Bionic antigen presenting cell targeting lipid nanoparticle for delivering nucleic acid and application of bionic antigen presenting cell targeting lipid nanoparticle

Abstract

The invention belongs to the technical field of medicines, and relates to bionic nanoparticles for delivering nucleic acid. The lipid nanoparticles comprise nucleic acid, at least one cationic lipid and at least one APCs targeting lipid, and the lipid and a buffer solution containing the nucleic acid are mixed in microfluidics to form the targeting lipid nanoparticles. By simulating an in-vivo physiological process, the uptake and transfection of nucleic acid in antigen presenting cells are improved, and the expression efficiency of antigen protein is improved, so that the immune effect is improved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefits of the following Chinese patent application filed with the State Intellectual Property Office of China: Chinese Patent Application No. 202311103792.0 filed on August 29, 2023, entitled: A biomimetic antigen presenting cell-targeted lipid nanoparticle for delivering nucleic acid and its application. The entire text of the above patent application is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of medical technology, and specifically relates to a bionic antigen-presenting cell-targeted lipid nanoparticle for delivering RNA and an application thereof in delivering antigen RNA. Background Art

[0004] mRNA has become a promising therapeutic tool in the field of vaccine development and protein replacement therapy. Compared with subunit, inactivated and attenuated live viruses and DNA-based vaccines, mRNA vaccines are non-infectious, non-integrating platforms without the potential risk of infection or insertion mutations, and can be naturally degraded in cells, so they have better safety. In addition to the mRNA encoding antigens that can stimulate immune responses, mRNA itself also has inherent immunostimulatory properties. Reasonable regulation of its immunogenicity to make it an adjuvant also makes mRNA have a more effective immune effect. Once the required mRNA sequence information is determined, rapid large-scale production can be achieved through in vitro transcription, which gives mRNA vaccines a shorter development cycle.

[0005] In recent years, although vaccine platforms based on mRNA technology have received widespread attention and research, the lack of a safe and effective delivery system is still one of the main reasons limiting its application in fields such as tumor immunity.

[0006] In view of the instability and electronegativity of mRNA, researchers have developed a series of delivery systems for mRNA delivery, including lipid-based delivery systems and polymer-based delivery systems. Among them, lipid-based lipid nanoparticles (LNPs) have become the most promising mRNA delivery system due to their good biosafety and delivery efficiency. They have been approved by the FDA for liver siRNA delivery and COVID-19 mRNA vaccine delivery. For tumor immunotherapy, more than 90% of LNPs will be delivered to the liver when administered systemically. Although these LNPs provide feasibility for mRNA treatment of liver diseases, their non-liver delivery is still challenging due to the liver tropism of LNPs, which greatly reduces the effectiveness of mRNA vaccines.

[0007] Charge-dependent organ-specific accumulation has been shown to improve non-liver targeting of mRNA, but it is somewhat inferior to the effect of LNPs modified with biomimetic functional groups, because the targeting effect of the latter better simulates monocyte- or macrophage-mediated antigen presentation under physiological conditions. Summary of the invention

[0008] The present invention simulates the specific recognition of antigens by antigen presenting cells (APCs) through a bionic strategy to better simulate the antigen presentation function in vivo, and is a new mRNA delivery strategy.

[0009] One of the purposes of the present disclosure is to provide a new nano-delivery system to improve the immune effect of nucleic acid vaccines by simulating the phagocytosis and presentation process of antigens by antigen presenting cells (APCs) under physiological conditions.

[0010] In order to achieve the above objectives, the present disclosure adopts the following technical solutions:

[0011] The present disclosure provides a targeted lipid nanoparticle, comprising:

[0012] (i) nucleic acids,

[0013] (ii) at least one cationic lipid, and

[0014] (iii) at least one APCs targeting lipid.

[0015] Specifically, the amino groups in cationic lipids are protonated and adsorb negatively charged antigen nucleic acids at low pH, and self-assemble to form stable nanoparticles through hydrophilic-hydrophobic supramolecular forces. By modifying LNP with one or more biomimetic APCs targeting lipids, the physiological process of endogenous antigen presentation can be simulated, the uptake and transfection of nucleic acids in antigen-presenting cells can be improved, the expression efficiency of antigen proteins can be improved, and the targeted delivery effect in target organs such as the spleen can be improved, thereby improving the immune effect.

[0016] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0017] (i) nucleic acids,

[0018] (ii) at least one cationic lipid, and

[0019] (iii) at least one APCs targeting lipid;

[0020] Targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by activating integrin receptors.

[0021] In some embodiments, the APCs targeting lipid is selected from lipids that activate integrin receptors.

[0022] In some embodiments, the integrin receptor activating lipid is selected from RGD-modified lipids.

[0023] In some embodiments, the RGD-modified lipid is selected from RGD-modified DSPE.

[0024] In some embodiments, the RGD-modified lipid is selected from RGD-DSPE, DSPE-PEG-RGD, or DSPE-PEG-iRGD.

[0025] In some embodiments, the APCs targeting lipid is selected from a combination of RGD-modified lipids and PS.

[0026] In some embodiments, the RGD-modified lipid is selected from a combination of any one of RGD-DSPE, DSPE-PEG-RGD or DSPE-PEG-iRGD and PS.

[0027] Specifically, the amino groups in cationic lipids are protonated and adsorb negatively charged antigen nucleic acids at low pH, and self-assemble into stable nanoparticles through hydrophilic-hydrophobic supramolecular forces. RGD activates integrin receptors and binds to PS through bridging molecules (such as MFG-E8), thereby improving the uptake and transfection of nucleic acids in antigen-presenting cells, improving the expression efficiency of antigen proteins, and enhancing the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0028] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0029] (i) nucleic acids,

[0030] (ii) at least one cationic lipid, and

[0031] (iii) at least one APCs targeting lipid;

[0032] Targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by binding to S1P receptors.

[0033] In some embodiments, the APCs targeting lipid is selected from lipids that bind to S1P receptors.

[0034] In some embodiments, the S1P receptor-binding lipid is selected from any one of sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod, or Ceramide.

[0035] In some embodiments, the APCs targeting lipid is selected from a combination of any one of sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod, and Ceramide and PS.

[0036] Specifically, the amino groups in cationic lipids are protonated at low pH to adsorb negatively charged antigen nucleic acids, and self-assemble to form stable nanoparticles through hydrophilic-hydrophobic supramolecular forces. Lipid nanoparticles containing APCs bind to S1P receptors, induce the tropism of macrophages, increase the uptake and transfection of nucleic acids in antigen-presenting cells, increase the expression efficiency of antigen proteins, and enhance the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0037] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0038] (i) nucleic acids,

[0039] (ii) at least one cationic lipid, and

[0040] (iii) at least one APCs targeting lipid;

[0041] Targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by binding to the G2A receptor.

[0042] In some embodiments, the APCs targeting lipid is selected from lipids that bind to the G2A receptor.

[0043] In some embodiments, the G2A receptor binding lipid is selected from any one of lysophosphatidylcholine (Lyso PC), 9-HODE or 13-HODE.

[0044] In some embodiments, the APCs targeting lipid is selected from a combination of lysophosphatidylcholine (Lyso PC), 9-HODE or 13-HODE and PS.

[0045] Specifically, the amino groups in cationic lipids are protonated at low pH to adsorb negatively charged antigen nucleic acids, and self-assemble to form stable nanoparticles through hydrophilic-hydrophobic supramolecular forces. Lipid nanoparticles containing APCs bind to G2A receptors, induce the tropism of macrophages, improve the uptake and transfection of nucleic acids in antigen-presenting cells, increase the expression efficiency of antigen proteins, and enhance the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0046] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0047] (i) nucleic acids,

[0048] (ii) at least one cationic lipid, and

[0049] (iii) at least one APCs targeting lipid;

[0050] Targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by binding to β2-GPI or vimentin with an apparent ionization constant (pKa) between 6 and 7.

[0051] In some embodiments, the APCs targeting lipid is selected from lipids with two or more negative charges; preferably, the lipid with two or more negative charges is selected from cardiolipin.

[0052] In some embodiments, the APCs targeting lipid is selected from cardiolipin.

[0053] In some embodiments, the cardiolipin (CL) includes 18:0CL, 18:1CL, 18:2CL.

[0054] In some embodiments, the targeted lipid nanoparticle delivers the nucleic acid to a target organ selected from the group consisting of spleen, lymph node, heart, brain, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, thymus, or spinal cord.

[0055] In some embodiments, the target organ is the spleen.

[0056] In some embodiments, the target organ is a lymph node.

[0057] In some embodiments, the APCs targeting lipid further comprises phosphatidylserine (PS).

[0058] In some embodiments, the APCs targeting lipid is selected from a combination of any one of a lipid that activates an integrin receptor, a lipid that binds to an S1P receptor, and a lipid that binds to a G2A receptor and phosphatidylserine (PS).

[0059] In some embodiments, the molar ratio of any one of the lipids that activate integrin receptors, the lipids that bind to S1P receptors, and the lipids that bind to G2A receptors to PS is 0.45-10:10-20.

[0060] In some embodiments, the APCs targeting lipid is selected from a combination of any one of RGD-modified DSPE, lysophosphatidylcholine (Lyso PC), 9-HODE, 13-HODE, sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod or Ceramide and phosphatidylserine (PS).

[0061] In some embodiments, the APCs targeting lipid is selected from a combination of RGD-DSPE, DSPE-PEG-RGD, DSPE-PEG-iRGD, lysophosphatidylcholine (Lyso PC), 9-HODE, 13-HODE, sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod or Ceramide and phosphatidylserine.

[0062] In some embodiments, the APCs targeting lipid is selected from a combination of any one of RGD-DSPE, Lyso PC, S1P and phosphatidylserine.

[0063] In some embodiments, the APCs targeting lipid is selected from a combination of any one of RGD-DSPE and Lyso PC and phosphatidylserine.

