Ionizable cationic lipids based on imidoboronic ester structure and methods of making the same

Abstract

The application provides an imino boronic ester structure-based ionizable lipidoid and a preparation method and application thereof in treating mouse choroidal neovascularization, has high mRNA delivery capacity, and is synthesized by a one-pot method of "Ax+By+Cz". The structure has dual sensitivity to slight acidity and ROS, and especially, the lipidoid lipidoid-A4B3C7 can be assembled into a complex with other lipid components and mRNA molecules, and has optimal mRNA delivery efficiency. In addition, in a mouse laser-induced choroidal neovascularization (CNV) model, by single intravitreal injection, an LNP-A4B3C7 co-encapsulated Cas9 mRNA (mCas9) and sgRNA (sgVEGFA) complex targeting a VEGFA gene (LNP-A4B3C7 / mCas9 / sgVEGFA complex) can successfully penetrate into the retinal RPE layer, effectively knock out the VEGFA gene in the RPE cell and significantly reduce the area of the neovascularization. The treatment effect is equivalent to that of the commercial fusion protein Aflibercept. These performances of the lipidoid make it have great development prospects in the field of biomedical materials, especially in the field of gene delivery and gene editing.

Description

Technical Field

[0001] This invention relates to the fields of liposome preparation and gene loading and delivery, specifically to a method for preparing ionizable cationic lipids based on iminoboronic acid ester structures with high mRNA delivery capability, and its application in the treatment of choroidal neovascularization in mice. Background Technology

[0002] Wet age-related macular degeneration (wAMD) is a neovascularization disease of the eye. Because newly formed blood vessels are more fragile and irregular than normal vessels, they are more prone to rupture, leading to recurrent exudation and hemorrhage. Photodynamic therapy is the main clinical treatment, but it can damage normal ocular tissues. In recent years, overexpression of vascular endothelial growth factor A (VEGFA) has been identified as one of the main causes of angiogenesis; therefore, various VEGFA inhibitors have been approved for the treatment of wet macular degeneration, such as bevacizumab, ranibizumab, and sunitinib. However, frequent or even lifelong injections can cause severe financial burdens and devastating ocular complications for patients. To achieve a "one-and-done" treatment, adeno-associated virus (AAV)-mediated CRISPR / Cas9 genome editing systems have shown success in preventing choroidal neovascularization (CNV) in wAMD mouse models. However, the immunogenicity of AAV and the safety issues caused by long-term Cas9 expression cannot be ignored.

[0003] In recent years, non-viral CRISPR / Cas9 vectors have been developed. Lipid nanoparticles (LNPs) have become the most widely used non-viral vectors due to their advantages such as high transfection efficiency, low cytotoxicity, and negligible immunogenicity. Scientists have developed several methods for delivering LNPs via CRISPR / Cas9 to treat both hereditary and non-hereditary diseases. It has been reported that encapsulating a Cas9 and sgVEGFA complex (RNP) in lipofectamine 2000 (LPF 2K) can knock out the VEGFA gene in RPE cells and reduce CNV area through a single subretinal injection. However, this requires high doses of RNP, and the high cytotoxicity of LPF 2K cannot be ignored. Therefore, developing a CRISPR / Cas9 delivery system based on LNPs with minimal invasiveness, high genome editing efficiency, and low cytotoxicity is of great significance for the treatment of wet macular degeneration. Summary of the Invention

[0004] The purpose of this invention is to provide an ionizable cationic lipid based on an iminoboronate structure. Liposomes assembled from this lipid and helper lipids can effectively encapsulate mRNA molecules, utilizing the high concentration of ROS within cells and the slightly acidic environment of lysosomes to trigger lipid structure degradation, promoting the intracellular release of nucleic acid molecules. Furthermore, it exhibits good biocompatibility and stability. The invention also provides a method for preparing the aforementioned ionizable cationic lipid based on an iminoboronate structure and its application in the treatment of choroidal neovascularization in mice.

[0005] This invention provides an ionizable cationic lipid based on an iminoboronic ester structure, synthesized via a one-pot "Ax + By + Cz" process, and subsequently formed into a liposome / mRNA complex through electrostatic self-assembly with auxiliary lipids (cholesterol, DOPE, DMG-PEG2000) and mRNA molecules. The ionizable lipid of this invention exhibits both ROS and acid responsiveness, and its molecule contains imine bonds and a borate ester structure.

[0006] The present invention adopts the following technical solution:

[0007] An ionizable lipid based on an iminoboronic ester structure, having the structure shown in formula (I):

[0008] .

[0009] In the structure shown in formula (I), R1 is an alkyl chain or other hydrophobic structure with different carbon atoms, and R2 is an amine molecule with different structures.

[0010] This invention provides a method for preparing the above-mentioned ionizable lipid having the structure of formula (I), comprising the following steps: using monomer A, monomer B, and monomer C as raw materials, reacting to prepare an ionizable cationic lipid based on an iminoboronic ester structure. Preferably, the molar ratio of monomer A, monomer B, and monomer C is 1 to 2:1:1, and more preferably, the molar ratio is 1:1:1. Monomer A, monomer B, and monomer C are reacted in a mixed solvent of methanol and chloroform at room temperature for 1 to 10 hours, preferably, monomer A, monomer B, and monomer C are reacted in a molar ratio of 1:1:1 in a mixed solvent of methanol and chloroform (methanol / chloroform = 1 / 10, V / V) for 6 to 8 hours.

[0011] In this invention, monomer A is an ionizable amino-containing compound having a primary, secondary, or tertiary amine structure, with the specific structure as follows:

[0012] In the formula: R2 represents a group with an ionizable structure such as a primary amine, secondary amine, or tertiary amine.

