Antioxidant ionizable cationic lipid and methods of making and using same

Abstract

The application belongs to the technical field of biological medicine, and discloses an antioxidative ionizable cationic lipid as well as a preparation method and application thereof. The ionizable cationic lipid with ionizable characteristics, antioxidative property and reactive oxygen species (ROS) scavenging capacity is efficiently synthesized by introducing a single-sulfur or double-sulfur heterocyclic structure unit through a four-component Ugi reaction. Based on the lipid, solid sterol, phospholipid and polyethylene glycol lipid form a lipid nanoparticle, which can be used for nucleic acid drug delivery, significantly improves the antioxidative property and nucleic acid transfection efficiency, and reduces the inflammatory response, and can meet the application requirements of mRNA therapy, nucleic acid vaccine and gene editing, and has very important significance for expanding the clinical application of mRNA therapy, nucleic acid vaccine and gene editing therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an antioxidant ionizable cationic lipid, its preparation method, and its application. Background Technology

[0002] Lipid nanoparticle (LNP) technology is currently the clinically approved nucleic acid drug delivery system. This technology has achieved breakthroughs in the application of siRNA drugs (patisiran), COIVD-19 mRNA vaccines (Comirnaty and Spikevax), and respiratory syncytial virus mRNA vaccines (mRNA-1345). Ionizable cationic lipids are key functional components of nucleic acid delivery systems, and their unique roles in pH responsiveness and intracellular transport make them the core delivery material for LNP technology. Ionizable cationic lipids are near-neutral at physiological pH to reduce toxicity, and after intracellular endocytosis into acidic endostomies, they become positively charged to promote lysosomal escape, thus achieving efficient encapsulation and excellent delivery efficiency. However, traditional ionizable cationic lipids face side effects such as in vivo oxidative stress and reactive oxygen species (ROS)-induced inflammation, and their inherent immunogenicity limits their widespread application. For example, Comirnaty and Spikevax vaccines have side effects after intramuscular injection, and systemic administration of patisiran can induce severe systemic inflammatory responses. These findings highlight the urgent need to develop low-immunogenic LNP technology to improve efficacy and reduce side effects.

[0003] Therefore, there is an urgent need to develop novel ionizable cationic lipids with antioxidant and ROS scavenging capabilities to overcome the safety bottleneck caused by oxidative stress, thereby developing nucleic acid delivery systems with low immunogenicity and high safety, and ultimately promoting the clinical application of more mRNA vaccines, protein replacement therapies, and CRISPR gene editing. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antioxidant ionizable cationic lipid, its preparation method and application. The ionizable cationic lipid of this invention has antioxidant and reactive oxygen species (ROS) scavenging capabilities.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an antioxidant ionizable cationic lipid, the structural formula of which is shown below: ; In the formula, m1 is independently selected , , , , , , ; The m2, m3, or m4 are independently selected from... , , , , , , , , , , , , , .

[0006] In a preferred embodiment of the antioxidant ionizable cationic lipids described in this invention, m2 is... At that time, m3 is independently selected from , , , The m4 is independently selected from , , , , , , , ; And / or, the m3 is At that time, m2 is independently selected from , , , , , , , The m4 is independently selected from , , , , , , , ; And / or, the m4 is At that time, m2 is independently selected from , , , , , , , The m3 is independently selected from , , , .

[0007] As a further preferred embodiment of the antioxidant ionizable cationic lipid described in this invention, its structural formula is as follows: Any one of them.

[0008] Secondly, the present invention provides a method for preparing the antioxidant ionizable cationic lipid, characterized in that aldehyde compounds and amine compounds are added to an organic solution, followed by the reaction, then a carboxylic acid compound is added, followed by the reaction, then an isonitrile compound is added, and the product is separated and purified after the reaction to obtain the ionizable cationic lipid.

[0009] In a preferred embodiment of the method for preparing the antioxidant ionizable cationic lipids of the present invention, the aldehyde compound is any one of A0-A4: ; And / or, the amine compound is any one of R0-R8: ; And / or, the carboxylic acid compound is any one of S1-S7: ; And / or, the isonitrile compound is any one of I0-I8: .

[0010] Thirdly, the present invention provides an antioxidant lipid nanoparticle, comprising at least one of the antioxidant ionizable cationic lipids, sterols, auxiliary lipids, and polymeric lipid derivatives described in the first aspect.