[0064] In some embodiments, the APCs targeting lipid is selected from a combination of any one of RGD-DSPE, Lyso PC, S1P and PS, wherein the molar ratio of any one of RGD-DSPE, Lyso PC, S1P to PS is 0.45-10:10-20.

[0065] In some embodiments, the APCs targeting lipid is selected from a combination of any one of RGD-DSPE, Lyso PC, S1P and PS, wherein the molar ratio of any one of RGD-DSPE, Lyso PC, S1P to PS is 0.45-9:18-20.

[0066] In some embodiments, the APCs targeting lipid is selected from a combination of PS and RGD-DSPE or a combination of PS and Lyso PC.

[0067] In some embodiments, the APCs targeting lipid is selected from a combination of PS and any one of RGD-DSPE and Lyso PC, wherein the molar ratio of any one of RGD-DSPE and Lyso PC to PS is 0.45-10:10-20.

[0068] In some embodiments, the APCs targeting lipid accounts for about 1 mol % to about 50 mol % of the total lipids in the particle.

[0069] In some embodiments, the APCs targeting lipid accounts for about 2 mol % to about 40 mol % of the total lipids in the particle.

[0070] In some embodiments, the APCs targeting lipid accounts for about 5 mol% to about 40 mol% of the total lipids in the particle.

[0071] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 30 mol % of the total lipids in the particle.

[0072] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 25 mol % of the total lipids in the particle.

[0073] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 20 mol % of the total lipids in the particle.

[0074] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 10 mol % of the total lipids in the particle.

[0075] In some embodiments, the APCs targeting lipid accounts for about 5 mol% to about 9 mol% of the total lipids in the particle.

[0076] In some embodiments, the APCs targeting lipid accounts for about 5 mol% to about 8 mol% of the total lipids in the particle.

[0077] In some embodiments, the APCs targeting lipid accounts for about 5 mol% to about 7 mol% of the total lipids in the particle.

[0078] In some embodiments, the APCs targeting lipid accounts for about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, about 70 mol%, about 71 %, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol% to about 40 mol%, or any range selectable therein, including but not limited to about 6 mol% to about 7 mol%, about 10 mol% to about 40 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 20 mol% to about 40 mol%, about 20 mol% to about 30 mol%.

[0079] In some embodiments, the cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ( DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, or any one thereof or a combination thereof.

[0080] In some embodiments, the cationic lipid is selected from any one of DOTMA, DLin-MC3-DMA, SM-102, ALC-0315 or a combination thereof.

[0081] In some embodiments, the cationic lipid is selected from any one of DLin-MC3-DMA, SM-102, and ALC-0315.

[0082] In some embodiments, the cationic lipid is selected from DLin-MC3-DMA.

[0083] In some embodiments, the cationic lipid is selected from SM-102.

[0084] In some embodiments, the cationic lipid comprises about 20 mol % to about 80 mol % of the total lipids in the particle.

[0085] In some embodiments, the cationic lipid comprises about 20 mol % to about 70 mol % of the total lipids in the particle.

[0086] In some embodiments, the cationic lipid comprises about 25 mol % to about 60 mol % of the total lipids in the particle.

[0087] In some embodiments, the cationic lipid comprises about 25 mol % to about 55 mol % of the total lipids in the particle.

[0088] In some embodiments, the cationic lipid comprises about 25 mol % to about 50 mol % of the total lipids in the particle.

[0089] In some embodiments, the cationic lipid comprises about 28 mol % to about 50 mol % of the total lipids in the particle.

[0090] In some embodiments, the cationic lipid accounts for about 28 mol%, 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, 43 mol%, about 44 mol%, 45 mol%, about 46 mol%, 47 mol%, about 48 mol%, 49 mol%, to about 50 mol%, or any range selectable therein, including but not limited to about 40 mol% to about 50 mol%, about 40 mol% to about 48 mol%, about 41 mol% to about 47 mol% of the total lipid in the particle.

[0091] In some embodiments, the molar ratio of the cationic lipid to the APCs targeting lipid is 4-10:1-10.

[0092] In some embodiments, the molar ratio of the cationic lipid to the APCs targeting lipid is 5-10:1-10.

[0093] In some embodiments, the molar ratio of the cationic lipid to the APCs targeting lipid is 7-10:1-10.

[0094] In some embodiments, the molar ratio of the cationic lipid to cardiolipin is 4-10:1-10.

[0095] On the other hand, the targeted lipid nanoparticles described in the present disclosure further include phospholipids.

[0096] In some embodiments, the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (D MPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), d Any one of itricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE).

[0097] In some embodiments, the phospholipid is selected from any one of DSPC, DOPC, DMPC, DOPE or DPPE.

[0098] In some embodiments, the phospholipid is selected from DSPC.

[0099] In some embodiments, the phospholipids comprise from about 2 mol % to about 50 mol % of the total lipids in the particle.

[0100] In some embodiments, the phospholipids comprise from about 2 mol % to about 40 mol % of the total lipids in the particle.

[0101] In some embodiments, the phospholipids comprise about 5 mol % to about 30 mol % of the total lipids in the particle.

[0102] In some embodiments, the phospholipids comprise about 5 mol % to about 20 mol % of the total lipids in the particle.

[0103] In some embodiments, the phospholipids comprise about 5 mol % to about 10 mol % of the total lipids in the particle.

[0104] In some embodiments, the phospholipids account for about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol% to about 10 mol% of the total lipids in the particle, or any range selectable therein, including but not limited to about 7 mol% to about 10 mol%, about 7 mol% to about 8 mol%, about 9 mol% to about 10 mol%.

[0105] In some embodiments, the molar ratio of the cationic lipid, APCs targeting lipid, and phospholipid is 4-10:1-10:1-2.

[0106] In some embodiments, the molar ratio of the cationic lipid, APCs targeting lipid, and phospholipid is 5-10:1-10:1-2.

[0107] On the other hand, the targeted lipid nanoparticles described in the present disclosure further include a conjugated lipid.

[0108] In some embodiments, the conjugated lipid is selected from any one of ALC-0159, DMG-PEG2000, and PEG2000.

[0109] In some embodiments, the conjugated lipid is selected from ALC-0159 or DMG-PEG2000.

[0110] In some embodiments, the conjugated lipid comprises about 1 mol % to about 40 mol % of the total lipid in the particle.

[0111] In some embodiments, the conjugated lipid comprises about 1 mol % to about 30 mol % of the total lipid in the particle.

[0112] In some embodiments, the conjugated lipid comprises about 1 mol % to about 20 mol % of the total lipid in the particle.

[0113] In some embodiments, the conjugated lipid comprises about 1 mol % to about 10 mol % of the total lipid in the particle.

[0114] In some embodiments, the conjugated lipid comprises about 1 mol % to about 5 mol % of the total lipid in the particle.

[0115] In some embodiments, the conjugated lipid comprises about 1 mol % to about 2 mol % of the total lipid in the particle.

[0116] In some embodiments, the conjugated lipid accounts for about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 3 mol%, about 4 mol% to about 5 mol% of the total lipid in the particle, or any range selectable therein, including but not limited to about 1 mol% to about 1.5 mol%, about 1.2 mol% to about 1.5 mol%, about 1.3 mol% to about 1.4 mol%.

[0117] On the other hand, the targeted lipid nanoparticles of the invention further include cholesterol or a cholesterol derivative.

[0118] In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol % to about 50 mol % of the total lipids in the particle.

[0119] In some embodiments, the cholesterol or cholesterol derivative comprises about 20 mol % to about 50 mol % of the total lipids in the particle.

[0120] In some embodiments, the cholesterol or cholesterol derivative comprises about 20 mol % to about 40 mol % of the total lipids in the particle.

[0121] In some embodiments, the cholesterol or cholesterol derivative comprises about 25 mol % to about 40 mol % of the total lipids in the particle.

[0122] In some embodiments, the cholesterol or cholesterol derivative accounts for about 25 mol%, about 26 mol%, about 27 mol%, 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, 32 mol%, about 33 mol%, 34 mol%, about 35 mol%, 36 mol%, about 37 mol%, 38 mol%, about 39 mol% to about 40 mol% of the total lipid in the particle, or any range selectable therein, including but not limited to about 28 mol% to about 30 mol%, about 28 mol% to about 36 mol%, about 30 mol% to about 36 mol%, about 35 mol% to about 36 mol%.

[0123] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0124] (i) nucleic acids, (ii) cationic lipids, (iii) APCs targeting lipids, and (iv) phospholipids.

[0125] In another aspect, the present disclosure provides a targeted lipid nanoparticle comprising:

[0126] (i) nucleic acids;

[0127] (ii) a cationic lipid, said cationic lipid comprising from about 20 mol % to about 80 mol % of the total lipids in the particle;

[0128] (iii) an APCs targeting lipid, wherein the APCs targeting lipid accounts for about 1 mol % to about 50 mol % of the total lipids in the particle; and

[0129] (iv) phospholipids, said phospholipids comprising from about 2 mol % to about 50 mol % of the total lipids in the particle.

[0130] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0131] (i) nucleic acids, (ii) cationic lipids, (iii) APCs targeting lipids, (iv) phospholipids, (v) conjugated lipids, and (vi) cholesterol or cholesterol derivatives.

[0132] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0133] (i) nucleic acids,

[0134] (ii) a cationic lipid, said cationic lipid comprising from about 20 mol % to about 80 mol % of the total lipids in the particle;

[0135] (iii) APCs targeting lipids, wherein the APCs targeting lipids account for about 1 mol % to about 50 mol % of the total lipids in the particle;

[0136] (iv) a phospholipid, said phospholipid comprising from about 2 mol % to about 50 mol % of the total lipids in the particle;

[0137] (iv) a conjugated lipid, wherein the conjugated lipid accounts for about 1 mol % to about 40 mol % of the total lipid in the particle;

[0138] and (vi) cholesterol or a cholesterol derivative, said cholesterol or cholesterol derivative comprising from about 25 mol % to about 40 mol % of the total lipids in the particle.