[0013] In this invention, monomer B is one of 2-formylphenylboronic acid, 3-formylphenylboronic acid, and 4-formylphenylboronic acid, and its specific structure is as follows:

[0014]

[0015] In this invention, the structure of monomer C can be represented by the following formula:

[0016]

[0017] In the formula: R1 represents an alkyl chain or other hydrophobic chain with different carbon chain lengths.

[0018] As a specific example, monomer A has one of the structures shown in the following formula:

[0019]

[0020] Monomer B has one of the structures shown in the following formula:

[0021]

[0022] Monomer C has one of the structures shown in the following formula:

[0023]

[0024] This invention discloses a method for preparing nanomedicines, comprising the following steps: using monomers A, B, and C as raw materials, an ionizable cationic lipid based on an iminoboronic ester structure is prepared by reaction; the ionizable cationic lipid, auxiliary lipid, and drug self-assemble to obtain nanomedicines; specifically, the ionizable lipid and auxiliary lipids (cholesterol, DOPE, DMG-PEG) are dissolved in anhydrous ethanol, and then an aqueous solution of nucleic acid is added. The two phases are mixed using a pipette, and then incubated at room temperature for 30 min, followed by dialyzing in PBS to obtain nanomedicines.

[0025] In this invention, monomers A, B, and C are dissolved in a methanol / chloroform mixed solvent and reacted at room temperature to prepare an ionizable cationic lipid based on an iminoboronic ester structure; the volume ratio of methanol to chloroform is 1:10, and the reaction time is 6 h.

[0026] In this invention, monomer C is prepared using acrylate and 2-amino-1,3-propanediol as raw materials. Preferably, monomer C is prepared in a reaction solvent using acrylate and 2-amino-1,3-propanediol as raw materials under sealed conditions at 70–100°C.

[0027] Specifically, the preparation method of the lipid compound having the structure of formula (I) of this invention is as follows:

[0028] (1) Monomer C was prepared by reacting acrylate and 2-amino-1,3-propanediol as raw materials;

[0029] (2) Using monomers A, B and C as raw materials, ionizable cationic lipids based on iminoboronic esters were prepared by reaction.

[0030] In the above technical solution: In step (1), toluene is used as the reaction solvent and the reaction condition is 48h under sealed conditions at 90℃; In step (2), a mixed solvent of methanol and chloroform is used as the reaction solvent and the reaction is carried out at room temperature for 6h.

[0031] The specific reaction described above can be represented as follows:

[0032]

[0033] This invention discloses the application of the above-mentioned ionizable lipids based on iminoboronate structures in the preparation of drugs, or in the preparation of drug carriers. It also discloses the application of the above-mentioned nanomedicines in the preparation of mRNA delivery or gene editing drugs.

[0034] In this invention, the drug is mRNA, for example, the mRNA encodes luciferase, green fluorescent protein, or Cas9 protein. Preferably, the nitrogen-to-phosphorus ratio of the ionizable lipid based on the iminoboronic ester structure to the mRNA is (1-50):1, more preferably (4-10):1, and even more preferably (6-8):1.

[0035] The present invention provides an ionizable cationic lipid based on an iminoboronic ester structure that can self-assemble with auxiliary lipids and nucleic acids to form nanomedicines. Therefore, the present invention discloses a nanomedicine obtained by combining the above-mentioned iminoboronic ester structure lipid with auxiliary lipids.

[0036] In this invention, the particle size of the nanomedicine is 100–200 nm, preferably 100–150 nm, and more preferably 110–150 nm. The zeta potential of the nanomedicine is 2–4 mV.

[0037] In this invention, the auxiliary lipids are cholesterol, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), and 1,2-dimyristoyl-RAC-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), etc.

[0038] This invention discloses the application of the above-mentioned iminoboronic acid ester structured lipids in the preparation of drug carriers or in the preparation of nanomedicines; or the application of the above-mentioned nanomedicines in the preparation of gene drugs. This invention also discloses the application of the above-mentioned iminoboronic acid ester structured lipids in the preparation of drugs for treating choroidal neovascularization in mice.

[0039] The main advantages of this invention are:

[0040] (1) The present invention introduces an iminoboronic acid ester structure into the lipid structure. This chemical structure has the following advantages: ① Various ionizable amine-containing groups can be connected by imine bonds, giving lipids different degrees of positive charge to effectively condense nucleic acid molecules; ② The ortho-dihydroxyl structure on formylphenylboronic acid can react with the diol structure of monomer C to introduce hydrophobic chains of different lengths to help form lipids with different hydrophobic tail structures; ③ The product is obtained by the "one-pot" method, and the head, linker and tail structure of the lipid can be flexibly adjusted to synthesize a large number of lipids; ④ The iminoboronic acid ester is formed under mild conditions, can be carried out at room temperature, has no other by-products except water, and has a short reaction time and high yield.

[0041] (2) The ionizable cationic lipid based on iminoboronic acid ester of the present invention, wherein the imino group can be broken in the weakly acidic environment of intracellular lysosomes; the boronic acid ester bond can be broken under the action of high ROS in the cell. Both promote lipid degradation, reduce material toxicity, promote intracellular release of nucleic acid, and improve transfection efficiency.

[0042] (3) The iminoboronic acid ester-based ionizable lipids of the present invention have ionizable heads, which can promote the interaction between liposomes / nucleic acid complexes and cell membranes, thereby promoting material endocytosis.

[0043] (4) The iminoboronic acid ester-based ionizable lipids and auxiliary liposomes of the present invention can effectively condense nucleic acid molecules at a low nitrogen-to-phosphorus ratio, avoid the degradation of nucleic acids by nucleases, and reduce the toxicity of materials.

[0044] (5) The iminoboronic acid ester-based ionizable lipids of the present invention can not only efficiently deliver mRNA, but also co-deliver Cas9 mRNA and sgRNA. In the mouse choroidal neovascularization model, the VEGFA gene was successfully knocked out in RPE cells, which successfully reduced the area of ​​choroidal neovascularization without causing significant toxic side effects to retinal tissue and the body. Attached Figure Description

[0045] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0046] Figure 1 The 1H NMR spectrum of monomer C7 is described.