[0011] In a preferred embodiment of the antioxidant lipid nanoparticles of the present invention, the sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives. And / or, the auxiliary lipid is a phospholipid; And / or, the polymer lipid derivative is at least one of polyethylene glycol lipid, polyzwitterionic lipid, or polysarcosine lipid derivative; And / or, the molar ratio of the antioxidant ionizable cationic lipids, sterols, cofactor phospholipids, and polymer lipid derivatives is (15-70):(20-60):(5-40):(0.1-20).

[0012] Preferably, the molar ratio of the antioxidant ionizable cationic lipid, sterol, cofactor phospholipid, and polymer lipid derivative is 50:38.5:10:1.5.

[0013] Fourthly, the present invention provides an antioxidant drug-loaded nanoparticle, comprising the antioxidant lipid nanoparticles and the drug described in the third aspect.

[0014] As a preferred embodiment of the antioxidant drug-loaded nanoparticles of the present invention, the drug includes at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

[0015] As a preferred embodiment of the antioxidant drug-loaded nanoparticles of the present invention, the nucleic acid molecule is at least one of messenger RNA, circular RNA, self-replicating RNA, transfer RNA, ssRNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, and miRNA. And / or, the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

[0016] As a further preferred embodiment of the antioxidant drug-loaded nanoparticles of the present invention, the mass ratio of the antioxidant ionizable cationic lipid to nucleic acid molecules is ionizable cationic lipid: nucleic acid molecules = (1-50):1.

[0017] Preferably, the mass ratio of the antioxidant ionizable cationic lipid to nucleic acid molecules is any one or a range of two of the following: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1.

[0018] Fifthly, the present invention provides a method for preparing the antioxidant drug-loaded nanoparticles described in the fourth aspect, comprising the following steps: (1) The antioxidant ionizable cationic lipids, cholesterol, cofactor phospholipids and polymer lipid derivatives in the antioxidant lipid nanoparticles are dissolved in an organic solution to obtain an organic phase; (2) Dissolve the drug in a buffer solution to obtain an aqueous phase; The volume of the aqueous phase : the volume of the organic phase = (1-6) : 1; Preferably, the volume ratio of the aqueous phase to the organic phase is any one or a combination of 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1.

[0019] (3) The aqueous phase and the organic phase are rapidly mixed evenly and dialyzed to obtain the antioxidant drug-loaded nanoparticles.

[0020] In a sixth aspect, the present invention applies the antioxidant ionizable cationic lipids described in the first aspect, the antioxidant lipid nanoparticles described in the third aspect, and the antioxidant drug-loaded nanoparticles described in the fourth aspect to the preparation of delivery or transport of molecular drugs or nucleic acid vaccines.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces monosulfide or disulfide heterocyclic structural units and efficiently synthesizes ionizable cationic lipids (LNPs) via a four-component Ugi reaction involving aldehydes, isonitriles, amines, and carboxylic acids. Integrating sulfur-containing heterocyclic structural units into these LNPs not only addresses the safety bottleneck caused by oxidative stress at the molecular level but also endows LNPs with antioxidant, ROS-capturing, and low immunogenicity properties, effectively scavenging ROS and thus reducing cellular stress and inflammatory responses, thereby improving safety. The screened ionizable cationic lipids, combined with sterols, phospholipids, and PEGylated lipids, form lipid nanoparticles suitable for drug delivery, including small molecule compounds, messenger RNA, circular RNA, self-replicating RNA, DNA, plasmid DNA, siRNA, and other nucleic acid molecules, protein / peptide molecules, and gene editing complexes such as mRNA / sgRNA and Cas9 / sgRNA. High in vivo mRNA transfection efficiency and high protein expression levels are achieved through intravenous or intramuscular injection, meeting the application needs of mRNA nucleic acid therapy, protein replacement therapy, and gene editing. This invention provides a key design direction for developing ionizable cationic lipids with antioxidant and low immunogenicity, and is of great significance for expanding the clinical applications of mRNA therapy, nucleic acid vaccines and gene editing therapy. Attached Figure Description

[0022] Figure 1 Mass spectrometry data for ionizable cationic lipid S2R0A2I2; Figure 2 Mass spectrometry data for ionizable cationic lipid S3R0A2I2; Figure 3 Mass spectrometry data for ionizable cationic lipid S4R0A2I2; Figure 4 Mass spectrometry data for ionizable cationic lipid S6R0A2I2; Figure 5 The ABTS free radical scavenging efficiency of ionizable cationic lipids; Figure 6 To quantitatively analyze intracellular ROS levels in RAW 264.7 cells using flow cytometry; Figure 7 This indicates the in vivo protein expression of Fluc mRNA-LNP based on S4R2A2I0 after intramuscular injection. Detailed Implementation

[0023] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0025] Example 1: Synthesis of ionizable cationic lipids Ionizable cationic lipids were synthesized from different aldehydes, isonitriles, amines, and carboxylic acids using the Ugi-4CR method. The synthetic route is as follows: ; Among them, carboxylic acid compounds It can be any one of S1-S7: ; amine compounds It can be any one of R0-R8: ; Aldehyde compounds It can be any one of A0-A4: ; Isonitrile compounds It can be any one of I0-I8: .

