Targeting lipid nanoparticles for nucleic acid delivery

Abstract

Targeting lipid nanoparticles for nucleic acid delivery, In one embodiment, the targeting lipid nanoparticle includes an ionizable lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a targeting lipid, and a nucleic acid. In other embodiments, the targeting lipid nanoparticle including an ionizable lipid, a cationic lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a targeting lipid; and a nucleic acid. In other embodiments, the targeting lipid nanoparticle including an ionizable lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a functionalized lipid; and a nucleic acid. In other embodiments, the targeting lipid nanoparticle including an ionizable lipid, a cationic lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a functionalized lipid; and a nucleic acid. Also provided are kits for preparing targeting lipid nanoparticles and functionalized lipid nanoparticles for nucleic acid delivery.

Description

TARGETING LIPID NANOPARTICLES FOR NUCLEIC ACID DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 579,838, filed August 31, 2023, the entire content of which is incorporated herein by reference.BACKGROUND

[0002] Nucleic acid therapeutics have shown tremendous potentials to treat a variety of undruggable disease including genetic diseases, immune diseases and organ disorders. For example, small interfering RNA, which can silent the expression of unwanted proteins, was used to treat polyneuropathy in patients with hereditary transthyretin-mediated amyloidosis; messenger RNA, which can induce expression of engineered proteins, proved to be efficient to control COVID-19 infections and immunotherapy. The challenges for nucleic acids as drugs remain as lack of cell permeability, easy degradation, off-target and poor pharmacokinetic and pharmacodynamic properties.

[0003] Lipid nanoparticles (LNPs) have proven to be powerful carriers to facilitate the drug capability of nucleic acids. The composition of lipid nanoparticles generally includes one or more cationic and / or ionizable lipids, phospholipid-based helper lipids, sterol-like structural lipids and PEG-modified lipids. Although a variety of lipid compositions have been studied, the efficiency, particularly specificity, must be improved to effectively deliver nucleic acids to cells. A need exists for improved lipid nanoparticles to deliver nucleic acids to specific cells.SUMMARY

[0004] In a first embodiment, a targeting lipid nanoparticle (LNP) is provided, the targeting LNP including an ionizable lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a targeting lipid and a nucleic acid. In various embodiments, the molar ratio of ionizable / cationic lipid: cholesterol:helper lipid: PEG lipid: targeting / functionalized lipid is in the range of (40-60): (20-40): (10-20): (0.5-5): (0.5-5).

[0005] In a second embodiment, the targeting lipid nanoparticles provided herein include an ionizable lipid, a cationic lipid, a helper lipid, a cholesterol or cholesterol derivative, a PEG lipid, a targeting lipid and a nucleic acid. In various embodiments the molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: targeting lipid is in the range from (30-50): (5-20): (10-25): (25-40): (0.5-5): (0.5-5).

[0006] In a third embodiment, the targeting lipid nanoparticles for nucleic acid delivery provided herein include an ionizable lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a functionalized lipid and a nucleic acid. In various embodiments, the molar ratio of ionizable lipid: cholesterol: helper lipid: PEG lipid: functionalized lipid is in the range of (40- 60): (20-40): (10-20) :(0.5-5): (0.5-5).

[0007] In a fourth embodiment, the targeting lipid nanoparticle for nucleic acid delivery includes an ionizable lipid, a cationic lipid, a helper lipid, a cholesterol or cholesterol derivative, a PEG lipid, a functionalized lipid and a nucleic acid. In various embodiments, the molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: functionalized lipid is in the range from (30-50): (5-20): (10-25): (25-40): (0.5-5): (0.5-5).

[0008] Also provided are kits for preparing targeting lipid nanoparticles based on the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows the targetability of lipid nanoparticles in HeLa and HEK 293 cells.

[0010] FIG. 2 shows size characterization of lipid nanoparticles which did encapsulate or did not encapsulate siRNA (eGFP siRNA).

[0011] FIG. 3 shows polydispersity of lipid nanoparticles which did encapsulate or did not encapsulate siRNA (eGFP siRNA).

[0012] FIG. 4 shows the GFP silencing results by siRNA-loaded lipid nanoparticles or lipofectamine.