[0139] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0140] (i) nucleic acids,

[0141] (ii) a cationic lipid, wherein the cationic lipid is selected from any one of DLin-MC3-DMA, SM-102, and ALC-0315;

[0142] (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from cardiolipin, a combination of PS and RGD-DSPE, and a combination of PS and Lyso PC;

[0143] (iv) a phospholipid selected from DSPC;

[0144] (iv) a conjugated lipid selected from ALC-0159 or DMG-PEG2000;

[0145] and (vi) cholesterol.

[0146] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0147] (i) nucleic acids,

[0148] (ii) a cationic lipid selected from DLin-MC3-DMA;

[0149] (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from cardiolipin;

[0150] (iv) a phospholipid selected from DSPC;

[0151] (iv) a conjugated lipid selected from ALC-0159;

[0152] and (vi) cholesterol.

[0153] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0154] (i) nucleic acids,

[0155] (ii) a cationic lipid selected from SM-102;

[0156] (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from a combination of PS and RGD-DSPE, and a combination of PS and Lyso PC;

[0157] (iv) a phospholipid selected from DSPC;

[0158] (iv) a conjugated lipid, wherein the conjugated lipid is selected from DMG-PEG2000;

[0159] and (vi) cholesterol.

[0160] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0161] (i) nucleic acids,

[0162] (ii) a cationic lipid, wherein the cationic lipid is selected from DLin-MC3-DMA, and the cationic lipid accounts for about 20 mol% to about 60 mol% of the total lipid in the particle;

[0163] (iii) an APCs targeting lipid, wherein the APCs targeting lipid is selected from cardiolipin, and the APCs targeting lipid accounts for about 5 mol% to about 40 mol% of the total lipids in the particle;

[0164] (iv) a phospholipid selected from DSPC, wherein the phospholipid accounts for about 5 mol% to about 20 mol% of the total lipids in the particle;

[0165] (iv) a conjugated lipid selected from ALC-0159, wherein the conjugated lipid accounts for about 1 mol% to about 10 mol% of the total lipid in the particle;

[0166] and (vi) cholesterol, said cholesterol comprising from about 20 mol % to about 40 mol % of the total lipids in the particle.

[0167] On the other hand, the present disclosure also provides a targeted lipid nanoparticle, comprising:

[0168] (i) nucleic acids,

[0169] (ii) a cationic lipid selected from SM-102, wherein the cationic lipid accounts for about 20 mol% to about 60 mol% of the total lipids in the particle;

[0170] (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from a combination of PS and RGD-DSPE, and a combination of PS and Lyso PC, and the APCs targeting lipid accounts for about 5 mol% to about 20 mol% of the total lipids in the particle;

[0171] (iv) a phospholipid selected from DSPC, wherein the phospholipid accounts for about 5 mol% to about 20 mol% of the total lipids in the particle;

[0172] (iv) a conjugated lipid, wherein the conjugated lipid is selected from DMG-PEG2000, wherein the conjugated lipid is selected from ALC-0159, and wherein the conjugated lipid accounts for about 1 mol% to about 10 mol% of the total lipid in the particle;

[0173] and (vi) cholesterol, said cholesterol comprising from about 20 mol % to about 40 mol % of the total lipids in the particle.

[0174] On the other hand, the present disclosure also provides a pharmaceutical composition, comprising the targeted lipid nanoparticles described above, and pharmaceutically acceptable excipients.

[0175] On the other hand, the present disclosure provides a pharmaceutical composition, comprising targeted lipid nanoparticles, and pharmaceutically acceptable excipients, wherein the targeted lipid nanoparticles include:

[0176] (i) a nucleic acid, (ii) at least one cationic lipid, and (iii) at least one APCs targeting lipid.

[0177] In another aspect, the present disclosure provides a pharmaceutical composition comprising targeted lipid nanoparticles and pharmaceutically acceptable excipients, wherein the targeted lipid nanoparticles comprise:

[0178] (i) nucleic acids, (ii) cationic lipids, (iii) APCs targeting lipids, and (iv) phospholipids.

[0179] On the other hand, the present disclosure provides a pharmaceutical composition, comprising targeted lipid nanoparticles, and pharmaceutically acceptable excipients, wherein the targeted lipid nanoparticles include:

[0180] (i) nucleic acids, (ii) cationic lipids, (iii) APCs targeting lipids, (iv) phospholipids, (v) conjugated lipids, and (vi) cholesterol or cholesterol derivatives.

[0181] In some embodiments, the nucleic acid is selected from RNA.

[0182] In some embodiments, the RNA includes one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, and tsRNA.

[0183] In some embodiments, the RNA is selected from mRNA. Preferably, the mRNA is a tumor antigen mRNA.

[0184] In some embodiments, the pharmaceutical composition is formulated for intravenous injection.

[0185] In another aspect, the present disclosure provides a method for non-liver targeted delivery of nucleic acids, the method comprising administering the above-mentioned targeted lipid nanoparticles comprising nucleic acids to a subject in need thereof, wherein the targeted lipid nanoparticles comprise (ii) cationic lipids and (iii) APCs targeting lipids.

[0186] In another aspect, the present disclosure provides a method of regulating gene expression comprising delivering a nucleic acid to a cell, the method comprising: contacting the cell with a targeted lipid nanoparticle described herein or a pharmaceutical composition thereof under conditions sufficient to cause the nucleic acid to be taken up into the cell.

[0187] In some embodiments, the cells are contacted in vitro or ex vivo.

[0188] In some embodiments, the cells are contacted in vivo.

[0189] In some embodiments, modulation of gene expression is sufficient to treat a disease or disorder.

[0190] In some embodiments, the disease or condition is cancer.

[0191] In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the above-mentioned targeted lipid nanoparticles or a pharmaceutical composition thereof to a subject in need of such treatment.

[0192] In some embodiments, the subject is a mammal, such as a human.

[0193] In another aspect, the present disclosure provides use of the above-mentioned targeted lipid nanoparticles or pharmaceutical compositions thereof in the preparation of drugs for treating cancer.

[0194] In another aspect, the present disclosure provides use of the above-mentioned targeted lipid nanoparticles or pharmaceutical compositions thereof in treating cancer.

[0195] In another aspect, the present disclosure provides the above-mentioned targeted lipid nanoparticles or pharmaceutical compositions thereof for treating cancer.

[0196] In another aspect, the present disclosure provides a method for preparing targeted lipid nanoparticles.

[0197] (1) dissolving a cationic lipid, an APCs targeting lipid, a phospholipid, a conjugated lipid, cholesterol or a cholesterol derivative in a first solvent to form a first solution;

[0198] (2) dissolving the nucleic acid in a buffer to form a second solution;

[0199] (3) Mixing the first solution and the second solution to form targeted lipid nanoparticles.

[0200] In some embodiments, the cationic lipid, APCs targeting lipid, phospholipid, conjugated lipid, cholesterol or cholesterol derivative is dissolved in an organic solvent or water according to its lipophilicity to finally form a first solution.

[0201] In some embodiments, cationic lipids, phospholipids, conjugated lipids, cholesterol or cholesterol derivatives are dissolved in organic solvents, such as ethanol; cardiolipin, PS are dissolved in citrate buffer, RGD-DSPE, Lyso PC, S1P are dissolved in organic solvents, such as ethanol.

[0202] In some embodiments, mixing the first solution and the second solution may be performed by microfluidics.

[0203] Any embodiment of any aspect of the present invention can be combined with other embodiments without contradiction. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical feature in other embodiments without contradiction.

[0204] Definitions and explanations of terms

[0205] Unless otherwise defined in the present disclosure, scientific and technical terms related to the present disclosure shall have the meanings understood by those of ordinary skill in the art. When a trade name appears in this document, it is intended to refer to its corresponding trade name or its active ingredient.

[0206] The term "mRNA" as used herein refers to messenger ribonucleic acid. mRNA can be naturally or non-naturally occurring or synthetic. For example, mRNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or joints. mRNA can include a cap structure, a 5' transcription leader, a 5' untranslated region, a start codon, an open reading frame, a stop codon, a chain terminating nucleoside, a stem loop, a hairpin, polyadenylic acid (polyA), a polyadenylation signal and / or one or more cis-regulatory elements. mRNA can have a nucleotide sequence encoding a polypeptide. Translation of mRNA, such as in vivo translation of mRNA in mammalian cells, can produce a polypeptide. Traditionally, the basic components of a natural mRNA molecule include at least one coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap and a polyA sequence.

[0207] The term "untranslated region" or "UTR" as used herein refers to the portion of the mRNA upstream of the start codon and downstream of the stop codon that is not translated and is therefore referred to as the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed with the coding region and are therefore exonic when present in the mature mRNA.

[0208] As used herein, the term "5' untranslated region, 5'UTR" generally refers to a part of mRNA that is located 5' (i.e., "upstream") of the open reading frame and is not translated into protein. 5'UTR is generally understood to be a specific segment of messenger RNA (mRNA) that is located at the 5' end of the open reading frame of the mRNA. Typically, 5'UTR starts at the transcription start site and terminates at one nucleotide before the start codon of the open reading frame. Preferably, the 5'UTR has a length of more than 20, 30, 40 or 50 nucleotides. 5'UTR may include elements for controlling gene expression, also referred to as regulatory elements. The regulatory element may be, for example, a ribosome binding site. 5'UTR may be post-transcriptionally modified, for example, by adding a 5'-cap modification. The 5'UTR of mRNA is not translated into an amino acid sequence. 5'UTR sequences are generally encoded by genes that are transcribed into individual mRNAs during gene expression. The genomic sequence is first transcribed into pre-mature mRNA, which includes optional introns. Pre-mature mRNA is then further processed into mature mRNA during maturation. The maturation process includes the following steps: 5' capping, splicing of pre-mature mRNA to remove optional introns and 3' terminal modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 5'UTR corresponds to the mature mRNA sequence located between the start codon and, for example, the 5'-cap. Preferably, the 5'UTR corresponds to the nucleotide located at the 3' side of the 5' cap, more preferably from the 3' side nucleotide immediately adjacent to the 5' cap, to the nucleotide located at the 5' side of the start codon of the protein coding region, preferably to the sequence extending to the nucleotide immediately adjacent to the 5' side of the start codon of the protein coding region. The nucleotide immediately adjacent to the 3' side of the mature mRNA 5' cap typically corresponds to the transcription start site. The term "corresponding to" means that the 5'UTR sequence can be an RNA sequence in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence.

