A lipid nanoparticle, its preparation method, and its application in immune cell delivery.

Abstract

This invention belongs to the field of biomedical technology, specifically relating to lipid nanoparticles, their preparation method, and their application in immune cell delivery. This invention introduces amino acid-derived lipids as a fifth component into a preferred four-component formulation. Furthermore, through screening of the nitrogen-to-phosphorus ratio, the proportion of amino acid-derived lipids introduced, and the type of amino acid-derived lipids, novel lipid nanoparticles capable of efficiently delivering primary immune cells are selected. The delivery carrier described in this invention can significantly improve the delivery efficiency to primary immune cells, especially NK cells, providing a safe and efficient tool for the engineering of immune cells and their clinical application in immune cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a lipid nanoparticle, its preparation method, and its application in immune cell delivery. Background Technology

[0002] Immunotherapy is a strategy that uses living cells as therapeutic agents. The core of immunotherapy lies in utilizing and modifying human immune cells to enable them to treat specific diseases, such as cancer and infectious diseases.

[0003] Currently, nucleic acid delivery to immune cells (such as NK cells) mainly occurs via viral (e.g., lentiviral and adeno-associated virus infection) or non-viral (including transposon systems, electroporation, and lipid nanoparticles (LNPs)). Viral systems carry the risk of insertional mutations and have strong immunogenicity; transposon systems depend on plasmid transfection efficiency; and electroporation systems can lead to partial cell death and may cause morphological changes, thus affecting biological function. Lipid nanoparticle (LNP) delivery systems are increasingly widely used in clinical practice, primarily for the prevention of viral infections, cancer, and autoimmune diseases. However, there are still some limitations to overcome in immune cell drug delivery. For example, in the LNP delivery process targeting non-professional endocytic cells such as T cells or NK cells, low endocytic activity limits the uptake of mRNA loaded in LNPs; simultaneously, the highly sensitive immune surveillance systems of T cells or NK cells (TLR, RIG-I, etc.) can block the translation of exogenous mRNA and degrade it, thereby significantly reducing mRNA expression efficiency.

[0004] NK cells, a subset of lymphocytes, possess biological characteristics that make them a safer and more promising immunocellular therapy compared to T-cell therapies with severe side effects. However, NK cells, especially primary NK cells, face significant challenges in delivering exogenous nucleic acids. This may be due to the abundance of pattern recognition receptors on the NK cell surface activating defense responses and hindering nucleic acid transfection. Recent studies have reported that adding cationic lipids (DOTAPs) to an FDA-approved four-component regimen (D-Lin-MC3-DMA, 1,2-distearate-sn-glycerol-3-phosphocholine DSPC, cholesterol, and PEG-DMG) can improve NK cell transfection efficiency. This LNP achieved a delivery efficiency of 62.7% in the NK-92MI cell line and a maximum delivery efficiency of 76.60% in primary NK cells. Another study disclosed an LNP delivery platform containing imidazole-modified ionizable lipids, achieving efficient nucleic acid delivery to both NK cell lines and primary NK cells, with a delivery efficiency of approximately 70% in the NK-92 cell line. However, the introduction of cationic lipids raises potential safety concerns, and the high positive charge under physiological conditions limits the in vivo application of LNPs. Furthermore, protein expression after delivery of therapeutic nucleic acid drugs needs to reach a certain threshold, and the efficiency of these two reported LNPs in delivering nucleic acids to NK cells and their protein expression efficiency still have room for further optimization.

[0005] In summary, current nucleic acid delivery systems for immune cells, especially those targeting NK cells, remain inadequate. There is an urgent need to develop an LNP delivery system that can safely improve delivery efficiency to immune cells and enhance their effector function. Summary of the Invention

[0006] The purpose of this invention is to provide a lipid nanoparticle, its preparation method, and its application in immune cell delivery. The specific technical solution adopted by this invention is as follows.

[0007] A first aspect of the present invention is to provide a novel lipid nanoparticle.

[0008] A lipid nanoparticle for delivering nucleic acids and drugs to immune cells, wherein the lipid nanoparticle is prepared from amino acid-derived lipids, ionizable lipids, cofactor phospholipids, sterols, and PEG lipids; wherein the molar ratio of the amino acid-derived lipids in the lipid nanoparticle ranges from 5% to 30%.

[0009] The amino acid-derived lipid comprises one or more materials selected from the group consisting of: stearoyl taurine, palmitoyl taurine, tauroursodeoxycholic acid, oleylglutamine, stearoyl leucine, oleyl phenylalanine, oleyl serine, oleyl alanine, palmitoyl glycine, palmitoyl aspartic acid, and linolenic acid tyrosine.

[0010] The amino acid-derived lipids can interact with the nucleic acid (e.g., mRNA) or drug to be delivered via van der Waals forces, thereby significantly improving the encapsulation efficiency of LNPs on mRNA. Simultaneously, the introduction of amino acid-derived lipids can significantly enhance the endocytosis of mRNA-LNPs by immune cells, thereby improving the translation efficiency of mRNA drugs and their protein expression levels in immune cells.

[0011] Furthermore, the amino acids in the amino acid-derived lipids comprise one or more materials selected from the group consisting of: glutamine, leucine, taurine, arginine, serine, glycine, tyrosine, aspartic acid, alanine, or phenylalanine.

[0012] The amino acid-derived lipids refer to synthetic lipid molecules formed by chemically bonding amino acids or their derivatives as hydrophilic heads with hydrophobic fatty acid chains or lipid backbones (such as sterols, fatty acids, etc.). For example, glutamine as the head compound covalently bonds with oleic acid via an amide bond to form oleylglutamine; leucine as the head compound covalently bonds with stearoyl via an amide bond to form stearoylleucine; taurine as the head compound covalently bonds with stearic acid via an amide bond to form stearoyltaurine; phenylalanine as the head compound covalently bonds with oleic acid via an amide bond to form oleylphenylalanine; serine as the head compound covalently bonds with oleic acid via an amide bond to form oleylserine; alanine as the head compound covalently bonds with oleic acid via an amide bond to form oleylalanine; glycine as the head compound covalently bonds with palmitic acid via an amide bond to form palmitoylglycine; aspartic acid as the head compound covalently bonds with palmitic acid via an amide bond to form palmitoylaspartic acid; tyrosine as the head compound covalently bonds with linolenic acid via an amide bond to form linolenic acid tyrosine; and taurine as the head compound covalently bonds with ursodeoxycholic acid via an amide bond to form tauroursodeoxycholic acid.

[0013] Furthermore, the ionizable lipids include DLin-MC3-DMA, ALC-0315, SM-102, Lipid 5, L319, C12-200, or FTT5.

[0014] Furthermore, the auxiliary phospholipids include DSPC, DOPE, DSPE, DOPC, or DOTAP.

[0015] Furthermore, the sterols include cholesterol, phytosterols, or bile acids.

[0016] Furthermore, the phytosterols include β-sitosterol or stigmasterol.

[0017] Furthermore, the PEG lipids include ALC-0159, DMG-PEG 2000, DSPE-PEG 2000, or DSG-PEG2000.

[0018] In some preferred embodiments, the ionizable lipid is ALC-0315.

[0019] In some preferred embodiments, the auxiliary phospholipid is DSPC (1,2-distearate-sn-glycerol-3-phosphocholine).

[0020] In some preferred embodiments, the phytosterol is β-sitosterol.

[0021] In some preferred embodiments, the PEG lipid is DMG-PEG 2000 (1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000).

[0022] Furthermore, the lipid nanoparticles have a particle size range of 80-125 nm and a positive zeta potential under acidic conditions.

[0023] Furthermore, the molar ratio of each component in the lipid nanoparticles is (10-35): (5-30): (5-20): (30-45): (0.5-3.0).

[0024] In some preferred embodiments, the molar ratio of the components in the lipid nanoparticles is 31.5: 10: 19: 38.5: 1 for ionizable lipids: amino acid-derived lipids: cofactor phospholipids: sterols: PEG lipids.