[0047] Figure 2 The 1H NMR spectrum of lipidoid-A1B3C7 was described.

[0048] Figure 3 The 1H NMR spectrum of lipidoid-A2B3C7 was described.

[0049] Figure 4 The 1H NMR spectrum of lipid-A4B3C7 was described.

[0050] Figure 5 Describes lipidoid-A 11 1H NMR spectrum of B3C7.

[0051] Figure 6 The gene transfection of four LNP / mLuc complexes on HeLa cells, B6F10 cells, A549 cells, and A673 cells (from left to right) is described.

[0052] Figure 7 The cytotoxicity of four LNP / mLuc complexes with different mRNA concentrations was described in HeLa, B6F10, A549 and A673 cells.

[0053] Figure 8 The particle size variations of four LNP / mLuc complexes in PBS at pH 7.4 were described.

[0054] Figure 9 The fluorescence images, EGFP-positive cell ratio, and mean EGFP fluorescence intensity of HeLa cells after transfection with the LNP-A4B3C7 / mEGFP complex are described.

[0055] Figure 10 Gel electrophoresis images of LNP-A4B3C7 / mluc complexes prepared under different nitrogen-to-phosphorus ratios are described.

[0056] Figure 11 Transmission electron microscopy images of the LNP-A4B3C7 / mLuc complex at a nitrogen-to-phosphorus ratio of 8 are described.

[0057] Figure 12 Gel electrophoresis images of LNP-A4B3C7 / mluc complexes prepared under different nitrogen-to-phosphorus ratios before and after ribonuclease A treatment are described.

[0058] Figure 13 Gel electrophoresis images of LNP-A4B3C7 / mluc complexes prepared under different nitrogen-to-phosphorus ratios after acid or H2O2 treatment are described.

[0059] Figure 14 Flow cytometry and quantitative fluorescence analysis of the uptake levels of the LNP-A4B3C7 / YOYO-1-mRNA complex on HeLa cells (left) and ARPE-19 cells (right) are shown.

[0060] Figure 15The graph depicts the uptake levels of the LNP-A4B3C7 / mRNA complex in HeLa cells (left) and ARPE-19 cells (right) after treatment with different endocytosis inhibitors.

[0061] Figure 16 The relative transfection levels of the LNP-A4B3C7 / mLuc complex or the LNP-A4B3C7 / mEGFP complex after incubation on VC-pretreated HeLa cells were described.

[0062] Figure 17 The relative transfection levels of the LNP-A4B3C7 / mLuc complex or the LNP-A4B3C7 / mEGFP complex incubated on H2O2-pretreated ARPE-19 cells were described.

[0063] Figure 18 The study described the relative intracellular VEGFA mRNA level and the VEGFA content in the culture medium after incubation of the LNP-A4B3C7 / mCas9 / sgVEGFA complex in H2O2-treated ARPE-19 cells.

[0064] Figure 19 The retinal distribution of YOYO-1-mRNA at different time points after intravitreal injection of the LNP-A4B3C7 / YOYO-1-mRNA complex was described.

[0065] Figure 20 This study describes fundus angiography and confocal images of the laser spot 7 days after a single intravitreal injection of the LNP-A4B3C7 / mCas9 / sgVEGFA complex in a mouse laser-induced CNV model.

[0066] Figure 21 This study describes the relative expression level of VEGFA mRNA and the VEGFA protein content in the RPE-choroid-sclera complex (RCS complex) 7 days after a single intravitreal injection of the LNP-A4B3C7 / mCas9 / sgVEGFA complex in a mouse CNV model; and the next-generation sequencing results of the VEGFA gene in RPE cells 7 days after a single intravitreal injection of the LNP-A4B3C7 / mCas9 / sgVEGFA complex in a mouse CNV model. Detailed Implementation

[0067] This invention discloses a method for preparing an ionizable cationic lipid based on an iminoboronic ester structure with high mRNA delivery efficiency and its application in the treatment of choroidal neovascularization in mice. The invention is further illustrated below with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Furthermore, various different embodiments of the invention can be combined in any way, as long as they do not depart from the spirit of the invention, and should also be considered as part of the disclosure of this invention.

[0068] This invention relates to an ionizable lipid gene delivery vector based on iminoboronic esters. The lipid has an ionizable amine-containing head, a phenylboronic acid intermediate linker, and a hydrophobic alkyl tail. This lipid can self-assemble with helper lipids and nucleic acid molecules to form liposome / nucleic acid complexes. The positively charged tertiary amine on the lipid can effectively condense negatively charged nucleic acid molecules through electrostatic interactions, while the helper lipids participate in the self-assembly of liposomes to form stable liposome / nucleic acid complexes. Endocytosis is primarily achieved through the interaction between the liposome surface and the cell membrane, thus enabling nucleic acid delivery. Studies have shown that, compared to other structures, the lipid of this invention can rapidly synthesize amphiphilic lipids with different amine-containing heads, intermediate linkers, and different hydrophobic tails at room temperature through imine bonds and the formation of borate esters. By screening lipids with specific structures, efficient gene transfection has been achieved. Furthermore, its dual responsiveness to acidic conditions and ROS allows for more efficient release of nucleic acid molecules.

[0069] In this embodiment of the invention, all raw materials are commercially available products, including Luciferase mRNA.

[0070] mLuc, EGFP mRNA (mEGFP), and Cas9 mRNA (mCas9) were purchased from APExBIO Inc. (USA). sgEGFP and sgVEGFA were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0071] As a specific example, the method for synthesizing ionizable cationic lipids based on iminoboronic ester structures prepared in this invention is illustrated below:

[0072]

[0073] Specific preparation methods are exemplified as follows:

[0074] (1) Octadecyl acrylate and 2-amino-1,3-propanediol were added to toluene and reacted under heating and sealing conditions. After the reaction was completed, the solvent was removed by vacuum distillation, and hexane was added to precipitate the product. The product was filtered and dried under vacuum to obtain a white powder monomer C7.