[0013] FIG. 5A shows the quantification results of GFP silencing efficacy by siRNA-loaded lipid nanoparticles after 24 hours.

[0014] FIG. 5B shows the quantification results of GFP silencing efficacy by siRNA-loaded lipid nanoparticles after 72 hours.

[0015] FIG. 6 shows the biocompatibility of lipid nanoparticles with or without siRNA loading.

[0016] FIG. 7 shows size characterization of lipid nanoparticles which did encapsulate or did not encapsulate mRNA (luciferase mRNA).

[0017] FIG. 8 shows polydispersity of lipid nanoparticles which did encapsulate or did not encapsulate mRNA (luciferase mRNA).

[0018] FIG. 9 shows the mRNA expression results by luciferase mRNA- loaded lipid nanoparticles.

[0019] FIG. 10 shows the biocompatibility of luciferase mRNA-loaded lipid nanoparticles.DETAILED DESCRIPTION

[0020] Combining the advances of targeting lipid nanoparticles, we herein describe the synthesis of targeting lipid nanoparticles to selectively deliver nucleic acids into target cells.

[0021] As used herein, the term ionizable lipid refers to a lipid that is capable of modulating its charge depending on environmental pH. Some non-limiting examples of ionizable lipids useful in the targeting lipid nanoparticles described herein include, e.g., 1 ,2-dioleyloxydioleyloxy-3-dimethylaminopropane (DODMA), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(DOBAQ), 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315).

[0022] In some embodiments of the targeting lipid nanoparticles described herein, the ionizable lipid may be combined with or replaced by a cationic lipid. Exemplary cationic lipids useful herein include, e.g., 2-di-O-octadecenyl-3- trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3- trimethylammonium-propane (chloride salt) (DOTAP) and dimethyldioctadecylammonium (bromide salt) (DDAB).

[0023] Cholesterol and cholesterol derivative, as used herein, refer to cholesterol molecules or derivatives that function to improve intracellular delivery and / or stability of the lipid nanoparticle. Exemplary cholesterol derivatives useful in the targeting lipid nanoparticles described herein include, but are not limited to cholesterol hemisuccinate and cholesterol NHS succinate.

[0024] The term helper lipid, as used herein, refers to lipid molecules that increase stability and fluidity of lipid nanoparticles, such as phospholipids. Some non-limiting examples of helper lipids include, e.g., 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0025] PEG lipids, or PEGylated lipids are PEG derivatives attached to a lipid moiety. Some PEG lipids useful in the targeting lipid nanoparticles described herein may include, but are not limited to, DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, wherein PE is a phosphoethanolamine such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1 ,3-dipalmitoyl-glycero-2-phosphoethanolamine (DSPE).

[0026] The term targeting lipid, as used herein, refers to a lipid that targets a specific organ, cell, cell type or subcellular organelle. Some exemplary targeting lipids useful in the targeting lipid nanoparticles described herein include, e.g., 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), DSPE-PEG2000 folate, DSPE-PEG2000-biotin,DSPE-PEG2000-DBCG, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, e.g., GALA, TAT, and so forth, and DSPE-PEG-antibody, e.g., transferrin.

[0027] The term functionalized lipid refers to a lipid functionalized with a PEG and another functional group. Some non-limiting examples of functional lipids include DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE- PEG2000-DBCG, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE- PEG2000-carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG- succinyl, and DGPE-PEG2000-azide, DGPE-PEG2000-amine, DOPE- PEG2000-carboxylic acid, and DGPE-PEG2000-HALO-TAG.

[0028] In one aspect, the provided herein are a nanoparticle compositions including (1) lipid nanoparticles containing an ionizable lipid, a PEG lipid, cholesterol or a cholesterol derivative, a helper lipid, and a targeting or functionalized lipid; and (2) nucleic acid, which may be siRNA or mRNA.

[0029] In another aspect, a method of delivering nucleic acid into target cells is provided, which involves encapsulating nucleic acid into targeting nanoparticles.

[0030] Advantages of the targeting lipid nanoparticles provided herein compared with conventional lipid nanoparticles include (1 ) increased cell- selective gene silencing or expression; (2) targetability or enhanced targetability; and (3) decreased cytotoxicity and instability during nucleic acid transfection.