[0026] Specific method: 1.0 mmol of aldehyde compound and 1.0 mmol of amine compound were added to 0.5 mL of methanol solution at room temperature. After reacting at room temperature for 60 min, 1.0 mmol of carboxylic acid compound was added. After reacting at room temperature for 60 min, 1.0 mmol of isonitrile compound was added. The reaction was carried out at 40 °C for 24 h. After the reaction was completed, the products were separated and purified by chromatography using a mixture of methanol and dichloromethane as the mobile phase.

[0027] The specific structures of 28 representative lipid compounds synthesized using the above methods are shown in Table 1: Table 1. Specific structural formulas of the synthesized antioxidant ionizable cationic lipids. Ionizable cationic lipid molecules are named SnRmAxIy according to the chemical combination of the reactants of carboxylic acids (Sn), amines (Rm), aldehydes (Ax), and isonitriles (Iy). In the formula, n, m, x, and y represent the numerical designations of the selected groups.

[0028] In addition, four lipid compounds were synthesized as controls, with the ionizable cationic lipid S7R0A2I2 as an example, as shown in Table 2: Table 2. Specific structural formulas of the synthesized control ionizable cationic lipids. The chemical structures of the above ionizable cationic lipids were characterized by mass spectrometry. Data for four representative ionizable cationic lipids, S2R0A2I2, S3R0A2I2, S4R0A2I2, and S6R0A2I2, are shown below. Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown, this indicates that the corresponding lipid product was successfully prepared.

[0029] Example 2: Preparation of drug-loaded nanoparticles The drug-loaded nanoparticles comprise ionizable cationic lipids, cholesterol, distearate phosphatidylcholine (DSPC), and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG). The molar ratio of ionizable cationic lipids:cholesterol:DSPC:DMG-PEG is (15-70):(20-60):(5-40):(0.1-20).

[0030] In this drug-loaded nanoparticle, the mass ratio of ionizable cationic lipids to nucleic acid drugs (messenger RNA (mRNA), circular RNA (circRNA), or small interfering RNA (siRNA)) is ionizable cationic lipids: nucleic acid drugs = (1~50):1.

[0031] Taking the preparation method of drug-loaded nanoparticles (Fluc mRNA-LNP) encoding firefly luciferase mRNA (Fluc mRNA) as an example: First, an organic phase containing lipids was prepared, with a fixed molar ratio of ionizable cationic lipids, cholesterol, DSPC, and DMG-PEG of 50:38.5:10:1.5, and a fixed mass ratio of ionizable cationic lipids to mRNA of 11:1. Next, an aqueous phase containing mRNA was prepared, buffered in 10 mM pH 4 sodium citrate buffer. Then, three volumes of the aqueous phase were rapidly added to the organic phase using a pipette and thoroughly mixed to obtain the LNP solution. After incubating the LNP solution at room temperature for 15 min, for animal experiments, the LNP solution was dialyzed against 300 volumes of 1×PBS at 4°C for 2 h, and then diluted to an appropriate volume with 1×PBS before use.

[0032] After dialysis, the LNP solution was diluted with 1×PBS to a mRNA concentration of 0.01 mg / mL. Particle size, polydispersity index (PDI), and zeta potential were measured using a nanoparticle size analyzer. The encapsulation of mRNA was assessed using the Quant-iT™ RiboGreen RNA Quantification Kit. A sample with LNP membrane disrupted by 1% Triton X-100 was used as a control for total mRNA content. Fluorescence intensity at excitation wavelength of 485 nm and emission wavelength of 528 nm was measured using a multi-mode microplate reader. The mRNA encapsulation efficiency was calculated as follows: Encapsulation efficiency (EE%) = 100 × (Total mRNA content of the disrupted sample – Sample mRNA content) / Total mRNA content of the disrupted sample.