[0075] (2) N,N-dimethylethylenediamine (monomer A4), 4-formylphenylboronic acid (monomer B3) and monomer C7 were added to a mixed solvent of chloroform and methanol and reacted at room temperature to obtain a lipid, named lipidoid-A4B3C7; lipidoid-A4B3C7 was used in the following examples.

[0076] To further understand the present invention, preferred embodiments are described below with reference to examples. These descriptions are only for further illustrating the features and advantages of the present invention and are not intended to limit the scope of the claims. The raw materials involved in this invention are all conventional products, commercially available or prepared conventionally according to existing technology; the specific operating methods involved, such as stirring and freeze-drying, are conventional methods, and the specific tests are also conventional methods in the art.

[0077] Example 1

[0078] Octadecyl acrylate (0.10 g, 0.3 mmol) and 2-amino-1,3-propanediol (0.01 g, 0.12 mmol) were added to toluene (5 mL), and the reaction was carried out at 90 °C under sealed conditions for 48 h. After the reaction was completed, the solvent was removed by vacuum distillation, and n-hexane (30 mL) was added to precipitate the product. The product was filtered and dried under vacuum to obtain a white powder monomer C7. Figure 1 The 1H NMR spectrum is for monomeric C7.

[0079] N,N-dimethylethylenediamine (A4, 2.8 μL, 0.02 mmol), 3-formylphenylboronic acid (B3, 3 mg, 0.02 mmol), and monomer C7 (14.5 mg, 0.02 mmol) were added to a mixed solvent of chloroform and methanol (v / v = 10 / 1, 200 μL) and reacted at room temperature for 6 h. The product was dried over MgSO4 for 12 h and then rotary evaporated to obtain lipidoid-A4B3C7. Figure 2 The 1H NMR spectrum of lipidoid-A4B3C7.

[0080] Comparative Example 1

[0081] N,N-dimethylethylenediamine (A1, 2.6 μL, 0.02 mmol), 3-formylphenylboronic acid (B3, 3 mg, 0.02 mmol), and monomer C7 (14.5 mg, 0.02 mmol) were added to a mixed solvent of chloroform and methanol (v / v = 10 / 1, 200 μL) and reacted at room temperature for 6 h. The product was dried over MgSO4 for 12 h and then rotary evaporated to obtain lipidoid-A1B3C7. Figure 3 The 1H NMR spectrum of lipidoid-A1B3C7.

[0082] Comparative Example 2

[0083] 1-(2-aminoethyl)pyrrolidine (A2, 2.7 μL, 0.02 mmol), 3-formylphenylboronic acid (B3, 3 mg, 0.02 mmol), and monomer C7 (14.5 mg, 0.02 mmol) were added to a mixed solvent of chloroform and methanol (v / v = 10 / 1, 200 μL) and reacted at room temperature for 6 h. The product was dried over MgSO4 for 12 h and then rotary evaporated to obtain lipidoid-A2B3C7. Figure 4 The 1H NMR spectrum of lipidoid-A2B3C7.

[0084] Comparative Example 3

[0085] 1-(3-aminopropyl)imidazolium (A) 10 3-Formylphenylboronic acid (B3, 3 mg, 0.02 mmol) and monomer C7 (14.5 mg, 0.02 mmol) were added to a mixed solvent of chloroform and methanol (v / v = 10 / 1, 200 μL) and reacted at room temperature for 6 h. The product was dried over MgSO4 for 12 h and then rotary evaporated to obtain lipidoid-A. 11 B3C7, Figure 5 For lipidoid-A 11 1H NMR spectrum of B3C7.

[0086] Example 2

[0087] Lipidoid (example or comparative example), cholesterol, DOPE, and DMG-PEG2000 were dissolved in anhydrous ethanol at a fixed molar ratio (lipidoid / cholesterol / DOPE / DMG-PEG=40 / 40 / 10 / 0.25); mluc (mRNA encoding the Luciferse protein sequence, purchased from APExBIO, USA) was dissolved in nuclease-free aqueous solution (0.1 mg / mL); the two phases were mixed at a nitrogen-to-phosphorus ratio (N / P) of 8 (ethanol phase / aqueous phase = 1 / 3, V / V), pipetted for 30 s, and then incubated at 37°C for 30 min to form an LNP / mRNA complex.

[0088] To compare the in vitro transfection efficiency of different LNP / mRNA complexes, HeLa cells, B16F10 cells, A549 cells, and A637 cells were transfected at 1×10⁻⁶ cells per well. 4Individual samples were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free DMEM, and LNP-A1B3C7 / mLuc complex, LNP-A2B3C7 / mLuc complex, LNP-A4B3C7 / mLuc complex, and LNP-A... were added to each well at a concentration of 0.1 µg mRNA / well. 11 B3C7 / mLuc complex. LPF 2K / mLuc complex (w / w=2) was used as a control. After incubation at 37 ℃ for 6 h, the culture medium was removed and replaced with fresh medium for another 20 h. Luciferase expression efficiency was evaluated by measuring luciferase expression using a luciferase kit. Intracellular total protein content was measured using a BCA kit to evaluate gene transfection efficiency. See details... Figure 6 The experimental results showed that the LNP-A4B3C7 / mLuc complex had the highest transfection efficiency in all four cell types. Especially in HeLa cells, its transfection efficiency was more than 20 times higher than that of the LPF 2K / mLuc complex, demonstrating that LNP-A4B3C7 has excellent mRNA transfection performance.