[0031] In a first embodiment, the targeting lipid nanoparticles for nucleic acid delivery provided herein include an ionizable lipid, a cholesterol or cholesterol derivative, a PEG lipid, a helper lipid, a targeting lipid; and a nucleic acid.

[0032] In various embodiments, the molar ratio of ionizable lipid: cholesterol: helper lipid: PEG lipid: targeting lipid is in the range of (40-60): (20-40): (10-20): (0.5-5): (0.5-5). In some preferred embodiments, the molar ratio of ionizable lipid: cholesterol hemisuccinate: helper lipid: PEG lipid: targeting lipid is in the range from 53:30:15:1.5:0.5 to 53:30:15:0.5:1.5.

[0033] In a preferred embodiment, the ionizable lipid may be selected from1.2-dioleyloxy-3-dimethylaminopropane (DODMA), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol),1.2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0034] In various embodiments, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

[0035] In various embodiments, the helper lipid is selected from 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

[0036] In various embodiments, the PEG lipid may be selected from PE- PEG500, PE-PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE- PEG1000, PE-PEG2000, wherein PE is selected from 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE) and 1,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE), and is preferably PE-PEG2000.

[0037] In various embodiments, the targeting lipid is selected from DSPE- PEG2000 folate, DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCO, DSPE- PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE- PEG-antibody, and is preferably DSPE-PEG2000-folate.

[0038] In various embodiments, the nucleic acid may be virtually any RNA. Exemplary nucleic acids include, for example, eGFP siRNA and luciferase mRNA.

[0039] In a second embodiment, the targeting lipid nanoparticles for nucleic acid delivery includes an ionizable lipid, a cationic lipid, a helper lipid, acholesterol or cholesterol derivative, a PEG lipid, a targeting lipid; and a nucleic acid.

[0040] In this embodiment, molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: targeting lipid is in the range from (30-50): (5-20): (10-25): (25-40): (0.5-5): (0.5-5). In some preferred embodiments, the molar ratio is in the range from 40:13:30:15:1.5:0.5 to 40:13:30:15:0.5:1.5.

[0041] In some preferred embodiments, the ionizable lipid may be selected from 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol),1 .2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0042] In preferred embodiments, the cationic lipid may be selected from 1 ,2- di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2- dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB), and combinations thereof.

[0043] In various embodiment, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

[0044] In various embodiment, the helper lipid is selected from selected from1 .2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

[0045] In various embodiments, the PEG lipid is selected from DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2-phosphoethanolamine (DSPE). In one preferred embodiment, the PEG lipid is DMG-PEG2000.

[0046] In various embodiments, the targeting lipid is selected from DSPE- PEG2000 folate, DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCG, DSPE- PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, e.g., GALA, TAT, and so forth, and DSPE-PEG-antibody, e.g., transferrin. In some preferred embodiments, the targeting lipid is DSPE-PEG2000 folate.

[0047] In various embodiments, the nucleic acid may be virtually any RNA. Exemplary nucleic acids include, for example, eGFP siRNA and luciferase mRNA.

[0048] Further provided is a kit for preparing a targeting lipid nanoparticle for nucleic acid delivery, the kit including an ionizable lipid, a cationic lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, and a targeting lipid. Each of the components can be as described above.

[0049] In some embodiments, the kit included: ionizable lipid may 1 ,2- dioleyloxy-3-dimethylaminopropane (DODMA), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0050] The cationic lipid may be 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB), and combinations thereof.

[0051] The cholesterol or cholesterol derivative may be, e.g., cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate or combinations thereof. The helper lipid may be 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) and combinations thereof.

[0052] The included PEG lipid may be, e.g DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, wherein PE isselected from 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and1 ,3-dipalmitoyl-glycero-2-phosphoethanolamine (DSPE).

[0053] The targeting lipid included with the kit may be, e.g., DSPE-PEG2000 folate, DSPE-PEG2000-biotin, DSPE-PEG2000-DBCG, DSPE-PEG2000- mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE-PEG- antibody, and combinations thereof.

[0054] In some embodiments, the kit includes instructions.