[0209] As used herein, the term "3' untranslated region, 3'UTR" generally refers to a part of mRNA that is located 3' (i.e., "downstream") of the open reading frame and is not translated into protein. Typically, 3'UTR is a part of the mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylic acid sequence of the mRNA. In the context of the present disclosure, the term 3'UTR may also include elements that are not encoded in the template, from which the RNA is transcribed, but added after transcription during the maturation process, such as polyadenylic acid sequences. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is typically encoded by a gene that is transcribed into a respective mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA comprising an optional intron. The pre-mature mRNA is then further processed into a mature mRNA during the maturation process. The maturation process comprises the following steps: 5' capping, splicing of pre-mature mRNA to remove optional introns and 3' terminal modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 3'-UTR corresponds to the polyadenylic acid sequence located between the 3' end of the protein coding region stop codon, preferably immediately following the 3' end of the protein coding region stop codon and the mRNA. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence. Preferably, the 3'UTR has a length of more than 20, 30, 40 or 50 nucleotides.

[0210] Mol % herein means molar percentage.

[0211] As used herein, the terms "comprise" or "comprises" and variations thereof such as comprises or comprising may be construed in an open, non-exclusive sense, ie, "including but not limited to."

[0212] The term "about" is used in this disclosure to mean approximately, around, roughly, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical range stated. Unless otherwise specified, the term "about" is used herein to modify the upper and lower limits of the numerical value stated by a 10% deviation.

[0213] As used herein, the term "therapeutically effective amount" means

[0214] An amount of a compound of the present disclosure that (i) treats a particular disease, condition or disorder, (ii) alleviates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition or disorder as described herein.

[0215] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0216] As used herein, the term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0217] The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure with suitable pharmaceutically acceptable excipients, for example, they can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

[0218] Typical routes of administration of the disclosed compounds or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

[0219] a. APCs targeting lipids

[0220] In some embodiments, the APCs targeting lipid is selected from lipids that bind to β2-GPI or vimentin.

[0221] In some embodiments, the lipid nanoparticles containing APCs targeting lipids have an apparent ionization constant (pKa) between 6 and 7.

[0222] In some embodiments, the APCs targeting lipid is selected from cardiolipin.

[0223] In some embodiments, the APCs targeting lipid is selected from lipids bound to β2-GPI or vimentin, and the apparent ionization constant (pKa) of the lipid nanoparticles containing the APCs targeting lipid is between 6 and 7, and the lipid bound to β2-GPI or vimentin is selected from cardiolipin.

[0224] Among them, cardiolipin (CL) includes but is not limited to 18:0CL, 18:1CL, and 18:2CL. Among them, the 18:0 cardiolipin (18:0CL), CAS number: 64069-64-3, has the following structural formula:

[0225]

[0226] The 18:1 cardiolipin (18:0CL), CAS No.: 115404-77-8, has the following structural formula:

[0227]

[0228] The 18:2 cardiolipin (18:2CL), CAS No.: 383907-10-6, has the following structural formula:

[0229]

[0230] In some aspects, the APCs targeting lipid is selected from lipids that bind to the G2A receptor. The lipid binds to the G2A receptor, thereby inducing the tropism of macrophages, improving the uptake and transfection of nucleic acids in antigen-presenting cells, improving the expression efficiency of antigen proteins, and enhancing the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0231] In some embodiments, the lipids that bind to the G2A receptor include lysophosphatidylcholine (Lyso PC), 9-HODE, 13-HODE.

[0232] Lysophosphatidylcholine (Lyso PC) includes, but is not limited to, 16:0Lyso PC, 18:0Lyso PC, 20:0Lyso PC, and 24:0Lyso PC.

[0233] 9-HODE, CAS: 98524-19-7, structural formula is as follows:

[0234]

[0235] 13-HODE, CAS: 5204-88-6, has the following structural formula:

[0236]

[0237] In some aspects, the APCs targeting lipid is selected from lipids that activate integrin receptors. By adding the above lipids, integrin receptors can be activated, and the bridging molecules can bind to PS, thereby improving the uptake and transfection of nucleic acids in antigen presenting cells, improving the expression efficiency of antigen proteins, and enhancing the phagocytosis of apoptotic cells by antigen presenting cells, thereby improving the immune effect.

[0238] In some embodiments, the integrin receptor activating lipid comprises an RGD-modified lipid.

[0239] In some embodiments, the RGD-modified lipid is selected from RGD-modified DSPE.

[0240] In some embodiments, the RGD-modified lipid is selected from RGD-DSPE, DSPE-PEG-RGD, or DSPE-PEG-iRGD.

[0241] Wherein, RGD-DSPE refers to polypeptide modified distearoyl phosphatidylethanolamine. Wherein, RGD refers to Arg-Gly-Asp. The RGD-DSPE, CAS number: 2260795-79-5, has the following structural formula:

[0242]

[0243] DSPE-PEG-RGD refers to phospholipid-polyethylene glycol-RGD, wherein RGD refers to Arg-Gly-Asp. DSPE-PEG-RGD includes but is not limited to DSPE-PEG2000-RGD, DSPE-PEG3400-RGD, and DSPE-PEG5000-RGD.

[0244] DSPE-PEG-iRGD refers to phospholipid-polyethylene glycol-iRGD, wherein the amino acid composition of iRGD is: c(CRGDKGPDC) or c(CRGDRGPDC). DSPE-PEG-iRGD includes but is not limited to DSPE-PEG2000-iRGD and DSPE-PEG5000-iRGD.

[0245] In some aspects, the APCs targeting lipid is selected from lipids that bind to S1P receptors. The lipids bind to S1P receptors, induce the tropism of macrophages, improve the uptake and transfection of nucleic acids in antigen-presenting cells, improve the expression efficiency of antigen proteins, and enhance the phagocytosis of apoptotic cells by antigen-presenting cells, thereby improving the immune effect.

[0246] In some embodiments, the S1P receptor binding lipid includes sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod, and Ceramide.

[0247] Sphingosine 1-phosphate (S1P) includes but is not limited to Sphingosine-1-phosphate (d17:0), Sphingosine-1-phosphate (d18:1), and Sphingosine-1-phosphate (d20:0).

[0248] Sphingosine (D-Sphingosine), CAS: 123-78-4, has the following structure:

[0249]

[0250] Fingolimod, CAS: 162359-56-0, has the following structure:

[0251]

[0252] Ozanimod, CAS: 1306760-87-1, has the following structure:

[0253]

[0254] Ceramide, CAS: 100403-19-8, has the following structure:

[0255]

[0256] In some embodiments, the APCs targeting lipid further comprises phosphatidylserine (PS).

[0257] In some embodiments, phosphatidylserine (PS) includes but is not limited to 10:0PS, 12:0PS, 17:0PS, 18:0PS.

[0258] The phosphatidylserine (PS) includes but is not limited to the following structural compounds: CAS number: 124262-93-7, the structural formula is as follows:

[0259]

[0260] In some embodiments, the APCs targeting lipid accounts for about 2 mol % to about 40 mol % of the total lipids in the particle.

[0261] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 40 mol % of the total lipids in the particle.

[0262] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 30 mol % of the total lipids in the particle.

[0263] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 20 mol % of the total lipids in the particle.

[0264] In some embodiments, the APCs targeting lipid accounts for about 5 mol % to about 10 mol % of the total lipids in the particle.

[0265] In some embodiments, the APCs targeting lipid accounts for about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol% of the total lipid in the particle. %, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol% to about 40 mol%, or any range selectable therein, including but not limited to about 6 mol% to about 7 mol%, about 10 mol% to about 40 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 20 mol% to about 40 mol%, about 20 mol% to about 30 mol%.

[0266] b. “Cationic lipids”

[0267] In some aspects of the disclosure, the cationic lipid is ionizable.

[0268] In some embodiments, the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated and are uncharged at pH above 8, 9, 10, 11, or 12. The ionizable cationic group can contain one or more protonatable amines that are capable of forming a cationic group at physiological pH.

[0269] In some embodiments, the cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315 or any one thereof or a mixture thereof.

[0270] The CAS number of the structural compound of DLin-MC3-DMA is 1224606-06-7, and the structural formula is as follows:

[0271]

[0272] The CAS number of the structural compound of SM-102 is 2089251-47-6, and the structural formula is as follows:

[0273]

[0274] The structural compound of ALC-0315 has a CAS number of 2036272-55-4 and a structural formula as follows:

[0275]

[0276] In some embodiments, the cationic lipid is selected from any one of DOTMA, DLin-MC3-DMA, SM-102, ALC-0315 or a mixture thereof.

[0277] In some embodiments, the cationic lipid is selected from any one of DLin-MC3-DMA, SM-102, and ALC-0315.

[0278] In some embodiments, the cationic lipid comprises about 20 mol % to about 80 mol % of the total lipids in the particle.

[0279] In some embodiments, the cationic lipid comprises about 20 mol % to about 70 mol % of the total lipids in the particle.

[0280] In some embodiments, the cationic lipid comprises about 25 mol % to about 60 mol % of the total lipids in the particle.

[0281] In some embodiments, the cationic lipid comprises about 25 mol % to about 55 mol % of the total lipids in the particle.

[0282] In some embodiments, the cationic lipid comprises about 25 mol % to about 50 mol % of the total lipids in the particle.

[0283] In some embodiments, the cationic lipid comprises about 28 mol % to about 50 mol % of the total lipids in the particle.