[0025] Furthermore, the nitrogen-to-phosphorus ratio (N / P ratio) of the lipid nanoparticles is 6-20:1.

[0026] In some preferred embodiments, the nitrogen-to-phosphorus ratio of the lipid nanoparticles is 6:1.

[0027] Another aspect of the present invention is to provide the application of lipid nanoparticles.

[0028] The application of lipid nanoparticles in the preparation of drug delivery carriers for immune cells or engineered immune cells, wherein the lipid nanoparticles are used to deliver nucleic acids and / or drugs to immune cells or engineered immune cells.

[0029] Furthermore, the immune cells include NK cells, T cells, macrophages, or NKT cells.

[0030] Furthermore, the engineered immune cells include chimeric antigen receptor natural killer cells, chimeric antigen receptor T cells, chimeric antigen receptor macrophages, or chimeric antigen receptor natural killer T cells.

[0031] Furthermore, the nucleic acid includes mRNA encoding cytokines, mRNA encoding gene editing proteins, or mRNA encoding chimeric antigen receptors.

[0032] Another aspect of the present invention is to provide a method for preparing lipid nanoparticles.

[0033] The above-mentioned method for preparing lipid nanoparticles for delivering nucleic acids and drugs to immune cells includes the following steps:

[0034] S01: Amino acid-derived lipids, ionizable lipids, cofactor phospholipids, sterols, and PEG lipids are dissolved in an organic solvent to form the lipid phase;

[0035] S02: Dissolve the nucleic acid or drug to be delivered in an acidic buffer solution as the aqueous phase;

[0036] S03: Using a microfluidic device, the lipid phase and the aqueous phase are mixed at a mixing flow rate ratio of 1:3 to prepare lipid nanoparticles for delivering nucleic acids and drugs to immune cells.

[0037] In another aspect, the present invention may provide an engineered immune cell.

[0038] An engineered immune cell comprises immune cells, lipid nanoparticles, and an exogenous drug; the exogenous drug is loaded in the lipid nanoparticles; when the lipid nanoparticles are brought into contact with the immune cells, the immune cells undergo endocytosis with the lipid nanoparticles to ingest the lipid nanoparticles into the cells, thereby obtaining the engineered (modified) immune cell.

[0039] Furthermore, the structure and function of the engineered immune cells are modified by endocytosis of the lipid nanoparticles.

[0040] Furthermore, the exogenous drug includes mRNA encoding cytokines, mRNA encoding gene-editing proteins, or mRNA encoding chimeric antigen receptors.

[0041] In some specific embodiments, when the exogenous drug loaded in the lipid nanoparticles is CAR mRNA, the engineered immune cells internalize the lipid nanoparticles, and the delivered CAR mRNA is translated into CAR protein inside the cell and expressed on the cell surface. The cell membrane surface characteristics and the ability to target tumor cells of the immune cells are fundamentally modified, thereby becoming CAR immune cells capable of attacking specific tumor cells.

[0042] It is understood that the present invention may also provide a method for modifying immune cells based on lipid nanoparticles to obtain engineered immune cells with specific structures or functions.

[0043] Beneficial technical effects:

[0044] This invention provides a novel lipid nanoparticle. By introducing amino acid-derived lipids as a fifth component into a preferred four-component formulation, and through screening based on the nitrogen-to-phosphorus ratio, the proportion of amino acid-derived lipids introduced, and the type of amino acid-derived lipids, novel lipid nanoparticles capable of efficiently delivering primary immune cells are selected. These lipid nanoparticles can enhance phagocytosis by immune cells and increase the expression level of proteins encoded by the delivered mRNA. Experiments have demonstrated that the lipid nanoparticles provided by this invention significantly improve the delivery efficiency to primary immune cells, especially NK cells, compared to commercially available transfection reagents and other lipid nanoparticles, providing a safe and efficient tool for the engineering of immune cells and their clinical application in immunotherapy. Simultaneously, it also provides a more clinically applicable engineered immune cell modified with the aforementioned lipid nanoparticles.

[0045] Specifically, the preferred five-component lipid synergistic system of this invention can significantly improve the delivery efficiency to human primary NK cells compared to the existing LNP system, reaching up to 91.32%. In this five-component lipid synergistic system, the encapsulation rate of mRNA, the delivery efficiency to immune cells, and the level of protein expression after delivery are significantly improved mainly by the introduction of specific types of amino acids (non-positively charged amino acid derivatives). Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0047] Figure 1 For sterol screening and LNP speciation characterization;

[0048] Figure 2 For screening ionizable lipids and auxiliary lipids;

[0049] Figure 3 To detect the delivery efficiency of the five-component mRNA-LNP;

[0050] Figure 4 A comparison of transfection efficiency between A1-LNP and commercial products;

[0051] Figure 5 The introduction of amino acid-derived lipids enhances the uptake of A1-LNP cells;

[0052] Figure 6 Transfection efficiency of A1-LNP under different nitrogen-phosphorus ratios;

[0053] Figure 7 The effect of the proportion of amino acid-derived lipids introduced into the five-component LNP on transfection efficiency;

[0054] Figure 8 A schematic diagram of the ADL-LNP structure, its particle size distribution, PDI, Zeta potential, and encapsulation efficiency are shown.

[0055] Figure 9 ADL-LNP delivers Luciferase mRNA to the NK-92 cell line;

[0056] Figure 10 ADL-LNP delivers Luciferase mRNA to primary NK cells;

[0057] Figure 11 Introducing amino acid-derived lipids improves the encapsulation efficiency of LNP for eGFP mRNA;

[0058] Figure 12 The delivery efficiency of ADL-LNP in hPBMC-NK cells;

[0059] Figure 13 ADL-LNP synthesized from amino acid-derived lipids with taurine as the head group

[0060] Delivery efficiency in hPBMC-NK cells;

[0061] Figure 14 ADL-LNP delivers Cas9 mRNA to the NK-92 cell line;

[0062] Figure 15 ADL-LNP delivers anti-hCD19-CAR mRNA to NK-92 cells;

[0063] Figure 16 ADL-LNP delivers Cas9 mRNA to hPBMC-NK cells;

[0064] Figure 17 ADL-LNP delivers anti-hCD19-CAR mRNA to hPBMC-NK cells;

[0065] Figure 18 Functional evaluation of CAR-NK cells prepared in vitro from ADL-LNP;

[0066] Figure 19ADL-LNP delivers eGFP mRNA to human primary T cells;

[0067] Figure 20 ADL-LNP delivers eGFP mRNA to human primary NKT cells. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0069] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0070] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0071] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0072] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0073] Detailed descriptions of some of the attached figures:

[0074] Figure 1 A compares the expression efficiency, mean fluorescence intensity (MFI), and survival rate of eGFP in NK-92 cells after transfection with the Mock group, Dlin-MC3-DMA+cholesterol group, and Dlin-MC3-DMA+β-sitosterol group; B shows a transmission electron microscope image of LNP in the Dlin-MC3-DMA+β-sitosterol group.

[0075] Figure 2 A represents the eGFP expression efficiency of LNP-transfected NK-92 cells in the Mock group, ALC+DSPC group, ALC+DOPE group, MC3+DSPC group, and MC3+DOPE group; B represents the mean fluorescence intensity (MFI) of eGFP to evaluate protein expression levels; and C represents the cell viability to evaluate the safety of nanoparticles.

[0076] Figure 3 A represents the eGFP expression efficiency of NK-92 cells transfected with the Mock group, A-LNP group, and A1-LNP group; B represents the mean fluorescence intensity (MFI) of eGFP used to evaluate protein expression levels; and C represents the cell viability used to evaluate the safety of the nanoparticles.

[0077] Figure 7 A represents the expression efficiency of eGFP in LNP-transfected NK-92 cells after the fifth component, lipid oleyl glutamine, was introduced at different ratios: A0: 0%, A1-1: 10%, A1-2: 20%, and A1-3: 30%; B represents the mean fluorescence intensity (MFI) of eGFP used to evaluate protein expression levels; and C represents the cell viability used to evaluate the safety of the nanoparticles.