[0089] Example 3

[0090] To investigate the cytotoxicity of the LNP / mRNA complex, HeLa cells, B16F10 cells, A549 cells, and A673 cells were cultured at 1.5 × 10⁻⁶ cells per well. 4 Individual samples were seeded into 96-well plates and cultured for 24 h. Then, the medium was replaced with serum-free medium, and different concentrations of LNP-A1B3C7 / mLuc complex, LNP-A2B3C7 / mLuc complex, LNP-A4B3C7 / mLuc complex, and LNP-A were added. 11 The B3C7 / mLuc complex was used as a control, with the same concentration of LPF 2K / mRNA as a control. Cell viability was determined by MTT assay after incubation at 37 °C for 24 h. See details... Figure 7 The experimental results showed that, at the same mRNA concentration, LNP-A1B3C7 / mLuc, LNP-A2B3C7 / mLuc, LNP-A4B3C7 / mRNA, and LNP-A 11 Cells treated with the B3C7 / mRNA complex showed significantly higher survival rates than those treated with the LPF 2K / mLuc complex, demonstrating that the LNP / mRNA complex exhibits lower cytotoxicity and better biocompatibility.

[0091] Example 4

[0092] The LNP-A1B3C7 / mLuc complex, LNP-A2B3C7 / mLuc complex, LNP-A4B3C7 / mLuc complex, and LNP-A 11 The B3C7 / mLuc complex was added to PBS buffer (pH = 7.4), and its particle size was measured using DLS at specific time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h). See details... Figure 8 The experimental results showed that the particle size of each LNP / mLuc complex did not change significantly within 24 h, proving that the LNP / mRNA complex has good stability.

[0093] Example 5

[0094] To further verify the ability of LNP-A4B3C7 to efficiently deliver other mRNAs, HeLa cells were used at 5 × 10⁶ cells per well. 4 Cells were seeded into 24-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free DMEM, and LNP-A4B3C7 / mEGFP complex (N / P=8) was added to each well at a concentration of 0.5 µg mEGFP (EGFP mRNA, encoding the enhanced green fluorescent protein sequence, purchased from APExBIO, USA). An LPF 2K / mEGFP complex (w / w=2) was used as a control. After incubation at 37 °C for 6 h, the medium was removed and replaced with fresh medium for another 20 h. Transfection efficiency was evaluated by observing the brightness of green fluorescence in cells under a fluorescence microscope, quantifying the green fluorescence intensity by flow cytometry, and determining the percentage of EGFP-positive cells. See details... Figure 9 The experimental results showed that HeLa cells treated with the LNP-A4B3C7 / mEGFP complex had significantly higher green fluorescence brightness, EGFP-positive cell percentage, and EGFP fluorescence intensity than HeLa cells treated with the LPF 2K / mEGFP complex, further demonstrating that LNP-A4B3C7 can efficiently deliver mRNA.

[0095] Example 6

[0096] Lipid-A4B3C7, cholesterol, DOPE, and DMG-PEG2000 were dissolved in anhydrous ethanol at a fixed molar ratio (lipidoid-A4B3C7 / cholesterol / DOPE / DMG-PEG = 50 / 40 / 10 / 0.25), and mluc was dissolved in nuclease-free water (0.1 mg / mL). The two phases were mixed at different nitrogen-to-phosphorus ratios (1 / 1, 2 / 1, 4 / 1, 8 / 1, and 10 / 1) (ethanol / water phase = 1 / 3, V / V), pipetted for 10 s, and then incubated at 37°C for 30 min to form a liposome / mRNA complex, named the LNP-A4B3C7 / mLuc complex. The encapsulation energy of LNP-A4B3C7 / mLuc under different nitrogen-to-phosphorus ratios was evaluated by agarose gel electrophoresis. See details... Figure 10 Experimental results show that when the nitrogen-to-phosphorus ratio of LNP-A4B3C7 to mLuc is greater than or equal to 4, LNP-A4B3C7 can effectively encapsulate mLuc.

[0097] Example 7

[0098] Lipid-A4B3C7, cholesterol, DOPE, and DMG-PEG2000 were dissolved in anhydrous ethanol at a fixed molar ratio (Lipid-A4B3C7 / Cholesterol / DOPE / DMG-PEG = 50 / 40 / 10 / 0.25), and mLuc was dissolved in nuclease-free aqueous solution (0.1 mg / mL). The two phases were mixed at a nitrogen-to-phosphorus ratio of 8 (ethanol / aqueous phase = 1 / 3, V / V), pipetted for 30 s, and then incubated at 37°C for 30 min to form the LNP-A4B3C7 / mLuc complex. The complex was then dropped onto a copper grid, allowed to dry naturally, and its morphology was observed using transmission electron microscopy (TEM). See [link to TEM for details]. Figure 11 Experimental results show that the LNP-A4B3C7 / mLuc complex has a circular structure with a particle size of about 110 nm.

[0099] Example 8

[0100] Freshly prepared LNP-A4B3C7 / mRNA complexes were divided into two groups: one group was pretreated with ribonuclease A solution (0.1 mg / mL, 30 min), and the other group was not pretreated. The complexes were then incubated with a ribonuclease inhibitor at 37°C. o Incubation at C for 0.5 h removed ribonuclease A. Heparin sodium was then added to the complex to displace the loaded mRNA. The mixture was then loaded onto a 0.5% agarose gel for electrophoresis at 90 V for 30 min. Ethidium bromide staining was performed, and the gel imaging system was used to determine the integrity of the mRNA. See details... Figure 12The experimental results showed that when the nitrogen-to-phosphorus ratio was 2, the mRNA was rapidly degraded by ribonuclease A because LNP-A4B3C7 could not effectively encapsulate it. When the nitrogen-to-phosphorus ratio was 4, LNP-A4B3C7 could just encapsulate the mRNA, so only a portion of the mRNA was degraded by ribonuclease A. However, when the nitrogen-to-phosphorus ratio was 8, LNP-A4B3C7 completely encapsulated the mRNA, thus ribonuclease A could not effectively degrade it. This demonstrates that LNP-A4B3C7 protects mRNA from degradation by ribonuclease.