[0284] In some embodiments, the cationic lipid accounts for about 28 mol%, 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, 43 mol%, about 44 mol%, 45 mol%, about 46 mol%, 47 mol%, about 48 mol%, 49 mol%, to about 50 mol%, or any range selectable therein, including but not limited to about 40 mol% to about 50 mol%, about 40 mol% to about 48 mol%, about 41 mol% to about 47 mol% of the total lipid in the particle.

[0285] c. Phospholipids

[0286] In some embodiments, the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC) , ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE).

[0287] In some embodiments, the phospholipid is selected from DSPC, DOPC, DMPC, DOPE, or DPPE.

[0288] In some embodiments, the phospholipid is selected from DSPC.

[0289] In some embodiments, the phospholipids comprise from about 2 mol % to about 50 mol % of the total lipids in the particle.

[0290] In some embodiments, the phospholipids comprise from about 2 mol % to about 40 mol % of the total lipids in the particle.

[0291] In some embodiments, the phospholipids comprise from about 5 mol % to about 30 mol % of the total lipids in the particle.

[0292] In some embodiments, the phospholipids comprise from about 5 mol % to about 20 mol % of the total lipids in the particle.

[0293] In some embodiments, the phospholipids comprise about 5 mol % to about 10 mol % of the total lipids in the particle.

[0294] In some embodiments, the phospholipids account for about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol% to about 10 mol% of the total lipids in the particle, or any range selectable therein, including but not limited to about 7 mol% to about 10 mol%, about 7 mol% to about 8 mol%, about 9 mol% to about 10 mol%.

[0295] d. Conjugated lipids

[0296] In some embodiments, the conjugated lipid is selected from ALC-0159, DMG-PEG2000, PEG2000.

[0297] In some embodiments, the conjugated lipid is selected from ALC-0159 or DMG-PEG2000.

[0298] In some embodiments, the conjugated lipid comprises from about 1 mol % to about 40 mol % of the total lipid in the particle.

[0299] In some embodiments, the conjugated lipid comprises from about 1 mol % to about 30 mol % of the total lipid in the particle.

[0300] In some embodiments, the conjugated lipid comprises about 1 mol % to about 20 mol % of the total lipid in the particle.

[0301] In some embodiments, the conjugated lipid comprises from about 1 mol % to about 10 mol % of the total lipid in the particle.

[0302] In some embodiments, the conjugated lipid comprises from about 1 mol % to about 5 mol % of the total lipid in the particle.

[0303] In some embodiments, the conjugated lipid comprises about 1 mol % to about 2 mol % of the total lipid in the particle.

[0304] In some embodiments, the conjugated lipid accounts for about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 3 mol%, about 4 mol% to about 5 mol% of the total lipid in the particle, or any range selectable therein, including but not limited to about 1 mol% to about 1.5 mol%, about 1.2 mol% to about 1.5 mol%, about 1.3 mol% to about 1.4 mol%.

[0305] e. Cholesterol or cholesterol derivatives

[0306] Cholesterol, CAS: 57-88-5, has the following structure:

[0307]

[0308] Examples of cholesterol derivatives include, but are not limited to, cholestanol and cholestanone.

[0309] In some embodiments, cholesterol or a cholesterol derivative comprises about 20 mol % to about 50 mol % of the total lipids in the particle.

[0310] In some embodiments, cholesterol or a cholesterol derivative comprises about 20 mol % to about 40 mol % of the total lipids in the particle.

[0311] In some embodiments, cholesterol or a cholesterol derivative comprises about 25 mol % to about 40 mol % of the total lipids in the particle.

[0312] In some embodiments, cholesterol or a cholesterol derivative accounts for about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, 32 mol%, about 33 mol%, 34 mol%, about 35 mol%, 36 mol%, about 37 mol%, 38 mol%, about 39 mol% to about 40 mol% of the total lipid in the particle, or any range selectable therein, including but not limited to about 28 mol% to about 30 mol%, about 28 mol% to about 36 mol%, about 30 mol% to about 36 mol%, about 35 mol% to about 36 mol%. BRIEF DESCRIPTION OF THE DRAWINGS

[0313] Figure 1 This is a diagram showing the in vitro macrophage transfection effect of the LNP prepared in Example 1.

[0314] Figure 2 This is a diagram showing the in vivo spleen distribution effect of the APCs-targeted lipid-modified LNP and PS-modified LNP (control group) prepared in Example 1.

[0315] Figure 3 This is a diagram showing the in vitro macrophage transfection effect of the APCs-targeted lipid-modified LNP prepared in Example 2 and the control group.

[0316] Figure 4 This is a diagram showing the in vivo transfected organ distribution effects of the APCs-targeted lipid-modified LNP prepared in Example 2 and the control group.

[0317] Figure 5 This is a diagram showing the in vivo spleen distribution effects of the APCs-targeted lipid-modified LNP and PS-modified LNP (control group) prepared in Example 2. DETAILED DESCRIPTION

[0318] The present disclosure is described in detail below in conjunction with examples and drawings, but the following examples should not be construed as limiting the scope of the present disclosure.

[0319] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0320] GFP-luciferase mRNA (5UTR-LUC-P2A-GFP-3UTR-polyA) was homemade, and the sequence is as follows:

[0321]

[0322] Other reagents (all reagents used in this disclosure are commercially available):

[0323] DLin-MC3-DMA, CAS number: 1224606-06-7

[0324] SM102, CAS number: CAS:2089251-47-6

[0325] ALC-0315, CAS No.: 2036272-55-4

[0326] DSPC, CAS No.: 816-94-4

[0327] ALC-0159,CAS No.:1849616-42-7

[0328] DMG-PEG2000,CAS No.:160743-62-4

[0329] Cholesterol, CAS No.: 57-88-5

[0330] Lyso PC, CAS No.: 9008-30-4

[0331] PS, CAS No.: 124262-93-7

[0332] S1P CAS No.: 26993-30-6

[0333] RGD-DSPE, CAS No.: 2260795-79-5

[0334] 18:0 Cardiolipin (18:0CL), CAS No.: 64069-64-3

[0335] 18:1 cardiolipin (18:1CL), CAS No.: 115404-77-8

[0336] 18:2 Cardiolipin (18:2CL), CAS No.: 383907-10-6

[0337] Example 1

[0338] 1. Preparation of biomimetic APCs-targeted lipid nanoparticles

[0339] (1) According to Table 1 below, DLin-MC3-DMA, DSPC, ALC-0159 and cholesterol were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, according to Table 1 below, 18:0CL (18:0Cardiolipin) was weighed and dissolved in citric acid buffer until the solution was clear and transparent, and GFP-luciferase mRNA was added to obtain an aqueous phase solution with a volume of three times ethanol. Positive control prescriptions 1-4 are also listed in Table 1. The difference between their preparation process and the aforementioned prescription is that 18:0CL is replaced by PS. Control prescription A is also listed in Table 1. Its preparation process refers to the aforementioned prescription.

[0340] Table 1 Formulation of targeted lipid nanoparticles

[0341]

[0342]

[0343]

[0344] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the obtained mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0345] 2. Physical and chemical characterization of LNP

[0346] (1) The LNP particle size, polydispersity index (PDI), and zeta potential (Zeta) of the aqueous solution were measured by Malvern particle size analyzer;

[0347] (2) Ribogreen fluorescence method (Quant-iT TM RNA Reagent and Kit, Invitorgen) measures the free RNA concentration and total RNA concentration in the solution of LNP, and then calculates the LNP free rate (%). According to the commercially available kit experimental scheme, the sample to be tested is diluted to different concentrations with TE buffer, and the RNA concentration before and after demulsification using Triton X-100 is measured respectively. Specifically, RiboGreen dye is added to the sample to be tested before and after the gradient concentration mRNA standard curve solution and the demulsification, and the fluorescence value under the excitation wavelength of 485nm and the emission wavelength of 530nm is measured using an enzyme marker. The standard curve is made with the mRNA gradient concentration and the corresponding fluorescence value, and the RNA concentration (free concentration) of the sample to be tested before demulsification and the RNA concentration (total RNA concentration) of the sample to be tested after demulsification are converted into the curve to obtain the LNP free rate.

[0348] The physical and chemical characterization data of LNP are summarized in Table 2. After 18:0CL modification, the potential of LNP decreases with the increase of modification ratio. The physical and chemical properties of LNP are good, the particle size is below 150nm, the PDI is below 0.04, and the free rate is below 10%.

[0349] Table 2 Physical and chemical characterization results of targeted lipid nanoparticles

[0350] prescription Particle size (nm) PDI Zeta(mV) RNA concentration (μg / mL) Free rate (%) Prescription 1 122.3±2.95 0.02±0.02 11.87±0.21 197.0 2.87 Prescription 2 119.0±0.54 0.02±0.01 9.59±0.94 197.0 3.51 Prescription 3 124.0±0.25 0.02±0.01 9.79±0.37 197.0 2.90 Prescription 4 122.8±1.92 0.04±0.01 9.94±1.52 197.0 2.99 Comparative Prescription A 122.9±1.81 0.03±0.01 16.35±1.50 197.0 3.93 Positive control prescription 1 124.1±1.70 0.09±0.03 2.92±0.24 170.0 2.66 Positive control prescription 2 131.5±0.65 0.11±0.02 -2.33±0.11 170.0 3.08 Positive control prescription 3 129.5±2.14 0.10±0.02 -3.06±0.40 170.0 2.69 Positive control prescription 4 133.9±2.14 0.10±0.01 -5.16±0.16 170.0 2.81

[0351] 3. Determination of RNase protection efficiency of LNP

[0352] The RNase protection efficiency of LNP was measured by agarose gel electrophoresis (AGE): mRNA (drug substance, DS) and LNP samples were diluted with 1X TE buffer, RNase A was added and incubated at room temperature for 30 minutes, and Proteinase K was added to stop the reaction. Triton-phenol mixed solution (50 μL Triton X-100 was added to 650 μL phenol: chloroform: isoamyl alcohol mixture (25:24:1; V / V / V)) was further used to break the emulsion and extract RNA to the aqueous phase. The aqueous supernatant was extracted and added with RNA loading dye together with 9KMarker, and then electrophoresed on 1% agarose gel. The electrophoresis picture was obtained on the Bio-rad gel imager, and the enzyme protection efficiency of LNP was semi-quantitated according to the gray value of the electrophoresis band. The results are shown in Table 3.

