of SM-102.

[0141] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A lipid nanoparticle based on a meldonium derivative, characterized in that, The raw material composition of the lipid nanoparticles includes: meldonium derivative, auxiliary lipid, and cholesterol. The meldonium derivative is prepared by condensation reaction of meldonium with an alcohol or amine containing a fatty chain. The alcohol containing an aliphatic chain is any one of 2-n-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecylhexadec-1-ol, 2-hexyl-1-decanol, and 8-hexadecanol; the amine containing an aliphatic chain is bis(tetradecylamine) or bis(dodecylamine). The auxiliary lipids include phospholipids and polyethylene glycol-functionalized lipids; the phospholipids are distearate phosphatidylcholine or dioleoylphosphatidylethanolamine, and the polyethylene glycol-functionalized lipids are dimyristylglycerol-polyethylene glycol 2000.

2. The lipid nanoparticles based on meldonium derivatives as described in claim 1, characterized in that, The method for preparing the meldonium derivative includes: dissolving meldonium and an alcohol or amine containing an aliphatic chain in an organic solvent, and conducting a condensation reaction under the catalysis of a catalyst, and purifying the product to obtain the meldonium derivative.

3. The lipid nanoparticles based on meldonium derivatives as described in claim 2, characterized in that, The organic solvent is N,N-dimethylformamide, and the catalyst is an EDC / DMAP system.

4. The lipid nanoparticles based on meldonium derivatives as described in claim 2, characterized in that, The conditions for the condensation reaction are: temperature of 40-90℃ and reaction time of 6-72 h.

5. The lipid nanoparticles based on meldonium derivatives as described in claim 1, characterized in that, The molar ratio of meldonium derivatives, phospholipids, cholesterol, and polyethylene glycol functionalized lipids is 20-70:5-25:15-55:1-2.

6. The use of meldonium-based lipid nanoparticles as a carrier in the preparation of nucleic acid delivery drugs, as described in any one of claims 1-5.

7. The application as described in claim 6, characterized in that, The application includes: adding meldonium derivatives, auxiliary lipids, and cholesterol to an acidic buffer containing nucleic acids, and self-assembling to form lipid nanoparticles carrying nucleic acids, thereby preparing the nucleic acid delivery drug.

8. The application as described in claim 7, characterized in that, The delivered nucleic acid drug is an mRNA vaccine, and the ratio of the total mass of lipid material to the mass of mRNA is 20-160:1.

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