[0078] Figure 8 A is a structural schematic diagram of ADL-LNP; B is the particle size of ADL-LNP; C is the PDI of ADL-LNP; D is the zeta potential of ADL-LNP in acidic buffer solution.

[0079] Figure 10 A compares the expression levels of luciferase in primary PBMC-NK cells transfected from the Mock group, A0-LNP group, and ADL-LNP group; B compares the expression levels of luciferase in primary UCB-NK cells transfected from the Mock group, A0-LNP group, and ADL-LNP group.

[0080] Figure 12 A compares the eGFP transfection efficiency of primary PBMC-NK cells in each ADL-LNP group; B compares the average fluorescence intensity (MFI) of eGFP at the protein expression levels of the above groups.

[0081] Figure 18 A represents the cytotoxicity of CAR-NK cells prepared with A3-LNP against Nalm-6 cells at different effector-to-target ratios; B represents the significant difference in cytotoxicity between CAR-NK and PBMC-NK against Nalm-6 cells at each specific effector-to-target ratio.

[0082] Materials and Methods:

[0083] The sequence information involved in this invention is summarized as follows.

[0084] SEQ ID NO:1:

[0085] ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

[0086] SEQ ID NO:2:

[0087]

[0088] SEQ ID NO:3:

[0089]

[0090] SEQ ID NO:4:

[0091]

[0092] (1) Lipid dilution:

[0093] Each lipid was diluted with anhydrous ethanol as a solvent, and then diluted to a certain concentration of storage solution according to the solubility of the lipid and stored at -20℃.

[0094] (2) Preparation of citrate buffer

[0095] Weigh 0.626 g of citrate monohydrate (C6H8O7·H2O) and 0.5935 g of sodium citrate dihydrate (C6H5Na3O7·2H2O) into a sterile, enzyme-free centrifuge tube. Add 40 mL of DEPC-treated water, dissolve, and transfer to the volumetric flask. Then, add DEPC-treated water to bring the volume to 100 mL. This yields a 50 mM citrate buffer solution with pH 4. Prepare this buffer solution immediately before use.

[0096] (3) Preparation of PBS buffer

[0097] Weigh out 35.81 g of Na₂HPO₄·12H₂O, 2.45 g of KH₂PO₄, 80.07 g of NaCl, and 2.01 g of KCl using an analytical balance. Pour the powdered reagents into a PBS preparation beaker and add 800 mL of DEPC water. After stirring to dissolve, transfer the solution to a volumetric flask and bring the volume to 1 L. Adjust the pH to 7.4.

[0098] (4) LNP synthesis

[0099] The basic formulation used for sterol screening in Example 1 below, namely the classic formulation of the first LNP drug, Onpattro, is used as an example. Its formulation is: Dlin-MC3-DMA (MCE, USA): DSPC (Avita, China): Cholesterol (Avita, China): DMG-PEG 2000 (MCE, USA) = 50:10:38.5:1.5 (molar ratio), with eGFP mRNA or Luciferase mRNA as the mRNA. All mRNAs are modified with N1-methylpseuuridine. The sequence of the reporter gene is shown in Table 1. The delivery and expression efficiency of the reporter gene are indicators used to evaluate LNP efficacy.

[0100] Table 1 Reporter gene sequences

[0101]

[0102] According to the designed molar ratio, the corresponding volumes of lipids were mixed in anhydrous ethanol. The nitrogen-to-phosphorus ratio was optimized based on A1-LNP. The molar ratio of this formulation was ionizable lipid: amino acid-derived lipid: cofactor phospholipid: sterol: PEG lipid = 31.5:20:19:38.5:1. Reporter gene mRNA (eGFP-mRNA or Luciferase mRNA) obtained through in vitro transcription was diluted in citrate buffer (50 mM, pH=4) at specific nitrogen-to-phosphorus ratios (4, 6, 8, 10, 12, or 20). The lipid phase and aqueous phase were diluted separately with anhydrous ethanol and citrate buffer at a volume ratio of 1:3. LNPs were synthesized using a microfluidic preparation system (Maianna, XNano Prime). After washing and draining the flow, LNPs were synthesized at a total flow rate of 12 mL / min and a lipid-to-aqueous phase flow rate of 1:3 to obtain eGFP-LNPs.

[0103] ADL-LNP synthesis: Ionizable lipids, helper phospholipids, sterols, PEG lipids, and amino acid-derived lipids were mixed in anhydrous ethanol at a molar ratio of 31.5:19:38.5:1:10 to form the lipid phase. Reporter gene mRNAs (eGFP-mRNA or Luciferase mRNA) or therapeutic mRNAs (Cas9 mRNA or anti-hCD19 CAR mRNA) obtained through in vitro transcription were diluted in citrate buffer (50 mM, pH=4) at specific nitrogen-to-phosphorus ratios (4, 6, 8, 10, 12, or 20). The lipid and aqueous phases were diluted separately with anhydrous ethanol and citrate buffer at a volume ratio of 1:3. LNPs were synthesized using a microfluidic preparation system. After washing and draining the liquid, LNPs were synthesized at a total flow rate of 12 mL / min and a lipid-to-aqueous phase flow rate ratio of 1:3 to obtain ADL-LNPs encapsulated with different mRNAs.

[0104] (5) LNP solvent displacement

[0105] The synthesized LNP suspension was diluted with PBS, and the solvent was replaced using 30-100 kDa ultrafiltration centrifuge tubes at 4°C and 3000 rpm. The solution was then recovered after ultrafiltration centrifugation to the appropriate volume.

[0106] (6) Encapsulation efficiency test

[0107] The encapsulation of eGFP-mRNA by LNPs was detected using the Quant-iT™ RiboGreen RNA Quantification Kit (Thermo Fisher, R11490). Demulsification was performed using 2% Triton X-100 (Macklin, T6328) according to the kit instructions. The mRNA concentration after demulsification is the total concentration, and the mRNA concentration before demulsification is the concentration of mRNA free outside the LNPs. The encapsulation efficiency was calculated using the following formula:

[0108] Encapsulation efficiency EE% = (c total - c free) / c total × 100%; where c represents the concentration of the target RNA.

[0109] (7) Particle characterization and potential characterization

[0110] The average particle size, dispersion (PDI), and zeta potential of LNPs were characterized using dynamic light scattering (DLS).

[0111] The average particle size, dispersibility (PDI), and particle concentration of LNPs were characterized using a nanoparticle tracker (NTA).

[0112] The morphology and structure of LNPs were characterized using transmission electron microscopy.

[0113] (8) Culture of NK cell lines

[0114] The NK-92 cell line was purchased from the cell bank of Shanghai Enzyme Research Biotechnology Co., Ltd.

[0115] NK-92 cell culture conditions: MEMα, 20% FBS, 0.2mM Inositol, 0.1mM β-mercaptoethanol, 0.02mM Folic Acid, 100-200 U / mL recombinant IL-2 and 1% P / S.

[0116] Culture environment: air, 95%; carbon dioxide (CO2), 5%; constant temperature of 37℃.

[0117] (9) Human primary immune cell culture

[0118] Human primary NK cell culture conditions: RPMI 1640 medium, 10% FBS, 100 IU / mL recombinant IL-2 and 1% P / S.

[0119] Human primary T cells and NKT cells culture conditions: RPMI 1640 medium, 10% FBS, 100 IU / mL recombinant IL-2 and 1% P / S.

[0120] Culture environment: air, 95%; carbon dioxide (CO2), 5%; constant temperature of 37℃.