[0101] Example 9

[0102] To verify that lipidoid-A4B3C7 exhibits dual ROS and acid responsiveness, lipidoid-A4B3C7, cholesterol, DOPE, and DMG-PEG2000 were dissolved in anhydrous ethanol at a fixed molar ratio (lipidoid-A4B3C7 / cholesterol / DOPE / DMG-PEG = 40 / 40 / 10 / 0.5). mRNA (encoding the Luciferse protein sequence, purchased from APExBIO, USA) was dissolved in DEPC aqueous solution (0.1 mg / mL). The two phases were mixed at different nitrogen-to-phosphorus ratios (1 / 1, 2 / 1, 4 / 1, 8 / 1, and 10 / 1) (ethanol / aqueous phase = 1 / 3, V / V), pipetted for 10 s, and then incubated at 37°C for 30 min to form a liposome / mLuc complex, which was named the LNP-A4B3C7 / mLuc complex. The LNP-A4B3C7 / mLuc complex was divided into two groups: one group was treated with acetate / sodium acetate buffer (0.01 M, pH = 5.2) for 4 h, and the other group was pretreated with H2O2 solution (100 μM) for 4 h. The samples were loaded into 0.5% agarose gel electrophoresis wells and run at 90 V for 30 min. Ethidium bromide staining was performed, and the gel imaging system was used to determine the mRNA encapsulation. See details... Figure 13 Experimental results showed that after treating the complex with acidic buffer, a higher N / P ratio was required for LNP-A4B3C7 to effectively encapsulate mLuc. This indicates that acid induces the breakage of the imine bond, resulting in the loss of the positively charged head amine, reducing the positive charge of lipidoid-A4B3C7, and consequently decreasing its ability to bind mRNA. Furthermore, the LNP-A4B3C7 / mLuc complex treated with H2O2, even at an N / P ratio of 10, could not effectively encapsulate mLuc. This indicates that H2O2 treatment causes the breakage of the borate ester bond, leading to the disintegration of the lipidoid-A4B3C7 matrix and complete loss of its ability to encapsulate mRNA. This demonstrates that lipidoid A4B3C7 exhibits dual acid and ROS responsiveness.

[0103] Example 10

[0104] To evaluate the internalization effect of the LNP-A4B3C7 / mRNA complex, mRNA was labeled with YOYO-1 (20 μM) (one dye molecule was labeled for every 50 nt of mRNA). HeLa cells and ARPE-19 cells were cultured at 5 × 10⁻⁶ cells per well. 4 Cells were seeded into 24-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free medium, and LNP-A4B3C7 / YOYO-1-mRNA complex (N / P=8) was added to each well at a concentration of 0.5 μg YOYO-1-mRNA / well. Free YOYO-1-mRNA and LPF 2K / YOYO-1-mRNA complex (w / w=2) served as controls. After incubation at 37°C for 6 h, cells were washed three times with PBS containing heparin sodium (20 U / mL) to remove any unadsorbed complex. YOYO-1-mRNA fluorescence intensity was quantified by flow cytometry (λex = 485 nm, λem = 530 nm) to evaluate cellular uptake levels. See details... Figure 14 The experimental results showed that, compared with free YOYO-1-mRNA, LNP-A4B3C7 / YOYO-1-mRNA exhibited significant internalization levels in both cell types, and its internalization level was higher than that of the LPF 2K / mRNA complex. This demonstrates that LNP-A4B3C7 can effectively promote mRNA internalization.

[0105] Example 11

[0106] To further investigate the internalization mechanism of the LNP-A4B3C7 / mRNA complex, HeLa cells and ARPE-19 cells were cultured at 1.5 × 10⁻⁶ mRNA per well. 4 Individual cells were inoculated into 96-well plates and cultured for 24 hours. The medium was then replaced with serum-free medium and pretreated for 30 min at 4°C or under the conditions of small molecule endocytosis inhibitors, including chlorpromazine (CPZ, inhibiting clathrin-mediated endocytosis, 10 μg / mL), genistein (GNT, inhibiting pituitary protein-mediated endocytosis, 100 μg / mL), methyl-β-cyclodextrin (mβCD, reducing cholesterol on the membrane and inhibiting lipid raft-mediated endocytosis, 50 μM), and wollamyl (WTM, inhibiting macropinocytosis, 50 nM). Then, LNP-A4B3C7 / mRNA complex (N / P=8) was added at a concentration of 0.1 μg YOYO-1-mRNA / well and cultured at 37°C. o Incubate at C for 6 h, discard the culture medium, wash three times with PBS containing heparin sodium (20 U / mL), and evaluate cell uptake levels using fluorescence spectrophotometry. The uptake of untreated cells is considered 100%. See details... Figure 15 The experimental results showed that at 4℃, the cellular uptake level of the LNP-A4B3C7 / mRNA complex decreased significantly, while CPZ treatment resulted in a slight decrease. GNT treatment significantly reduced cellular uptake, indicating that most of the complex was taken up by cells via energy-dependent endocytosis, primarily through caveolin-mediated endocytosis, with some uptake via clathrin-mediated endocytosis. Furthermore, in HeLa cells, it is possible that some uptake occurs via macropinocytosis.