[0353] The results showed that after APCs targeted lipid modification, the RNase protection efficiency of LNP was good.

[0354] Table 3 RNase protection rate of targeted lipid nanoparticles

[0355] prescription RNase protection efficiency 5% 18:0 Cardiolipin, DLin-MC3-DMA (prescription 1) 100％ 10% 18:0 Cardiolipin, DLin-MC3-DMA (Prescription 2) 92％ 20% 18:0 Cardiolipin, DLin-MC3-DMA (prescription 3) 93％ 30% 18:0 Cardiolipin, DLin-MC3-DMA (prescription 4) 84％ DLin-MC3-DMA (Control Prescription A) 100％ 5% PS, DLin-MC3-DMA (positive control prescription 1) 95％ 10% PS, DLin-MC3-DMA (positive control prescription 2) 92％ 20% PS, DLin-MC3-DMA (positive control prescription 3) 93％ 30% PS, DLin-MC3-DMA (positive control prescription 4) 51％

[0356] 4. Determination of LNP apparent ionization constant pKa (TNS titration method)

[0357] A series of buffer solutions (150 mM NaCl, 10 mM boric acid solution, 10 mM phosphoric acid solution, and 10 mM citric acid solution) with different pH values ​​(pH = 2, 3, 4.5, 6.2, 6.9, 7.7, 10, 12) were prepared. Blank nanoparticles of different formulations in Example 1 were prepared (the preparation method was exactly the same as in Example 1 except that mRNA was not contained), and diluted in each buffer solution so that the final concentration of DLin MC3 DMA was 75 μM. 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt, TNS, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in DMSO at a concentration of 300 μM. Then 2 μL of TNS solution was mixed evenly with 200 μL of blank nanoparticle-buffer solution mixed solution. The fluorescence value under the conditions of excitation light 321 nm and emission light 435 nm was measured, and the pH of the final solution was measured at the same time. After obtaining the pH-fluorescence intensity scatter plot, a four-parameter sigmoid function was fitted to obtain a fitting curve. The pH value at the inflection point of the curve is the apparent ionization constant pKa of LNP.

[0358] Using the above method, the pKa of control prescription A was measured to be 6.26, the pKa of positive control prescription 1 was 6.32, and the pKa of prescription 3 was 6.25.

[0359] 5. In vitro cell level study on the macrophage transfection effect of APCs-targeted lipid-modified LNP

[0360] On day 0, THP-1 cells in good culture state were mixed with 100 nM PMA (phorbol 12-myristate 13-acetate, Stemcell, cat: 74044) in RPMI1640 (Gibco, 61870127) medium and cultured at 2×10 4cells / well were plated in a white opaque 96-well plate to induce THP-1 into macrophages; on the third day, the expression of CD14 was determined by flow cytometry to verify the successful induction. First, the control prescription A and the positive control prescriptions 1-3 were mixed with Opti-MEM (Gbico, 2492867) and β2-GPI (Probio, PB-BTA-02) at 0.25 μg / mL (high dose), so that the final concentration of β2-GPI in the culture medium was 1 for the mass ratio of β-GPI to lipid in LNP, and incubated at 37°C for 2 hours. 10 minutes before the end of the incubation, the THP-1 cells induced by PMA for 3 days were replaced with new RPMI1640 culture medium. After the incubation, the mixed sample after incubation was added to the 96-well plate, and 2 replicate wells were set. On the 4th day, prepare the luciferin substrate, take 1mL of luciferin bioluminescent substrate (D-luciferin), mix it evenly with 9mL PBS, prepare it into working solution, add 30μL / well of luciferin substrate to the cells transfected with LNPs sample, and react at room temperature for 10 minutes in the dark. After the reaction is completed, use the microplate reader to read the results, set the detection mode to chemiluminescence, and set the detection light source to Luminescence. Put the 96-well plate into the microplate reader to read directly, export the data for result analysis, and the results Figure 1 As shown in a. The control group with the most obvious increase in β2-GPI after incubation was selected as the subsequent positive control prescription 1.

[0361] Subsequently, the LNPs sample (prescription 3) and the control group (positive control prescription 1) were mixed with Opti-MEM (Gbico, 2492867) and β2-GPI (Probio, PB-BTA-02) at 0.25 μg / mL (high dose) or 0.025 μg / mL (low dose), respectively, so that the final concentration of β2-GPI in the culture medium was 1, and the mass ratio of β-GPI to lipid in LNP was 1. The culture medium was incubated at 37°C for 2 hours. Ten minutes before the end of the incubation, the THP-1 cells induced by PMA for 3 days were replaced with new RPMI1640 culture medium. After the incubation, the mixed samples after incubation were added to a 96-well plate, and 2 replicate wells were set up. On the 4th day, prepare the luciferin substrate, take 1mL of luciferin bioluminescent substrate (D-luciferin), mix it evenly with 9mL PBS, prepare it into working solution, add 30μL / well of luciferin substrate to the cells transfected with LNPs sample, and react at room temperature for 10 minutes in the dark. After the reaction is completed, use the microplate reader to read the results, set the detection mode to chemiluminescence, and set the detection light source to Luminescence. Put the 96-well plate into the microplate reader to read directly, export the data for result analysis, and the results Figure 1 As shown in b.

[0362] The results showed that by characterizing the changes in transfection effects of different proportions of PS (0%~20%) combination prescriptions (control prescription A, positive control prescription 1, positive control prescription 2, positive control prescription 3) after β-GPI incubation, it was found that 5% PS (positive control prescription 1) had the most obvious transfection improvement (2.27 times) after β2-GPI incubation. Therefore, positive control prescription 1 was subsequently used as the positive control prescription in subsequent characterizations.

[0363] Since PS can increase mRNA presentation through PSR-mediated pathways expressed by other APCs in addition to the β-GPI pathway, the positive control group PS-MC3 LNP (positive control prescription 1) that was not incubated with β-GPI expressed higher mRNA than 18:0CL-MC3LNP (prescription 3). However, after incubation with β-GPI, 18:0CL-MC3 LNP (prescription 3) showed significantly increased mRNA expression. In the low-dose group, after incubation with β-GPI, the mRNA expression of 18:0CL-MC3 LNP was higher than that of PS-MC3LNP. The ζ potential of prescription 3 was 9.79±0.37mV, and the ζ potential of positive control prescription 1 was 2.92±0.24mV, indicating that the transfection characteristics of this prescription do not depend on the increase in the overall negative charge of LNP, but are related to the strong binding of 18:0CL and β-GPI. Therefore, the "eat me" signal can be provided to APCs through the physiological process of strong binding of β-GPI and CL, enhancing the targeted delivery ability of antigens.

[0364] 6. In vivo animal level study on the APCs targeting ability of LNPs modified with APCs targeting lipids

[0365] The animal model was female BALB / c mice of 8 weeks (SHANGHAI SLAC); after tail vein injection of LNP sample (prescription 3) and control LNP (positive control prescription 1), spleens were taken for in vitro imaging at 3h, 6h, 48h and 72h, and the fluorescence intensity was measured and the area under the fluorescence intensity-time curve (3h-72h) was calculated. The results are shown in Figure 2 shown.

[0366] The results showed that the 18:0CL prescription (prescription 3) disclosed in the present invention significantly increased the AUC (4 times) of luciferase mRNA in the spleen enriched with APCs compared to the positive control prescription 1 (5% PS-DLinMC3DMA), and had a significantly improved spleen / APCs targeting effect.

[0367] Example 2

[0368] 1. Preparation of nanoparticles containing biomimetic APCs targeting lipid combinations

[0369] (1) According to Table 4 below, SM102, DSPC, DMG-PEG2000, cholesterol and DSPE-RGD (or Lyso PC) were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, according to Table 4 below, PS was weighed and dissolved in citric acid buffer until the solution was clear and transparent, and GFP-luciferase mRNA was added to obtain an aqueous phase solution containing mRNA in a volume of three times ethanol. Positive control prescription 5 is also listed in Table 4, and its preparation process refers to the above prescription.

[0370] Table 4 Prescription of targeted lipid nanoparticles

[0371]

[0372]

[0373] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the obtained mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0374] 2. Physical and chemical characterization of LNP

[0375] The determination method as described in Example 1 was adopted, and the results are shown in Table 5.

[0376] The physical and chemical characterization data of LNP are summarized in Table 5. The potential of LNP modified with Lyso PC and PS, or RGD-DSPE and PS decreases with the increase of modification ratio. The physical and chemical properties of LNP are good, with a particle size below 120 nm, a PDI below 0.2, and a free rate below 10%.

[0377] Table 5 Physical and chemical characterization results of targeted lipid nanoparticles

[0378] prescription Particle size (nm) PDI zeta(mv) Total RNA concentration (μg / mL) Free rate (%) Prescription 5 111.0±1.03 0.12±0.02 -19.14±0.40 197.0 4.05 Prescription 6 110.0±0.48 0.13±0.02 -21.37±1.30 197.0 3.98 Prescription 7 109.8±1.78 0.13±0.04 -26.75±1.37 197.0 4.62 Prescription 8 112.0±1.79 0.14±0.06 8.90±0.62 197.0 3.80 Prescription 9 113.0±1.86 0.14±0.01 10.72±0.82 197.0 4.40 Prescription 10 110.0±2.28 0.15±0.02 12.10±0.21 197.0 5.60 Positive control prescription 5 111.6±1.07 0.10±0.03 13.84±1.12 197.0 2.78

[0379] 3. Determination of RNase protection efficiency of LNP

[0380] The determination method described in Example 1 was used to obtain electrophoresis images on a bio-rad gel imager, and the enzyme protection efficiency of LNP was semi-quantitatively evaluated based on the grayscale value of the electrophoresis band. The results are shown in Table 6. After APCs targeted lipid modification, the RNase protection efficiency of LNP was good.