[0121] (10) LNP transfection of immune cells

[0122] Collect cultured immune cells (NK-92 cell line, PBMC-derived immune cells, or umbilical cord blood-derived immune cells) and transfer them to 15 mL centrifuge tubes. Centrifuge at 800 rpm for 5 min at room temperature. Discard the supernatant and resuspend in 1-2 mL of culture medium as described in Materials and Methods (8) and (9). After cell counting, take 2 × 10⁶ cells. 5 Cells were seeded in 48-well plates. For LNP transfection groups, mRNA-LNP dissolved in PBS was slowly added dropwise. The transfection dose was 0.4–0.6 μg / mL.

[0123] Collect cultured T cells or NKT cells and transfer them to 15 mL centrifuge tubes. Centrifuge at 800 rpm for 5 min at room temperature. Discard the supernatant and resuspend the cells in 1-2 mL of culture medium. The culture medium is as described in Materials and Methods (8) and (9). After cell counting, take 2 × 10⁶ cells. 5 Cells were seeded into 48-well plates. eGFP mRNA-LNP dissolved in PBS was slowly added to the LNP transfection group. The transfection dose was 0.4–0.6 μg / mL.

[0124] (11) Analysis of the endocytosis efficiency of lipid nanoparticles:

[0125] DiD was mixed with each component lipid at a molar ratio of 0.5% (relative to total lipids) in the ethanol phase. DiD-labeled LNPs were synthesized using a microfluidic preparation system. LNP particle count was quantified using NTA. LNPs were transfected into NK-92 cells 16 h after cell seeding. The LNPs were then added at a rate of 5 × 10⁶ cells per cell. 9 One LNP particle per milliliter of culture medium was used for transfection. Cells were collected 24 h after transfection, and the endocytosis efficiency of different LNP formulations in NK-92 cells was analyzed by flow cytometry.

[0126] (12) Lipid nanoparticles encapsulate mRNA drugs for delivery to NK cells:

[0127] The selected lipid nanoparticles were used to deliver mRNA drugs to NK cells, and the expression of the mRNA drug-encoded proteins was analyzed. The encapsulated mRNA drugs included Cas9 mRNA and Anti-hCD19-CAR mRNA. Sequence information of the mRNA drugs is shown in Table 2. The specific mRNA drug information used in these examples is merely illustrative and not limiting.

[0128] Table 2. Sequences of mRNA drugs

[0129]

[0130] (13) Luciferase detection method

[0131] Samples were collected into 1.5 mL centrifuge tubes 24 h after LNP delivery of Luciferase mRNA. The tubes were washed once with PBS. Centrifuged at 350×g for 5 min at room temperature, and the supernatant was discarded. 100 μL of thoroughly mixed reporter gene cell lysis buffer was added. The tubes were incubated on ice for 20 min to lyse. The firefly luciferase assay reagent was thawed on ice until room temperature was reached. After complete lysis, the tubes were centrifuged at 12000×g for 5 min, and 50 μL of the supernatant was used for measurement. 100 μL of firefly luciferase assay reagent was added to each well, mixed, and the RLU (Relative Light Unit) was measured.

[0132] (14) Flow cytometry analysis

[0133] After collecting cells, centrifuge to remove the culture medium, and wash once with PBS (containing 2% FBS). Resuspend again in PBS (containing 2% FBS) and adjust the concentration to 1×10⁻⁶. 6 100 μL / cells (or according to antibody instructions). Add flow cytometry antibody and mix with a pipette. Incubate on ice in the dark, shaking on a horizontal shaker. After 1 h, wash with 200 μL PBS (containing 2% FBS) to remove unbound antibody. Resuspend in PBS (containing 2% FBS) after staining. Filter the cell suspension through a flow cytometer into flow cytometry tubes, and then analyze using a Beckman CytoFLEX flow cytometer.

[0134] (15) Western blotting analysis of Cas9 mRNA transfection results

[0135] Collect NK-92 cells treated with mRNA-LNP for 48 h into 1.5 mL centrifuge tubes. Centrifuge at 400×g for 5 min and discard the supernatant. Add RIPA lysis buffer containing PMSF protease inhibitor, mix thoroughly, and incubate on ice for 30 min. Mix well and incubate on ice for 30 min. After lysis, centrifuge at 13000×g for 15 min at 4 °C. Transfer the supernatant to a new centrifuge tube. Quantify protein concentration using BCA. Add WB loading buffer to the remaining sample. Incubate at 98 °C for 10 min. Perform SDS-PAGE electrophoresis. Voltage: 80 V, 30 min, then 120 V, 1 h. After electrophoresis, transfer the protein to a PVDF membrane. Block with 5% skim milk powder for 2 h, then incubate with the corresponding primary antibody. Incubate overnight at 4 °C, then aspirate the antibody and wash three times with 1×TBST for 5 min each time. Horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies were diluted with 5% skim milk-TBST. The membranes were incubated in the secondary antibody dilution solution at room temperature for 1 h, followed by washing three times with TBST for 5 min each time. The membranes were then blotted clean with filter paper, ECL luminescent buffer was added, and the results were observed using a luminescence imaging system. The expression of the delivered mRNA-encoded protein was analyzed.

[0136] (16) Cytotoxicity of CAR-NK cells

[0137] Collect Nalm-6 cells and centrifuge at 400 g for 5 min to obtain cell pellet. Discard the culture medium and wash the cells with calcium- and magnesium-free PBS. Add Incucyte® Cytolight Rapid Red Dye (4706) to a final concentration of 1 x 10⁻⁶. 5 Add cells / mL to the cell suspension for staining. Incubate at 37°C in the dark for 20 minutes. Immediately after staining, add 6 volumes of serum-containing cell culture medium to the cell suspension to neutralize excess dye. Centrifuge at 400 g for 5 min and remove the supernatant. Resuspend the cells, count them, and seed them into 96-well plates at 10,000 cells / well. Collect hPBMC-NK cells and CAR-NK cells prepared with ADL-LNP, centrifuge (400 g, 5 min), and remove the supernatant. Resuspend the cells in 1640 complete medium and count them. Add effector cells to the corresponding wells for co-culture according to different effector-target ratios (E:T=0:1, E:T=0.2:1, E:T=0.4:1, E:T=0.8:1, E:T=1.6:1, and E:T=3.2:1). Set up 5 replicates per group. After incubation for 4 h, label dead cells with DAPI. Flow cytometry analysis - Incucyte® Cytolight Rapid Red Dye (4706) positive cell mortality rate.

[0138] Example 1

[0139] This embodiment mainly provides a method for screening highly effective LNP formulations based on immune cells.

[0140] First, using NK-92 cells as a model, Dlin-MC3-DMA was selected as the ionizable lipid, DMG-PEG2000 as the PEG lipid, and DSPC as the helper phospholipid. Under these conditions, suitable LNP sterol components for NK cell delivery were screened. The ionizable lipid, helper phospholipid, sterol, and PEG lipid were mixed in a molar ratio of 50:10:38.5:1.5. The LNP synthesis method is described in Materials and Methods (4).

[0141] Group design: NK-92 cells were divided into three groups: a blank control group (Mock) without treatment, an eGFP mRNA-LNP transfection group (sterol was cholesterol), and an eGFP mRNA-LNP transfection group (sterol was β-sitosterol). Transfection efficiency, mean fluorescence intensity (MFI), and cell viability were analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0142] Table 3

[0143]

[0144] Experimental results are as follows Figure 1 As shown, the delivery efficiency and mean fluorescence intensity (MFI) of LNPs with β-sitosterol as the sterol component in NK-92 cells were significantly higher than those with cholesterol as the sterol component. After incubation with both LNPs, the proportion of 7-AAD negative cells (cell viability) was not significantly different from the control group. Based on these screening results, β-sitosterol was selected as the sterol component of the LNP in subsequent experiments.

[0145] Furthermore, using NK-92 cells as a model, combinations of ionizable lipids and helper lipids that could improve NK cell delivery efficiency were screened. Ionizable lipids, helper phospholipids, sterols, and PEG lipids were mixed in a molar ratio of 50:10:38.5:1.5. The delivery efficiency of β-sitosterol combined with different ionizable lipids (ALC-0315 or Dlin-MC3-DMA) and different helper phospholipids (DSPC or DOPE) was analyzed.