[0107] Example 12

[0108] To investigate the effect of intracellular ROS levels on the transfection efficiency of the LNP-A4B3C7 / mRNA complex, HeLa cells were transfected at a density of 1 × 10⁶ mRNA per well. 4 Cells were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free DMEM, and cells were pretreated with vitamin C (VC, 2 h, to remove pre-existing ROS). Following this, LNP-A4B3C7 / mRNA complex (N / P=8) was added at a concentration of 0.1 µg mRNA per well. After incubation at 37 °C for 6 h, the medium was removed, and cells were cultured in fresh medium for another 20 h. Luciferase expression was then measured using a luciferase kit. The luciferase expression level in the group without VC pretreatment was considered 100%. See details... Figure 16 The experimental results showed that VC treatment reduced the expression level of luciferase in HeLa cells by approximately 40%, indicating that the reduction of intracellular ROS hindered mRNA expression. This is because borate esters in lipidoid-A4B3C7 cells were not degraded, thus hindering lipid degradation and consequently reducing transfection efficiency. This demonstrates that the ROS sensitivity of borate esters plays an important role in improving the mRNA transfection efficiency of LNP-A4B3C7 cells.

[0109] Example 13

[0110] To further verify that increased ROS in ARPE-19 cells promotes an increase in LNP-A4B3C7 / mRNA complex-mediated mRNA expression levels, ARPE-19 cells were cultured at 1 × 10⁻⁶ cells per well. 4Cells were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS for 24 h. The medium was then replaced with serum-free DMEM, and cells were pretreated with H2O2 (200 μM, to increase the original intracellular ROS concentration) for 2 h to construct an in vitro wet macular degeneration (wAMD) model. Subsequently, LNP-A4B3C7 / mluc complex (N / P=8) was added to each well at a concentration of 0.1 µg mRNA / well. After incubation at 37 °C for 6 h, the medium was removed, replaced with fresh medium, and cultured for another 20 h. Luciferase expression was measured using a luciferase kit. The luciferase expression level in the group without H2O2 pretreatment was taken as 100%. See details... Figure 17 The experimental results showed that after H2O2 treatment, the expression level of luciferase in ARPE-19 cells was three times that of the control group, further demonstrating that high intracellular ROS concentrations promote the mRNA transfection efficiency of the LNP-A4B3C7 / mRNA complex.

[0111] Example 14

[0112] To investigate the knockout ability of LNP-A4B3C7 on VEGFA in an in vitro wAMD model, ARPE-19 cells were grown at a density of 1.0 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and cultured for 24 h. Cells were pretreated with H2O2 (200 μM) for 2 h, then the medium was replaced with serum-free medium. Free mCas9 / sgVEGFA, LPF 2K / mCas9 / sgVEGFA complex (w / w / w=3 / 1 / 0.5), and LNP-A4B3C7 / mCas9 / sgVEGFA complex (N / P=8) were then added to each well at concentrations of 1 µg mCas9 / well and 0.5 µg sgVEGFA / well, respectively. After incubation at 37 ℃ for 12 h, the medium was removed and replaced with fresh medium for another 48 h. VEGFA mRNA expression levels were detected by real-time quantitative PCR.

[0113] ARPE-19 cells were planted at 1.0 × 10⁶ cells per well. 4Cells were seeded into 96-well plates and cultured for 24 h. Cells were pretreated with H2O2 (200 μM) for 2 h, then the medium was replaced with serum-free medium. Free mCas9 / sgVEGFA, LPF 2K / mCas9 / sgVEGFA complex (w / w / w=3 / 1 / 0.5), and LNP-A4B3C7 / mCas9 / sgVEGFA complex (N / P=8) were then added to each well at concentrations of 0.1 µg mCas9 / well and 0.05 µg sgVEGFA / well, respectively. After incubation at 37 °C for 12 h, the medium was removed and replaced with fresh medium for another 48 h. The VEGFA content in the medium was detected by enzyme-linked immunosorbent assay (ELISA).

[0114] ARPE-19 cells were planted at 1.0 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and cultured for 24 h. Cells were pretreated with H2O2 (200 μM) for 2 h, then the medium was replaced with serum-free medium. Subsequently, LNP-A4B3C7 / mCas9 / sgVEGFA complex (N / P=8) was added to each well at a concentration of 1 µg mCas9 / well and 0.5 µg sgVEGFA / well. After incubation at 37 ℃ for 12 h, the medium was removed and replaced with fresh medium for another 48 h. Genomic DNA was extracted using a cell / tissue whole-genome DNA extraction kit, and gene editing efficiency was analyzed by next-generation sequencing (NGS).

[0115] See details Figure 18 Experimental results showed that the LNP-A4B3C7 / mCas9 / sgVEGFA complex significantly reduced the expression level of VEGFA mRNA in H2O2-treated ARPE-19 cells, and the reduction was significantly better than that of the LPF 2K / mCas9 / sgVEGFA complex group. Furthermore, the LNP-A4B3C7 / mCas9 / sgVEGFA complex significantly reduced the VEGFA concentration in the supernatant of H2O2-pretreated ARPE-19 cells, and the reduction was significantly greater than that of the LPF 2K / mCas9 / sgVEGFA complex group, verifying the ability of the LNP-A4B3C7 / mCas9 / sgVEGFA complex to knock out VEGFA. The A4B3C7 / mCas9 / sgVEGFA complex successfully knocked out 71% of the VEGFA gene in H2O2-pretreated ARPE-19 cells, demonstrating its excellent gene editing ability.

[0116] Example 15

[0117] To investigate the penetration of the LNP-A4B3C7 / mRNA complex into the retina after intravitreal injection, C57BL / 6 mice were anesthetized, and then 2 μL of free YOYO-1-mRNA, LPF 2K / YOYO-1-mRNA complex, and LNP-A4B3C7 / YOYO-1-mRNA complex were injected intravitreally. The mRNA dose was 0.5 μg per eye. Eyes were enucleated at 6, 12, 24, and 48 hours, and the retinal distribution of YOYO-mRNA was observed by frozen section analysis. See details... Figure 19 Experimental results showed that no fluorescence signal of free YOYO-1-mRNA was detected in the retina, while in the LPF 2K / YOYO-1-mRNA complex group, YOYO-1-mRNA fluorescence was observed only in the retinal ganglion cell layer (GCL). In the LNP-A4B3C7 / YOYO-1-mRNA complex group, however, the fluorescence of YOYO-1-mRNA gradually distributed from the retinal ganglion cell layer to the RPE layer of the retina over time. This demonstrates that the LNP-A4B3C7 / YOYO-1-mRNA complex can successfully deliver mRNA to the basal layer of the retina via intravitreal injection.