[0381] Table 6 RNase protection rate of targeted lipid nanoparticles

[0382] prescription RNase protection efficiency Prescription 5 100％ Prescription 6 86％ Prescription 7 85％ Prescription 8 100％ Prescription 9 84％ Prescription 10 85％ Positive control prescription 5 98％

[0383] 4. In vitro cell level study on the macrophage transfection effect of APCs-targeted lipid-modified LNP

[0384] Day 0, well-cultured THP-1 cells were mixed with 100 nM PMA (phorbol 12-myristate 13-acetate, Stemcell, cat: 74044) using RPMI1640 (Gibco, 61870127) medium and cultured at 2×10 4 cells / well were plated in a white opaque 96-well plate to induce THP-1 into Macrophage; Day 3, the expression of CD14 was measured by flow cytometry to verify the successful induction. Then, the LNPs samples (prescriptions 5-7) and the control group (LNPs without APC targeting lipids, only containing 5% phosphatidylserine, positive control prescription 5) were mixed with Opti-MEM and β-GPI (1g / LNP lipid) at 0.025μg / mL, respectively, so that the final concentration of β2-GPI in the culture medium was 1 for the mass ratio of β-GPI to lipid in LNP, and incubated at 37°C for 2h. 10 minutes before the end of the incubation, the THP-1 cells induced by PMA for 3 days were replaced with new RPMI1640 culture medium. After the incubation, the mixed samples after incubation were added to the 96-well plate, and 2 duplicate wells were set. Day 4, prepare the luciferin substrate, take 1mL of luciferin bioluminescent substrate (D-luciferin, Yisheng Biotechnology, cat: 115144-35-9), mix it evenly with 9mL PBS, and prepare it into working solution. Add the luciferin substrate to the cells transfected with LNPs sample at 30μL / well, and react at room temperature for 10 minutes in the dark. After the reaction is completed, use an ELISA reader to read the results, set the detection mode to chemiluminescence, and set the detection light source to Luminescence. Put the 96-well plate into the ELISA reader to read directly, export the data for result analysis, and the results are as follows: Figure 3 shown.

[0385] The results showed that before incubation with β-GPI, as the ratio of RGD increased, the ζ potential decreased, but mRNA transfection increased. As the ratio of RGD increased, the transfection increased further after incubation with β-GPI, but the increase factor decreased (from 4.35 times to 2.65 times), that is, the combination of PS and RGD-DSPE lipids showed an increase in transfection efficiency that was independent of the β-GPI pathway, which was related to the RGD-targeted integrin receptor binding to PS through MFG-E8 as a bridging molecule, enhancing the phagocytosis of apoptotic cells by macrophages. Therefore, by modifying LNP with RGD (Arg-Gly-Asp) peptide targeting integrin receptors, the antigen presentation of macrophages can be further enhanced. Lysophosphatidylcholine (Lyso PC) is an inflammatory mediator, a "find me" signal, recruiting phagocytes, inducing cell phagocytosis and antigen presentation. Lyso PC combined with PS has a similar effect, which is related to the mimicking effect of the "eat me" signal lipid combination on apoptotic cells.

[0386] Therefore, other natural “eat me” or “find me” signals, such as sphingosine-1-phosphate (S1P) or lysophosphatidylcholine (Lyso PC) or RGD-DSPE combined with PS in a certain proportion to modify LNP can better mimic apoptotic cells under physiological conditions, thereby enhancing antigen presentation and increasing its targeting effect.

[0387] 5. In vivo animal level study on the APCs targeting ability of LNPs modified with APCs targeting lipids

[0388] The animal model was: female, BALB / c mice, 8 weeks (SHANGHAI SLAC); after tail vein injection of prescription 7, prescription 10 and control prescription positive control prescription 1 (5% PS-DLin MC3 DMA), the liver, lung and spleen were taken for in vitro imaging at 3h, 6h and 72h. The results are shown in Table 7 and Figure 4 As shown, Figure 4 a. Figure 4 b. Figure 4 c represents the in vitro imaging results at 3h, 6h, and 72h of drug administration. After measuring the fluorescence intensity, the area under the fluorescence intensity-time curve of the spleen (3h-72h) was calculated. The results are shown in Figure 5 shown.

[0389] Table 7 Distribution of targeted liposomes delivered to different organs

[0390]

[0391] The results showed that the combination of RGD-DSPE or Lyso PC and PS significantly increased the AUC of luciferase mRNA in the spleen enriched with APCs compared with the combination of PS alone. Specifically, the combination of RGD-DSPE or Lyso PC and PS had significantly improved the spleen / APCs targeting effect, especially in the long-term (72 hours) delivery of spleen targeting.

[0392] Furthermore, other natural “eat me” or “find me” signals, such as sphingosine-1-phosphate (S1P) or lysophosphatidylcholine (LysoPC) or RGD-DSPE, alone or in combination with CL (or PS) in a certain ratio, can be used to modify LNPs to better mimic apoptotic cells under physiological conditions, thereby enhancing antigen presentation and increasing their targeting effect.

[0393] Example 3

[0394] 1. Preparation of nanoparticles containing biomimetic APCs-targeting lipids

[0395] (1) ALC-0315 (or SM102), DSPC, DMG-PEG2000 (or ALC-0159) and cholesterol were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, 18:0CL was weighed and dissolved in citric acid buffer according to Table 8 to obtain a clear and transparent solution, and GFP-luciferase mRNA was added to obtain an aqueous phase solution containing mRNA in a volume of three times that of ethanol.

[0396] Table 8 Prescription of targeted lipid nanoparticles

[0397]

[0398]

[0399] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the obtained mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0400] The physical and chemical characterization method of LNP is the same as that of Example 1. The physical and chemical characterization of the prepared LNP is good, and the RNase protection efficiency of LNP is greater than 80%. The results of in vitro THP-1-induced macrophage transfection are similar to those of Prescription 3 in Example 1, and have a good effect of targeting APCs through the β-GPI pathway.

[0401] Example 4

[0402] 1. Preparation of nanoparticles containing biomimetic APCs-targeting lipids

[0403] (1) ALC-0315 (or DLin-MC3-DMA or SM102), DSPC, DMG-PEG2000 (or ALC0159) and cholesterol were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, 18:1CL was weighed and dissolved in citric acid buffer according to Table 9 until the solution was clear and transparent, and GFP-luciferase mRNA was added to obtain an aqueous phase solution containing mRNA with a volume of three times that of ethanol.

[0404] Table 9 Prescription of targeted lipid nanoparticles

[0405]

[0406]

[0407]

[0408]

[0409] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the resulting mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0410] The physical and chemical characterization method of LNP is the same as that of Example 1, and the physical and chemical characterization results of the prepared LNP are similar to those of prescriptions 1-4 in Example 1, and the enzyme protection efficiency is >80%. The results of in vitro THP-1-induced macrophage transfection are similar to those of prescription 3 in Example 1, and have a good effect of targeting APCs through the β-GPI pathway.

[0411] Example 5

[0412] 1. Preparation of nanoparticles containing biomimetic APCs-targeting lipids

[0413] (1) ALC-0315 (or DLin-MC3-DMA), DSPC, DMG-PEG2000 (or ALC0159) and cholesterol were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, 18:2CL with a gradient concentration was weighed and dissolved in a citric acid buffer solution until the solution was clear and transparent, and GFP-luciferase mRNA was added to obtain an aqueous phase solution containing mRNA with a volume of three times the ethanol volume.

[0414] Table 10 Prescription of targeted lipid nanoparticles

[0415]

[0416]

[0417]

[0418] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the resulting mixed solution was diluted with PBS and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0419] The physicochemical characterization method of LNP is the same as that of Example 1. The prepared LNP has good physicochemical characterization, and the enzyme protection efficiency is above 80%. The results of in vitro THP-1-induced macrophage transfection are similar to those of Formula 3 in Example 1, and have good effect of targeting APCs through the β-GPI pathway.

[0420] Example 6

[0421] 1. Preparation of nanoparticles containing biomimetic APCs targeting lipid combinations

[0422] (1) SM102, DSPC, DMG-PEG2000, cholesterol and RGD-DSPE (or Lyso PC) were weighed and dissolved in ethanol until the solution was clear and transparent to obtain an organic phase solution. At the same time, PS was weighed and dissolved in citric acid buffer according to Table 11 until the solution was clear and transparent, and GFP-luciferase mRNA was added to obtain an aqueous phase solution containing mRNA in a volume of three times that of ethanol.

[0423] Table 11 Prescription of targeted lipid nanoparticles

[0424]

[0425]

[0426] (2) The aqueous phase solution and the organic phase solution were mixed in a microfluidic system, and the obtained mixed solution was diluted with PBS (pH = 7.4) and concentrated by ultrafiltration to a final mRNA concentration of 0.2 mg / mL to obtain the final LNP solution, which was stored in a -80°C refrigerator.

[0427] 2. The physical and chemical characterization method of LNP is the same as that of Example 1. The results are shown in Table 12.

[0428] Table 12 Physical and chemical characterization results of targeted lipid nanoparticles

[0429] prescription Particle size (nm) PDI Zeta(mV) Total RNA concentration (μg / mL) Free rate (%) Prescription 43 133.3±0.70 0.19±0.02 25.04±4.05 197.0 16.60 Prescription 44 139.4±2.18 0.19±0.02 31.23±1.89 197.0 19.40 Prescription 45 142.1±1.41 0.21±0.05 38.24±3.00 197.0 19.60 Prescription 46 132.03±0.45 0.17±0.01 16.97±0.9 197.0 16.2 Prescription 47 139.2±0.85 0.2±0.01 14.78±0.7 197.0 19.1 Prescription 48 145.33±0.4 0.22±0.01 10.72±0.71 197.0 25.5 Positive control prescription 6 134.7±1.76 0.11±0.01 10.75±1.21 197.0 3.38

[0430] The results showed that the potential of LNP modified with Lyso PC and PS, or RGD-DSPE and PS decreased with the increase of modification ratio, and the physical and chemical properties of LNP were good. The particle size of the prepared LNP was below 150nm, PDI < 0.25, and the free rate < 30%.