[0146] DSPC: 1,2-distearyl-sn-glycerol-3-phosphocholine;

[0147] DOPE: 1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine.

[0148] Encapsulating eGFP Human NK-92 cells were transfected with the LNP of mRNA. The method for mRNA-LNP synthesis is described in Materials and Methods (4). The method for LNP transfection is described in Materials and Methods (10).

[0149] Group design: NK-92 cells were used as an untreated blank control group (Mock), eGFP mRNA-LNP transfection group 1 (ALC-0315 and DSPC combination), eGFP mRNA-LNP transfection group 2 (ALC-0315 and DOPE combination), eGFP mRNA-LNP transfection group 3 (Dlin-MC3-DMA and DSPC combination), and eGFP mRNA-LNP transfection group 4 (Dlin-MC3-DMA and DOPE combination). Transfection efficiency, mean fluorescence intensity, and cell viability were analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0150] Table 4

[0151]

[0152] Experimental results are as follows Figure 2 As shown, when β-sitosterol is the sterol component, the LNP containing ALC-0315 and DSPC is more effective in nucleic acid delivery in NK-92 cells. The delivery efficiency in NK-92 cells is 90.2%. The average fluorescence intensity is also significantly higher than the other three LNP formulations. Furthermore, after 24 h of treatment with this LNP formulation, the cell viability of NK-92 cells was not significantly different from that of the control group.

[0153] Based on these screening results, β-sitosterol, ALC-0315, DMG-PEG 2000 and DSPC were selected as the basic components of LNP, and this combination was preferred as the basic formulation for subsequent screening.

[0154] Furthermore, to improve LNP delivery efficiency and enhance protein expression levels after mRNA delivery, optimization was performed based on the four-component LNP selected in the previous step. An amino acid-derived lipid (oleoylglutamine, N-oleoylglutamine) was introduced as the fifth component into the four-component LNP construction system. Ionizable lipids, helper phospholipids, sterols, PEG lipids, and amino acid-derived lipids were mixed in a molar ratio of 21.5:19:38.5:1:20. Oleoylglutamine was purchased from MCE (HY-139006). Specifically, 20% (total lipid molar ratio) of the fifth component was introduced into the four-component lipid system. eGFP was then encapsulated. Human NK-92 cells were transfected with the LNP of mRNA. The method for mRNA-LNP synthesis is described in Materials and Methods (4). The method for LNP transfection is described in Materials and Methods (10).

[0155] Group design: NK-92 cells were used as an untreated blank control group (Mock), a four-component LNP transfection group (A0-LNP) obtained from the previous screening step, and a five-component LNP transfection group (A1-LNP) with oleylglutamine introduced. Transfection efficiency, mean fluorescence intensity, and cell viability were analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0156] Table 5

[0157]

[0158] Experimental results are as follows Figure 3 As shown, the introduction of oleylglutamine significantly enhances protein expression levels after LNP delivery. It is well known in the art that enhanced protein expression levels are crucial for the efficacy of therapeutic mRNA-LNPs.

[0159] Furthermore, the delivery efficiency of A1-LNP was compared with that of the LNP (denoted as B-LNP) delivery vector used in Pfizer's vaccine product BNT162b2 and the commercial mRNA transfection reagent (Lipofectamine MessengerMAX) in NK-92 cells. The dosage of eGFP mRNA transfected by both delivery methods was consistent. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10).

[0160] Group design: NK-92 cells were used as an untreated blank control group (Mock), transfected with the commercial mRNA transfection reagent MessengerMAX, transfected with the LNP delivery vector B-LNP used in Pfizer's vaccine product BNT162b2, and transfected with five LNPs containing an amino acid derivative (oleylglutamine) obtained from the previous screening step (A1-LNP). Transfection efficiency was analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0161] Table 6

[0162]

[0163] Experimental results are as follows Figure 4 As shown, the LNP formulation used in Pfizer's vaccine product BNT162b2 has excessively low delivery efficiency to NK cells. MessengerMAX delivers less than 1% of mRNA to NK-92 cells. In contrast, A1-LNP achieves a delivery efficiency of 95.40% at 24 h post-transfection. Compared to commercial transfection reagents, A1-LNP has a significant advantage in transfecting NK-92 cells.

[0164] The changes in endocytosis efficiency of A1-LNP after introducing amino acid-derived lipids into a five-component system were analyzed. A0-LNP and A1-LNP labeled with DiD fluorescent dye were synthesized using a microfluidic instrument. The endocytosis efficiency of LNPs was compared in NK-92 cells. Specific methods are detailed in Materials and Methods (11).

[0165] Group design: NK-92 cells were used as an untreated blank control group (Mock), a four-component LNP transfection group (A0-LNP), and a five-component LNP transfection group (A1-LNP) with an amino acid derivative (oleylglutamine). The endocytosis efficiency of DiD-LNP was analyzed by flow cytometry. The flow cytometry method is described in Materials and Methods (14).

[0166] Figure 5 Experimental results showed that the introduction of amino acid-derived lipids significantly improved the endocytosis efficiency of LNPs in NK-92 cells. Compared with the A0-LNP group, the DiD fluorescence intensity of the A1-LNP group increased by 3.62 times. These results indicate that the introduction of amino acid-derived lipids helps overcome the barrier of immune cells, enabling them to efficiently endocytose LNPs, thus providing a basis for the gene regulation and engineering of immune cells.

[0167] Furthermore, to screen for optimal nitrogen-phosphorus ratios, the transfection efficiency of A1-LNP in NK-92 cells was analyzed at different nitrogen-phosphorus ratios (6:1 or 20:1). The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10).

[0168] In this embodiment, the nitrogen-phosphorus ratio was adjusted based on the A1-LNP ratio. Group design: untreated NK-92 cells (Mock control group), LNP transfection group 1 (A1-LNP, N:P=6:1) with a nitrogen-phosphorus ratio of 6, and LNP transfection group 2 (A1-LNP, N:P=20:1) with a nitrogen-phosphorus ratio of 20. Transfection efficiency, mean eGFP fluorescence intensity, and cell viability were analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (13).

[0169] Table 7

[0170]

[0171] Experimental results are as follows Figure 6 As shown, the transfection efficiency of A1-LNP was not significantly different when the NPK ratio was 6 compared to that when it was 20. However, the former resulted in higher protein expression levels after delivery, indicating that an NPK ratio of 6 is more preferable.

[0172] Furthermore, the transfection efficiency of LNP was analyzed after introducing the fifth component, lipid oleylglutamine, at different proportions (10%, 20%, or 30%). The molar ratios of ionizable lipids, helper phospholipids, sterols, PEG-derived lipids, and amino acid-derived lipids were 11.5 / 21.5 / 31.5:19:38.5:1:10 / 20 / 30, respectively. Delivery efficiency was analyzed in NK-92 cells. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10).

[0173] Group design: NK-92 cells were used as an untreated blank control group (Mock), an optimized four-component LNP transfection group (A0-LNP), A1-LNP (A1-1, 10% amino acid-derived lipids) with 10% oleylglutamine, A1-LNP (A1-2, 20% amino acid-derived lipids) with 20% oleylglutamine, and A1-LNP (A1-3, 30% amino acid-derived lipids) with 30% oleylglutamine. Transfection efficiency, mean eGFP fluorescence intensity, and cell viability were analyzed by flow cytometry. Flow cytometry analysis methods are described in Materials and Methods (14).

[0174] Table 8

[0175]

[0176] Experimental results are as follows Figure 7 As shown, the transfection efficiency of A1-LNP with 10% or 20% oleylglutamine was not significantly different from that of the four-component A0-LNP in terms of delivery efficiency. However, the eGFP fluorescence intensity was significantly enhanced. Among them, A1-1 LNP transfection produced the highest eGFP fluorescence intensity, up to 5.71 times. Based on this screening result, the preferred total lipid molar ratio of amino acid-derived lipids was 10%. In addition, cell viability experiments showed that A1-LNP still had good safety after the introduction of different molar ratios of oleylglutamine.