[0033] The results are shown in Table 3: Table 3 Physicochemical properties of Fluc mRNA-LNP (the names of the corresponding Fluc mRNA-LNPs are replaced with the names of the ionizable cationic lipids used). Example 3: Determination of ABTS radical scavenging activity of ionizable cationic lipids The free radical scavenging capacity of different antioxidant ionizable cationic lipids was evaluated using the experimental protocol provided in the ABTS Free Radical Scavenging Activity Assay Kit. Specifically, an ABTS solution was mixed with an equal volume of oxidant solution to prepare a working solution, which was then incubated in the dark for 12 hours. The solution was then diluted 50-fold with phosphate-buffered saline (PBS) and added to 96-well plates at a volume of 200 μL per well. Subsequently, using Trolox solution as a reference, 10 μL volumes of different concentrations of ionizable cationic lipid solutions (S2R0A2I2, S3R0A2I2, S4R0A2I2, S6R0A2I2, S7R0A2I2, and C1R0A2I2) were added to the 96-well plates. After 2 hours of incubation, the absorbance (A) of each sample at 734 nm was recorded using a microplate reader. The ABTS free radical scavenging rate was calculated using the following formula: ABTS free radical scavenging rate (%) = (A / 2) * ... 空白 -A 测定 )÷A 空白 ×100%.

[0034] The results are as follows Figure 5 As shown, compared with the control group of ionizable cationic lipids (C1R0A2I2), ionizable cationic lipids containing monosulfide or disulfide heterocyclic structures (S2R0A2I2, S3R0A2I2, S4R0A2I2, S6R0A2I2 and S7R0A2I2) have better free radical scavenging ability at higher concentrations due to the introduction of sulfur-containing atomic structures, indicating that they have better antioxidant effects.

[0035] Example 4: Determination of the Intracellular Reactive Oxygen Species (ROS) Scavenging Performance of Drug-Loaded Nanoparticles The intracellular ROS scavenging capacity of various antioxidant drug-loaded nanoparticles was evaluated using RAW 264.7 cells: 1×10 4 RAW 264.7 cells were seeded in 96-well plates. After 24 hours of incubation, different mRNA-LNPs (S3R0A2I2, S4R0A2I2, S6R0A2I2, S7R0A2I2, and C1R0A2I2) at a concentration of 0.5 μg / mL were added to each well. After another 3 hours of incubation, 0.5 μg / mL lipopolysaccharide (LPS) was added to each well to stimulate macrophages. After another 3 hours of incubation, the cells were washed three times with PBS. Finally, 10 μM of the ROS detection probe DCFH-DA was added to each well and the cells were incubated for 0.5 hours. After washing the cells with PBS, to quantify the intracellular reactive oxygen species (ROS) level, RAW 264.7 cells stained with DCFH-DA were collected for flow cytometry analysis. A stronger average fluorescence intensity indicated a higher intracellular ROS level.

[0036] The results are as follows Figure 6As shown, compared with the positive group (LPS stimulation produces ROS), the mRNA-LNP based on the control group C1R0A2I2 did not significantly inhibit ROS production. However, the mRNA-LNP based on ionizable cationic lipids (S3R0A2I2, S4R0A2I2, S6R0A2I2 and S7R0A2I2) containing monosulfide or disulfide heterocyclic structures could significantly inhibit intracellular ROS production, indicating that they have antioxidant and anti-inflammatory functions.

[0037] Example 5: Animal experiments to detect in vivo mRNA expression levels Flux mRNA was used as a reporter gene model; drug-loaded nanoparticles (Fluc mRNA-LNP) were prepared using the ionizable cationic lipids of Example 1 according to the method of Example 2.

[0038] The specific procedures for in vivo animal experiments are as follows: C57BL / 6 mice were administered Fluc mRNA at a dose of 0.125 mg / kg mouse body weight, with a total LNP solution volume of 100 μL, via intravenous or intramuscular injection. Six hours after administration, the mice were anesthetized and intraperitoneally injected with D-fluorescein potassium solution at a dose of 150 mg / kg mouse body weight. Seven minutes later, the mice were placed in a small animal in vivo imaging system for bioluminescence imaging. For isolated organ protein expression analysis, the mice were euthanized, dissected, and major organs were removed for isolated organ bioluminescence imaging. The total bioluminescence intensity (p / s) selected in the in vivo imaging software was used as the quantitative statistical standard.