[0431] 3. The method for determining the RNase protection rate of LNP is the same as in Example 2. The results are shown in Table 13.

[0432] Table 13 RNase protection rate of targeted lipid nanoparticles

[0433] prescription RNase protection rate Prescription 43 100％ Prescription 44 96％ Prescription 45 90％ Prescription 46 100％ Prescription 47 81％ Prescription 48 80％ Positive control prescription 6 97％

[0434] 4. In vitro cell level study of the macrophage transfection effect of LNP modified with APCs targeting lipids The method was the same as in Example 1. The results showed that LNPs combined with RGD-DSPE or Lyso PC and PS lipids had a good effect of targeting APCs through a non-β-GPI pathway that simulates antigen presentation in vivo. It has a good effect of targeting APCs in vivo and improving antigen presentation.

[0435] Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A targeted lipid nanoparticle, comprising: (i) nucleic acids, (ii) at least one cationic lipid, and (iii) at least one APCs targeting lipid.

2. A targeted lipid nanoparticle according to claim 1, comprising: (i) nucleic acids, (ii) at least one cationic lipid, and (iii) at least one APCs targeting lipid; Targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by activating integrin receptors; Alternatively, targeted lipid nanoparticles containing APCs-targeting lipids can deliver nucleic acids to target organs other than the liver by binding to S1P receptors; Alternatively, targeted lipid nanoparticles containing APCs-targeting lipids can deliver nucleic acids to target organs other than the liver by binding to the G2A receptor; Alternatively, targeted lipid nanoparticles containing APCs-targeting lipids deliver nucleic acids to target organs other than the liver by binding to β2-GPI or vimentin with an apparent ionization constant (pKa) between 6 and 7.

3. The targeted lipid nanoparticle according to claim 1, wherein The targeted lipid nanoparticle delivers the nucleic acid to a target organ selected from the group consisting of spleen, lymph node, heart, brain, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, thymus, or spinal cord; Preferably, the target organ is the spleen.

4. The targeted lipid nanoparticle according to claim 2, wherein The APCs targeting lipid is selected from lipids that activate integrin receptors; preferably, the lipid that activates integrin receptors is selected from lipids modified with RGD; preferably, the RGD-modified lipid is selected from RGD-modified DSPE; more preferably, the RGD-modified lipid is selected from any one of RGD-DSPE, DSPE-PEG-RGD or DSPE-PEG-iRGD.

5. The targeted lipid nanoparticle according to claim 2, wherein The APCs targeting lipid is selected from lipids that bind to S1P receptors; preferably, the lipid that binds to S1P receptors is selected from any one of sphingosine 1-phosphate (S1P), D-Sphingosine, Fingolimod, Ozanimod or Ceramide.

6. The targeted lipid nanoparticle according to claim 2, wherein The APCs targeting lipid is selected from lipids that bind to the G2A receptor; preferably, the lipid that binds to the G2A receptor is selected from any one of lysophosphatidylcholine (Lyso PC), 9-HODE or 13-HODE.

7. The targeted lipid nanoparticle according to claim 1 or 2, wherein The APCs targeting lipid is selected from cardiolipin; preferably, the cardiolipin (CL) includes 18:0CL, 18:1CL, and 18:2CL.

8. The targeted lipid nanoparticle according to any one of claims 2 to 6, wherein The APCs targeting lipid also includes phosphatidylserine (PS).

9. The targeted lipid nanoparticle according to any one of claims 1 to 8, wherein The cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTA P), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, or any one thereof or a combination thereof; Preferably, the cationic lipid is selected from any one of DLin-MC3-DMA, SM-102, ALC-0315 or a combination thereof.

10. The targeted lipid nanoparticle according to any one of claims 1 to 9, wherein The molar ratio of the cationic lipid to the APCs targeting lipid is 4-10:1-10.

11. The targeted lipid nanoparticle according to any one of claims 1 to 10, wherein The targeted lipid nanoparticles further include phospholipids; The phospholipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditri Any one of cosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE); Preferably, the phospholipid is selected from any one of DSPC, DOPC, DMPC, DOPE or DPPE; More preferably, the phospholipid is selected from DSPC.

12. The targeted lipid nanoparticle according to any one of claim 11, wherein The molar ratio of the cationic lipid, the APCs targeting lipid and the phospholipid is 4-10:1-10:1-2.

13. The targeted lipid nanoparticle according to any one of claims 1 to 12, wherein The targeted lipid nanoparticles further include conjugated lipids; the conjugated lipids are selected from any one of ALC-0159, DMG-PEG2000, and PEG2000; Preferably, the conjugated lipid is selected from ALC-0159 or DMG-PEG2000.

14. The targeted lipid nanoparticle according to any one of claims 1 to 13, wherein The targeted lipid nanoparticles further include cholesterol or cholesterol derivatives; preferably, the targeted lipid nanoparticles further include cholesterol.

15. The targeted lipid nanoparticle according to any one of claims 1 to 14, wherein The APCs targeting lipid accounts for about 1 mol% to about 50 mol% of the total lipids in the particle; Preferably, the APCs targeting lipid accounts for about 2 mol% to about 40 mol% of the total lipids in the particle; Preferably, the APCs targeting lipid accounts for about 5 mol% to about 40 mol% of the total lipids in the particle; Preferably, the APCs targeting lipid accounts for about 5 mol% to about 30 mol% of the total lipid in the particle; Preferably, the APCs targeting lipid accounts for about 5 mol% to about 20 mol% of the total lipids in the particle; More preferably, the APCs targeting lipid accounts for about 5 mol% to about 10 mol% of the total lipids in the particle; or, The cationic lipid accounts for about 20 mol% to about 80 mol% of the total lipids in the particle; Preferably, the cationic lipid accounts for about 20 mol% to about 70 mol% of the total lipids in the particle; Preferably, the cationic lipid accounts for about 20 mol% to about 70 mol% of the total lipids in the particle; Preferably, the cationic lipid accounts for about 25 mol% to about 60 mol% of the total lipids in the particle; Preferably, the cationic lipid accounts for about 25 mol% to about 55 mol% of the total lipids in the particle; Preferably, the cationic lipid accounts for about 25 mol% to about 50 mol% of the total lipids in the particle; More preferably, the cationic lipid accounts for about 28 mol% to about 50 mol% of the total lipids in the particle; Alternatively, the phospholipids comprise from about 2 mol% to about 50 mol% of the total lipids in the particle; Preferably, the phospholipids account for about 2 mol% to about 40 mol% of the total lipids in the particle; Preferably, the phospholipids account for about 5 mol% to about 30 mol% of the total lipids in the particle; Preferably, the phospholipids account for about 5 mol% to about 20 mol% of the total lipids in the particle; More preferably, the phospholipids account for about 5 mol% to about 10 mol% of the total lipids in the particle; or, The conjugated lipids account for about 1 mol% to about 40 mol% of the total lipids in the particle; Preferably, the conjugated lipid accounts for about 1 mol% to about 30 mol% of the total lipid in the particle; Preferably, the conjugated lipid accounts for about 1 mol% to about 20 mol% of the total lipid in the particle; Preferably, the conjugated lipid accounts for about 1 mol% to about 10 mol% of the total lipid in the particle; Preferably, the conjugated lipid accounts for about 1 mol% to about 5 mol% of the total lipid in the particle; More preferably, the conjugated lipid accounts for about 1 mol% to about 2 mol% of the total lipid in the particle; or, The cholesterol or cholesterol derivative accounts for about 15 mol% to about 50 mol% of the total lipids in the particle; Preferably, the cholesterol or cholesterol derivative accounts for about 20 mol% to about 50 mol% of the total lipids in the particle; Preferably, the cholesterol or cholesterol derivative accounts for about 20 mol% to about 40 mol% of the total lipids in the particle; More preferably, the cholesterol or cholesterol derivative comprises about 25 mol % to about 40 mol % of the total lipids in the particle.

16. The targeted lipid nanoparticle according to claim 1, wherein Targeted lipid nanoparticles, including: (i) nucleic acids, (ii) a cationic lipid, wherein the cationic lipid is selected from any one of DLin-MC3-DMA, SM-102, and ALC-0315; (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from cardiolipin, a combination of PS and RGD-DSPE, and a combination of PS and LysoPC; (iv) a phospholipid selected from DSPC; (iv) a conjugated lipid selected from ALC-0159 or DMG-PEG2000; and (vi) Cholesterol.

17. The targeted lipid nanoparticle according to claim 1, wherein Targeted lipid nanoparticles, including: (i) nucleic acids, (ii) a cationic lipid selected from DLin-MC3-DMA; (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from cardiolipin; (iv) a phospholipid selected from DSPC; (iv) a conjugated lipid selected from ALC-0159; and (vi) Cholesterol.

18. The targeted lipid nanoparticle according to claim 1, wherein Targeted lipid nanoparticles, including: (i) nucleic acids, (ii) a cationic lipid selected from SM-102; (iii) APCs targeting lipid, wherein the APCs targeting lipid is selected from a combination of PS and RGD-DSPE, and a combination of PS and Lyso PC; (iv) a phospholipid selected from DSPC; (iv) a conjugated lipid selected from DMG-PEG2000; and (vi) Cholesterol.

19. The targeted lipid nanoparticle according to any one of claims 1 to 18, wherein The nucleic acid is selected from RNA; preferably, the RNA includes one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, and tsRNA; more preferably, the RNA is mRNA.

20. A pharmaceutical composition comprising the targeted lipid nanoparticles according to claims 1-19, and pharmaceutically acceptable excipients.

21. Use of the targeted lipid nanoparticle according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20 in the preparation of a drug for treating cancer.

Images