[0177] Furthermore, 10 amino acid-derived lipids were screened as a library for further screening. The five-component LNP containing amino acid-derived lipids was named ADL-LNP. The structural model of ADL-LNP is shown below. Figure 8As shown in the figure. Ten ADL-LNPs were synthesized using a microfluidic instrument and characterized. The characterization methods are described in Materials and Methods (6) and (7). The fifth component of A1-LNP is oleylglutamine, the fifth component of A2-LNP is stearoylleucine (Yusi Pharmaceutical), the fifth component of A3-LNP is stearoyl taurine (MCE, HY-120964), the fifth component of A4-LNP is palmitoyl arginine (Yusi Pharmaceutical), the fifth component of A5-LNP is oleyl phenylalanine (MCE, HY-138207), the fifth component of A6-LNP is oleyl serine (MCE, HY-124081), the fifth component of A7-LNP is oleyl alanine (MCE, HY-N7831), the fifth component of A8-LNP is palmitoyl glycine (MCE, HY-W074890), the fifth component of A9-LNP is palmitoyl aspartic acid (Targetmol, T25918), and the fifth component of A10-LNP is linolenic acid tyrosine (glpbio, GC44270). To facilitate screening, Luciferase mRNA was encapsulated. Ten ADL-LNPs were compared with the optimized four-component basic formulation (molar ratio of ionizable lipids, cofactor phospholipids, sterols, and PEG lipids was 31.5:19:38.5:1).

[0178] Group design: Optimized four-component LNP transfection group (A0-LNP), A1-LNP with 20% oleylglutamine introduced, and ADL-LNP transfection groups (A2-A10) synthesized with lipids derived from the other nine amino acids respectively. The synthesis method of ADL-LNP is as described in Materials and Methods (4) and (5). The material encapsulation efficiency detection method is as described in Materials and Methods (6). The particle size, PDI, and Zeta potential detection methods are as described in Materials and Methods (7).

[0179] Table 9

[0180]

[0181] Figure 8 To characterize ADL-LNP using DLS (DelsaNano C, Beckman coulter). Based on... Figure 8The results showed that the particle size range of the 10 synthesized ADL-LNPs was between 80 and 125 nm. The Zeta potential of ADL-LNPs was measured at pH 4, indicating that ADL-LNPs are positively charged under acidic conditions. Furthermore, A4-LNPs exhibited flocculent precipitation during encapsulation, indicating that A4-LNPs could not form a stable complex with mRNA and were therefore excluded from subsequent NK cell delivery efficiency analysis. Further analysis revealed that A4, being a positively charged amino acid derivative, exhibits excessively strong electrostatic interactions with RNA, which may be the reason for the instability of the formed complex. Therefore, the LNP system of this invention is suitable for the introduction of non-positively charged amino acid derivatives.

[0182] Furthermore, the delivery efficiency of nine ADL-LNPs to the NK-92 cell line was analyzed. Luciferase mRNA was delivered to the NK-92 cell line, and the expression level of luciferase was analyzed using a firefly luciferase assay kit 24 h later. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10).

[0183] Group design: NK-92 cells untreated blank control group (Mock), optimized four-component LNP transfection group (A0-LNP), and nine ADL-LNP transfection groups with 20% amino acid-derived lipids introduced (the amino acid-derived lipids represented by A1-A3 and A5-A10 are the same as above).

[0184] Transfection efficiency was evaluated by the expression level of luciferase in NK-92 cells after transfection. Analysis was performed using a luciferase assay kit (Beyotime, RG009S). Specific experimental methods are described in Materials and Methods (13).

[0185] Experimental results are as follows Figure 9 As shown, the introduction of amino acid-derived lipids improved the delivery efficiency of ADL-LNP in NK-92 cells to varying degrees compared to the four-component LNP. Among them, A10-LNP showed a significant advantage in delivery efficiency in NK-92 cells compared to A1-LNP, with a measured RLU 1.64 times that of A1-LNP. These results indicate that introducing amino acid-derived lipids into an optimized four-component formulation for immune cells can construct a series of LNP formulations with highly efficient delivery capabilities to immune cells.

[0186] Furthermore, to analyze the delivery efficiency of ADL-LNP to primary NK cells, Luciferase mRNA was delivered to NK cells derived from human peripheral blood or human umbilical cord blood, and the expression level of luciferase was analyzed using firefly luciferase assay reagent after 24 h. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10).

[0187] Group design: Untreated blank control group (Mock) of human peripheral blood NK cells or umbilical cord blood NK cells, optimized four-component LNP transfection group (A0-LNP), A1-LNP with 20% oleylglutamine introduced and 8 other amino acid-derived lipids replaced the ionizable lipids respectively to obtain ADL-LNP transfection group (the amino acid-derived lipids represented by A2, A3 and A5-A10 are the same as above). The transfection efficiency was evaluated by the expression level of luciferase in primary NK cells after transfection. The luciferase detection kit (Beyotime, RG009S) was used for analysis. The specific experimental methods are as described in Materials and Methods (13).

[0188] Experimental results are as follows Figure 10 As shown, compared with the four-component LNP, the introduction of amino acid-derived lipids improved the delivery efficiency of ADL-LNP in primary NK cells to varying degrees. Among them, A1-LNP, A3-LNP, A5-LNP, A9-LNP, and A10-LNP showed higher luciferase expression levels in primary NK cells. In human peripheral blood NK (hPBMC-NK) cells, compared with the A0-LNP group, the luciferase expression level of the A1-LNP group increased by 2.14 times; the luciferase expression level of the A3-LNP group increased by 1.89 times; the luciferase expression level of the A5-LNP group increased by 2.58 times; the luciferase expression level of the A9-LNP group increased by 2.14 times; and the luciferase expression level of the A10-LNP group increased by 3.89 times.

[0189] ADL-LNP can efficiently deliver mRNA to primary NK cells from various sources and significantly improve protein expression levels after delivery. These results indicate that the ADL-LNP delivery system can overcome the challenges of NK cell delivery and has great potential in enhancing the efficacy of therapeutic mRNA drugs.

[0190] In summary, the preferred lipid nanoparticle formulation provided by this invention is as follows: ALC-0315 as the ionizable lipid; DMG-PEG 2000 as the PEG phospholipid; β-sitosterol as the sterol; and DSPC as the cofactor phospholipid. Simultaneously, based on the four-component LNP, 10%-30% amino acid-derived lipids are introduced, preferably 10%. ADL-LNP is synthesized using a microfluidic instrument. The synthesis procedure is as follows: the volume ratio of lipid phase to aqueous phase is 1:3; the total flow rate is 12 mL / min. During the synthesis of ADL-LNP, the molar ratio of ionizable lipids to nucleic acids is 6:1.

[0191] Analysis of the experimental results revealed differences in the delivery efficiency of different ADL-LNPs to NK cells. Among the 10 ADL-LNPs screened in this invention, five ADL-LNPs enhanced the delivery to primary NK cells compared to A0-LNP. These are: A1-LNP, A3-LNP, A5-LNP, A9-LNP, and A10-LNP (the amino acid-derived lipids represented by A1-A10 are consistent with those described above).

[0192] Example 2

[0193] This embodiment mainly utilizes the ADL-LNP formulation screened in Example 1 to deliver eGFP mRNA and accurately analyze the proportion of ADL-LNP transfected immune cells.

[0194] To analyze the delivery efficiency of ADL-LNPs in primary NK cells and accurately determine the proportion of successfully transfected cells and fluorescence intensity, three types of ADL-LNPs (A1, A3, and A10-LNPs) encapsulating eGFP mRNA were synthesized using microfluidics. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP encapsulation efficiency detection method is described in Materials and Methods (6). The LNP transfection method is described in Materials and Methods (10). The three types of ADL-LNPs were delivered to peripheral blood NK cells, and their delivery efficiency was compared with that of A0-LNP after 24 h.