[0118] Example 16

[0119] The therapeutic effect of the LNP-A4B3C7 / mCas9 / sgVEGFA complex of the present invention on laser-induced choroidal neovascularization (CNV) in mice. On day 0, Bruch membranes in the eyes of C57BL / 6 mice were cauterized for 0.05 s using a laser with a wavelength of 659 nm and a power of 250 mW to induce CNV formation. On day 1, mice were randomly divided into 5 groups: (1) PBS group, (2) LNP-A4B3C7 / mCas9 / sgEGFP complex group, (3) LPF 2K / mCas9 / sgVEGFA complex group, (4) Aflibercept group, and (5) LNP-A4B3C7 / mCas9 / sgVEGFA complex group. Each group of mice was injected intravitreally with 2 μL of PBS, aflibercept, and each liposome complex. The dose of aflibercept was 0.1 mg / kg. The concentrations of mCas9 in the LNP-A4B3C7 / mCas9 / sgEGFA, LPF 2K / mCas9 / sgVEGFA, and LNP-A4B3C7 / mCas9 / sgVEGFA complex were 0.8 μg, and the concentrations of sgVEGFA and sgEGFP were 0.4 μg. Normal mice served as controls. On day 7, the fundus of the mice was monitored using fluorescein angiography (FFA) and indocyanine green angiography (ICGA). After fundus angiography, the choroid of the mice was removed, and the size of the CNV region was observed by immunofluorescence staining. See details... Figure 20 The experimental results showed that the leakage and damage area of ​​CNV sites in the LNP-A4B3C7 / mCas9 / sgVEGFA complex treatment group were significantly reduced, and the areas of the green fluorescently labeled CNV region and the red fluorescently labeled endothelial cell region in the laser spot were also significantly reduced. Its therapeutic effect was comparable to that of the commercial VEGFA inhibitor aflibercept, while the other groups did not show significant therapeutic effects, proving that the LNP-A4B3C7 / mCas9 / sgVEGFA complex effectively inhibits the formation of choroidal neovascularization.

[0120] Example 17

[0121] To evaluate the knockout ability of the LNP-A4B3C7 / mCas9 / sgVEGFA complex in RPE cells in a CNV model, on day 0, Bruch's membrane in the eyes of C57BL / 6 mice was cauterized for 0.05 s using a laser with a wavelength of 659 nm and a power of 250 mW to induce CNV formation. On day 1, mice were randomly divided into 5 groups: (1) PBS group, (2) LNP-A4B3C7 / mCas9 / sgEGFP complex group, (3) LPF 2K / mCas9 / sgVEGFA complex group, (4) Aflibercept group, and (5) LNP-A4B3C7 / mCas9 / sgVEGFA complex group. Each group of mice was injected intravitreally with 2 μL of PBS, aflibercept, and various liposome complexes. The dose of aflibercept was 0.1 mg / kg. The dosage of mCas9 in the LNP-A4B3C7 / mCas9 / sgEGF, LPF 2K / mCas9 / sgVEGFA, and LNP-A4B3C7 / mCas9 / sgVEGFA complexes was 0.8 μg, and the dosages of sgVEGFA and sgEGFP were 0.4 μg. On day 7, the RPE-choroid-sclera complex (RCS complex) of mice was removed, and the expression of VEGFA mRNA and the content of VEGFA in the RCS complex were detected by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. RPE cells were isolated from the LNP-A4B3C7 / mCas9 / sgVEGFA complex group, and genomic DNA was extracted using a cell / tissue genomic DNA extraction kit for next-generation sequencing (NGS). See details... Figure 21 Experimental results showed that a single intravitreal injection of the LNP-A4B3C7 / mCas9 / sgVEGFA complex significantly reduced the relative VEGFA mRNA level and VEGFA concentration in the RCS complex in the CNV model. Furthermore, it successfully knocked out 56% of the VEGFA gene in RPE cells.

Claims

1. An ionizable lipid based on an iminoboronic ester structure, characterized in that: The ionizable lipid based on the iminoboronic ester structure has the structure shown in formula (I): ； In the structure shown in equation (I), R1 is one of the following structures: ； R2 can be one of the following structures: 。 2. The method for preparing ionizable lipids based on iminoboronic ester structures according to claim 1, characterized in that, Includes the following steps: Using monomers A, B, and C as raw materials, an ionizable lipid based on an iminoboronic ester structure was prepared by reaction. Monomer A has one of the structures shown in the following formula: ； Monomer B has one of the structures shown in the following formula: ； Monomer C has one of the structures shown in the following formula: 。 3. The method for preparing ionizable lipids based on iminoboronic ester structures according to claim 2, characterized in that, Monomers A, B, and C were reacted in a mixed solvent of methanol and chloroform at room temperature for 1–10 hours.

4. The use of the ionizable lipid based on the iminoboronic ester structure as described in claim 1 in the preparation of a drug, or in the preparation of a drug carrier.

5. A nanomedicine, characterized in that, The drug is self-packaged from ionizable lipids and auxiliary lipids based on iminoboronic acid ester structure as described in claim 1.

6. The nanomedicine according to claim 5, characterized in that, The drug is mRNA.

7. The nanomedicine according to claim 5, characterized in that, The nitrogen-to-phosphorus ratio of the ionizable lipids based on the iminoboronic acid ester structure to the mRNA is (1-50):

1.

8. The use of the nanomedicine of claim 5 in the preparation of mRNA delivery or gene editing drugs.

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