[0039] Following intravenous injection of Fluc mRNA-LNP, proteins were expressed in the liver. The expression levels are shown in Table 4. Compared with the control group of three drug-loaded nanoparticles, S7R9A2I2, S7R10A2I2, and S7R0A5I2, the drug-loaded nanoparticles S2R0A2I2, S3R0A2I2, S4R0A2I2, S6R0A2I2, S7R0A2I2, and S4R2A0I2 produced orders of magnitude higher protein expression levels. Their performance was superior to or comparable to that of commercial DLin-MC3.

[0040] Table 4. Expression levels of Fluc mRNA-LNP in the liver after intravenous injection (the names of the corresponding Fluc mRNA-LNPs are replaced with the names of the ionizable cationic lipids used). In addition, the protein expression level of Fluc mRNA-LNP after intramuscular injection was evaluated, taking S4R2A2I0-based Fluc mRNA-LNP as an example. Figure 7The results show that drug-loaded nanoparticles can achieve protein expression with high efficiency in the local muscle area.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antioxidant ionizable cationic lipid, characterized in that, Its structural formula is shown below: ； In the formula, m1 is independently selected , , , , , , ; The m2, m3, or m4 are independently selected from... , , , , , , , , , , , , , .

2. The antioxidant ionizable cationic lipid according to claim 1, characterized in that, m2 is At that time, m3 is independently selected from , , , The m4 is independently selected from , , , , , , , ; And / or, the m3 is At that time, m2 is independently selected from , , , , , , , The m4 is independently selected from , , , , , , , ; And / or, the m4 is At that time, m2 is independently selected from , , , , , , , The m3 is independently selected from , , , .

3. The antioxidant ionizable cationic lipid according to claim 1 or 2, characterized in that, Its structural formula is Any one of them.

4. A method for preparing an antioxidant ionizable cationic lipid according to any one of claims 1-3, characterized in that, Aldehydes and amines are added to an organic solution, followed by the reaction of a carboxylic acid compound, then an isonitrile compound, and finally the product is separated and purified to obtain the ionizable cationic ester.

5. The preparation method according to claim 4, characterized in that, The aldehyde compound is any one of A0-A4: ； And / or, the amine compound is any one of R0-R8: ； And / or, the carboxylic acid compound is any one of S1-S7: ； And / or, the isonitrile compound is any one of I0-I8: 。 6. An antioxidant lipid nanoparticle, characterized in that, It includes at least one of the antioxidant ionizable cationic lipids, sterols, auxiliary lipids, and polymeric lipid derivatives as described in any one of claims 1-3.

7. The antioxidant lipid nanoparticles according to claim 6, characterized in that, The sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives; And / or, the auxiliary lipid is a phospholipid; And / or, the polymer lipid derivative is at least one of polyethylene glycol lipid, polyzwitterionic lipid, or polysarcosine lipid derivative; And / or, the molar ratio of the antioxidant ionizable cationic lipids, sterols, cofactor phospholipids, and polymer lipid derivatives is (15-70):(20-60):(5-40):(0.1-20).

8. An antioxidant drug-loaded nanoparticle, characterized in that, Includes the antioxidant lipid nanoparticles and drugs as described in claim 6 or 7.

9. The antioxidant drug-loaded nanoparticles according to claim 8, characterized in that, The drug comprises at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

10. The antioxidant drug-loaded nanoparticles according to claim 8, characterized in that, The nucleic acid molecule is at least one of messenger RNA, circular RNA, self-replicating RNA, transfer RNA, ssRNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, and miRNA. And / or, the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

11. The antioxidant drug-loaded nanoparticles according to claim 10, characterized in that, The mass ratio of the antioxidant ionizable cationic lipids to nucleic acid molecules is ionizable cationic lipids: nucleic acid molecules = (1-50):

1.

12. A method for preparing antioxidant drug-loaded nanoparticles according to any one of claims 8-11, characterized in that, Includes the following steps: (1) The antioxidant ionizable cationic lipids, cholesterol, cofactor phospholipids and polymer lipid derivatives in the antioxidant lipid nanoparticles are dissolved in an organic solution to obtain an organic phase; (2) Dissolve the drug in a buffer solution to obtain an aqueous phase; The volume of the aqueous phase : the volume of the organic phase = (1-6) : 1; (3) The aqueous phase and the organic phase are rapidly mixed evenly and dialyzed to obtain the antioxidant drug-loaded nanoparticles.

13. The use of the antioxidant ionizable cationic lipids according to any one of claims 1-3, the antioxidant lipid nanoparticles according to claim 6 or 7, and the antioxidant drug-loaded nanoparticles according to any one of claims 7-11 in the preparation of delivery or transport of molecular drugs or nucleic acid vaccines.

Images