[0195] Group design: Untreated peripheral blood NK cells blank control group (Mock), optimized four-component LNP transfection group (A0-LNP), A1-LNP group with amino acid-derived lipid oleylglutamine, A3-LNP group with amino acid-derived lipid stearoyl taurine, and A10-LNP group with amino acid-derived lipid linolenic acid. Transfection efficiency was analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0196] Experimental results are as follows Figure 11 As shown, the ADL-LNP constructed in this invention can improve the encapsulation efficiency of mRNA. In an acidic preparation environment, the amino acid-derived lipid head can further enhance the binding of ADL-LNP to mRNA through various van der Waals forces, thereby improving the mRNA encapsulation efficiency. The abundant groups on the amino acid branches can form a secondary bond network with the polar regions of mRNA. This multiple interaction can enhance the thermodynamic stability of the lipid-mRNA complex and promote the self-assembly of lipid molecules to form structures conducive to nucleic acid encapsulation.

[0197] Experimental results are as follows Figure 12As shown, the ADL-LNP constructed in this invention can be effectively delivered to primary NK cells. Furthermore, the delivery efficiency of ADL-LNP to human primary NK cells varies with the introduction of different amino acid-derived lipids. Specifically, the delivery efficiency of A3-LNP to primary NK cells is 91.32%. The delivery efficiency of the ADL-LNP constructed in this invention to human primary NK cells is higher than the delivery efficiency (76.6%) of DOTAP-containing LNPs currently reported.

[0198] The delivery efficiency of ADL-LNPs synthesized from different amino acid-derived lipids in hPBMC-NK cells varies. Taurine-derived lipid-synthesized ADL-LNPs show a certain advantage in hPBMC-NK cell delivery. Furthermore, tauroursodeoxycholic acid was introduced as a taurine-derived lipid to synthesize ADL-LNPs for delivery of eGFP mRNA to hPBMC-NK cells for verification. A11-LNPs encapsulating eGFP mRNA were synthesized using microfluidics. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10). A11-LNPs were delivered to peripheral blood NK cells, and after 24 h, they were compared with the A3-LNP transfection group.

[0199] Group design: Untreated human peripheral blood NK cells (Mock) group, A3-LNP group, and A11-LNP group (amino acid-derived lipid: tauroursodeoxycholic acid). Transfection efficiency was analyzed by flow cytometry. (See Materials and Methods 14 for flow cytometry analysis methods).

[0200] Experimental results are as follows Figure 13 As shown, the delivery efficiency of A11-LNP decreased compared to A3-LNP, reaching 87.9% of that of A3-LNP. However, this result indicates that A11-LNP constructed from tauroursodeoxycholic acid can be effectively delivered to primary NK cells with an efficiency of 75.2%.

[0201] In summary, the ADL-LNP constructed in this invention significantly improves the encapsulation efficiency of mRNA through the introduction of amino acid-derived lipids. The ADL-LNP provided by this invention achieves a delivery efficiency of up to 91.32% to human primary NK cells. Importantly, the ADL-LNP constructed in this invention can significantly increase protein expression levels, which is of great significance for immunotherapy.

[0202] Example 3

[0203] This embodiment provides validation of the effectiveness of ADL-LNP-encapsulated mRNA drug delivery to immune cells as screened in Example 1.

[0204] LNP drug delivery systems primarily deliver nucleic acid drugs, such as gene editing systems (Cas9 mRNA + sgRNA), chimeric antigen receptors (CAR mRNA), small interfering RNA (siRNA), oligonucleotides, etc.

[0205] To verify the efficiency of the ADL-LNP gene editing system in delivering nucleic acids to NK cells, Cas9 mRNA was encapsulated using the A1-LNP formulation described above and analyzed in NK-92 cells. The LNP transfection method is described in Materials and Methods (10). Cas9 protein expression was analyzed by Western blotting 48 h after transfection. The analytical methods are as described in Materials and Methods (15).

[0206] Group design: The NK-92 cell delivery experiment included an untreated blank control group (Mock) and an A1-LNP transfection group.

[0207] Experimental results are as follows Figure 14 As shown, A1-LNP can deliver Cas9 mRNA into NK-92 cells. The ADL-LNP delivery system constructed in this invention can precisely engineer human NK-92 cells through the delivery of gene editing systems, providing a transient modification method for strategies using NK-92 cells as a source of cell therapy. This is beneficial for developing "off-the-shelf" NK cell therapy products.

[0208] Furthermore, this study provides the CAR-NK cell therapy's pleiotropic killing mechanism of NK cells and the targeting strategy of CAR technology. To verify the delivery efficiency of ADL-LNP in delivering chimeric antigen receptor mRNA (CAR mRNA) to NK cells, anti-CD19-CAR mRNA was encapsulated using A1-LNP and analyzed in NK-92 cells using flow cytometry. The mRNA dosage was 0.8 μg / mL. The flow cytometry detection method is described in Materials and Methods (14).

[0209] Group design: NK-92 cells untreated blank control group (Mock) and A3-LNP-CAR transfection group (A3-LNP).

[0210] Experimental results are as follows Figure 15 As shown, CAR expression was detectable in NK-92 cells 24 h after A3-LNP-CAR transfection. The measured anti-hCD19 CAR expression efficiency was 82.10%.

[0211] Currently, NK cell sources for NK cell therapy include NK cells derived from human peripheral blood, NK cells derived from umbilical cord blood, NK cells derived from pluripotent induced cellular stem cells, and the NK-92 cell line. The NK-92 cell line is widely used in clinical research and is easily engineered. However, the NK-92 cell line carries a risk of tumorigenesis. Therefore, it must be irradiated before clinical infusion to disable its ability to proliferate in vivo. Primary NK cells, on the other hand, exhibit higher cytotoxic activity, safety, and longer in vivo survival. Therefore, primary NK cells are a more ideal cell source. Thus, developing a highly efficient primary NK cell delivery system can further enhance the clinical application of therapeutic NK cells.

[0212] To verify the efficiency of the ADL-LNP gene editing system in delivering nucleic acids to peripheral blood-derived NK cells (hPBMC-NK cells), Cas9 mRNA was encapsulated using A3-LNP and analyzed in hPBMC-NK cells. The mRNA dosage was 1 μg / mL. Cas9 protein expression was analyzed by Western blotting 48 h after transfection. The analytical methods are as described in Materials and Methods (15).

[0213] Group design: The hPBMC-NK cell delivery experiment included an untreated hPBMC-NK cell blank control group (Mock) and an A3-LNP transfection group.

[0214] Experimental results are as follows Figure 16 As shown, A3-LNP can deliver Cas9 mRNA into hPBMC-NK cells and efficiently express Cas9 protein. The ADL-LNP delivery system constructed in this invention can precisely engineer human peripheral blood-derived NK cells through a gene-editing delivery system, enhancing the anti-tumor function of NK cells, thereby providing a highly efficient and advantageous tool for NK cell therapy.

[0215] Furthermore, the efficiency of ADL-LNP delivery of CAR mRNA to hPBMC-NK cells was analyzed. AntihCD19-CAR mRNA was encapsulated using A3-LNP and analyzed in hPBMC-NK cells by flow cytometry. The mRNA dosage was 2 μg / mL. Flow cytometry detection methods are described in Materials and Methods (14).

[0216] Group design: untreated hPBMC-NK cells blank control group (Mock) and A3-LNP-CAR transfection group (A3-LNP).

[0217] Figure 17The results showed that CAR protein expression was detectable in hPBMC-NK cells 24 h after A3-LNP-CAR transfection. The measured anti-hCD19-CAR expression efficiency was 53.40%. Compared with viral vectors, transient expression of CAR protein in primary NK cells via ADL-LNP delivery significantly shortened the CAR-NK cell preparation time. It can serve as a tool for rapid screening of CAR protein structure and functional verification. Importantly, the strategy of LNP delivery vector and transient mRNA modification is beneficial to improving the safety of CAR-NK cells in clinical applications.

[0218] To analyze the specific cytotoxicity of CAR-NK cells prepared in vitro using ADL-LNP, the B-cell acute lymphoblastic leukemia cell line Nalm-6 was selected as the target cell. Anti-hCD19-CAR mRNA was encapsulated using A3-LNP to prepare CAR-NK cells (hPBMC-NK cells) in vitro. NK cells and Nalm-6 cells were co-cultured at effector-to-target ratios of E:T = 0:1, E:T = 0.2:1, E:T = 0.4:1, E:T = 0.8:1, E:T = 1.6:1, and E:T = 3.2:1. Specific experimental methods are detailed in Materials and Methods (16).

[0219] Group design: Untreated hPBMC-NK cells and CAR-NK cells prepared with A3-LNP-CAR. Each group had 6 effector-target ratios, with 5 replicates per effector-target ratio.

[0220] Experimental results are as follows Figure 18 As shown, CAR-NK cells prepared by A3-LNP significantly enhanced specific cytotoxicity against Nalm-6 cells at different effector-to-target ratios. This result further demonstrates that A3-LNP can effectively deliver CAR mRNA to primary NK cells, and successfully prepare CAR-NK cells with specific killing function.

[0221] In summary, the delivery strategy of the ADL-LNP "five-component" lipid synergistic system established in this invention improves the mRNA drug delivery efficiency of LNP to NK cells and the expression level of the target protein, while exhibiting good safety. Currently, no other studies have reported improving the delivery efficiency of LNP to NK cells and other immune cells or the post-delivery protein expression level through the introduction of amino acid-derived lipids.

[0222] Example 4

[0223] This embodiment provides verification of the effectiveness of ADL-LNP in delivering reporter gene mRNA to primary T cells or NKT cells as screened in Example 1.

[0224] T cells are a core component of adaptive immunity, possessing crucial anti-infection and anti-tumor functions. They have been widely used clinically in adoptive immunotherapy. NKT cell therapy, leveraging the inherent dual characteristics of T and NK cells, plays a unique bridging immune role in tumor immune surveillance, anti-infection, and autoimmune regulation.

[0225] To verify the delivery efficiency of the ADL-LNP delivery strategy for human peripheral blood-derived T cells, ADL-LNPs (specifically A3-LNPs) encapsulating eGFP mRNA were synthesized using microfluidics. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10). Delivery efficiency was analyzed by flow cytometry 24 h after LNP delivery into hPBMC-T cells. The flow cytometry analysis method is described in Materials and Methods (14).

[0226] Group design: primary T cells untreated blank control group (Mock) and A3-LNP transfection group. Transfection efficiency was analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0227] Experimental results are as follows Figure 19 As shown, the A3-LNP constructed in this invention can effectively deliver to primary T cells. The delivery efficiency of A3-LNP to primary T cells is 83.10%. This result indicates that the ADL-LNP delivery system can efficiently transfect hPBMC-derived T cells, which is beneficial for the transient and efficient expression of therapeutic proteins in T cells. The ADL-LNP delivery system helps reduce preparation costs and time, thereby improving the accessibility and safety of T cell therapy.

[0228] Furthermore, to verify the delivery efficiency of the ADL-LNP delivery strategy for human peripheral blood-derived NKT cells, ADL-LNP (A3-LNP) encapsulating eGFP mRNA was synthesized using microfluidics. The mRNA-LNP synthesis method is described in Materials and Methods (4). The LNP transfection method is described in Materials and Methods (10). LNPs were delivered to primary NKT cells. After 24 h, the delivery efficiency was analyzed by flow cytometry. The flow cytometry analysis method is described in Materials and Methods (14).

[0229] Group design: primary NKT cells untreated blank control group (Mock) and A3-LNP transfection group.

[0230] The results are as follows Figure 20As shown, the ADL-LNP constructed in this invention can effectively deliver primary NKT cells. The delivery efficiency of A3-LNP to primary NKT cells is 80.70%. NKT cells have unique antigen recognition mechanisms and immunomodulatory functions, and can be used as a combination therapy. Modifying primary NKT cells using the ADL-LNP delivery system is beneficial for enhancing the efficacy of NKT cell therapy.

[0231] In summary, the ADL-LNP delivery strategy established in this invention can be used for the efficient delivery of immune cells derived from human peripheral blood, including NK cells, T cells, and NKT cells. It provides a highly efficient tool for the engineering and modification of primary immune cells or difficult-to-transfect lymphocytes.

[0232] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0233] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A lipid nanoparticle for delivering nucleic acids and drugs to immune cells, characterized in that, Lipid nanoparticles were prepared from amino acid-derived lipids, ionizable lipids, cofactor phospholipids, sterols, and PEG lipids. The ionizable lipid is ALC-0315 or DLin-MC3-DMA, the auxiliary phospholipid is DSPC or DOPE, the sterol is cholesterol or β-sitosterol, and the PEG lipid is DMG-PEG 2000. The amino acid-derived lipids comprise one or more materials selected from the group consisting of: stearoyl taurine, palmitoyl taurine, tauroursodeoxycholic acid, oleylglutamine, stearoyl leucine, oleyl phenylalanine, oleyl serine, oleyl alanine, palmitoyl glycine, palmitoyl aspartic acid, and linolenic acid tyrosine. The molar ratio of each component in the lipid nanoparticles is (10-35): (5-30): (5-20): (30-45): (0.5-3.0). The nitrogen-to-phosphorus ratio of the lipid nanoparticles is 6-20:

1.

2. The lipid nanoparticles as described in claim 1, characterized in that, The lipid nanoparticles have a particle size range of 80-125 nm and a positive zeta potential under acidic conditions.

3. The lipid nanoparticles as described in claim 1, characterized in that, The molar ratio of each component in the lipid nanoparticles is 31.5: 10: 19: 38.5: 1 for ionizable lipids: amino acid-derived lipids: cofactor phospholipids: sterols: PEG lipids.

4. The use of the lipid nanoparticles according to any one of claims 1-3 in the preparation of drug delivery carriers for immune cells or engineered immune cells, characterized in that, The lipid nanoparticles are used to deliver nucleic acids and / or drugs to immune cells or engineered immune cells.

5. The application as described in claim 4, characterized in that, The engineered immune cells include chimeric antigen receptor natural killer cells, chimeric antigen receptor T cells, chimeric antigen receptor macrophages, or chimeric antigen receptor natural killer T cells.

6. The application as described in claim 5, characterized in that, The nucleic acids include mRNA encoding cytokines, mRNA encoding gene-editing proteins, or mRNA encoding chimeric antigen receptors.

7. The method for preparing lipid nanoparticles according to any one of claims 1-3, characterized in that, Includes the following steps: S01: An amino acid-derived lipid, an ionizable lipid, a cofactor phospholipid, a sterol, and a PEG lipid are dissolved in an organic solvent to form a lipid phase; the ionizable lipid is ALC-0315 or DLin-MC3-DMA, the cofactor phospholipid is DSPC or DOPE, the sterol is cholesterol or β-sitosterol, and the PEG lipid is DMG-PEG 2000; the amino acid-derived lipid comprises one or more materials selected from the group consisting of: stearoyl taurine, palmitoyl taurine, tauroursodeoxycholic acid, oleylglutamine, stearoylleucine, oleylphenylalanine, oleylserine, oleylalanine, palmitoylglycine, palmitoyl aspartic acid, and linolenic acid tyrosine. S02: Dissolve the nucleic acid or drug to be delivered in an acidic buffer solution as the aqueous phase; S03: Using a microfluidic device, the lipid phase and the aqueous phase are mixed at a mixing flow rate ratio of 1:3 to prepare lipid nanoparticles for delivering nucleic acids and drugs to immune cells.

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