[0055] In some embodiments, the kit may include a single ionizable lipid, a single cationic lipid, a single cholesterol or cholesterol derivative, a single helper lipid, a single PEG lipid and a single targeting lipid. In other embodiments, the kit may include more than one of any of the components. In still other embodiments, the kit may further include one or more nucleic acids.

[0056] In a third embodiment, the targeting lipid nanoparticles for nucleic acid delivery provided herein include an ionizable lipid, a cholesterol or cholesterol derivative, a PEG lipid, a helper lipid, a functionalized lipid; and a nucleic acid.

[0057] In various embodiments, the molar ratio of ionizable lipid: cholesterol: helper lipid: PEG lipid: functionalized lipid is in the range of (40-60):(20-40):(10- 20):(0.5-5): (0.5-5). In some preferred embodiments, the molar ratio of ionizable lipid: cholesterol hemisuccinate: helper lipid: PEG lipid: functionalized lipid is in the range from 53:30:15:1.5:0.5 to 53:30:15:0.5:1.5.

[0058] In a preferred embodiment, the ionizable lipid may be selected from1.2-dioleyloxy-3-dimethylaminopropane (DODMA), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol),1 .2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0059] In various embodiments, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

[0060] In various embodiments, the helper lipid is selected from 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

[0061] In various embodiments, the PEG lipid may be selected from PE- PEG500, PE-PEG1000, PE-PEG2000, PE-PEG5000, wherein PE is selected from DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE- PEG1000, PE-PEG2000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE), and is preferably PE-PEG2000.

[0062] In various embodiments, the functionalized lipid is selected from DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000- DBCO, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE-PEG2000- carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG-succinyl, and DOPE-PEG2000-azide, DOPE-PEG2000-amine, DOPE-PEG2000-carboxylic acid, and DOPE-PEG2000-HALO-TAG.

[0063] In various embodiments, the nucleic acid may be virtually any RNA. Exemplary nucleic acids include, for example, eGFP siRNA and luciferase mRNA.

[0064] In a fourth embodiment, the targeting lipid nanoparticles for nucleic acid delivery includes an ionizable lipid, a cationic lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a functionalized lipid; and a nucleic acid.

[0065] In this embodiment, molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: functionalized lipid is in the range from (30-50): (5- 20): (10-25): (25-40): (0.5-5): (0.5-5). In some preferred embodiments, the molar ratio is in the range from 40:13:30:15:1.5:0.5 to 40:13:30:15:0.5:1.5.

[0066] In preferred embodiments, the ionizable lipid may be selected from 1 ,2- dioleyloxy-3-dimethylaminopropane (DODMA), 3fS-[N-(N', N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol),1 .2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0067] In preferred embodiments, the cationic lipid may be selected from 1 ,2- di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2- dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB), and combinations thereof.

[0068] In various embodiment, the cholesterol or cholesterol derivative is selected from cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

[0069] In various embodiment, the helper lipid is selected from selected from1 .2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

[0070] In various embodiments, the PEG lipid is selected from DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2-phosphoethanolamine (DSPE). In one preferred embodiment, the PEG lipid is DMG-PEG2000.

[0071] In various embodiments, the functionalized lipid is selected from DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000- DBCO, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE-PEG2000- carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG-succinyl, and DOPE-PEG2000-azide, DOPE-PEG2000-amine, DOPE-PEG2000-carboxylic acid, and DOPE-PEG2000-HALO-TAG.

[0072] In various embodiments, the nucleic acid may be virtually any RNA. Exemplary nucleic acids include, for example, eGFP siRNA and luciferase mRNA.

[0073] Further provided is a kit for preparing a targeting lipid nanoparticle for nucleic acid delivery, the kit including an ionizable lipid, a cationic lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, and a functionalized lipid. Each of the components can be as described above.

[0074] In some embodiments, the kit included: ionizable lipid may 1 ,2- dioleyloxy-3-dimethylaminopropane (DODMA), 3f3-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol),1 .2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

[0075] The cationic lipid may be 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (Bromide Salt) (DDAB), and combinations thereof. The cholesterol or cholesterol derivative may be, e.g., cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate or combinations thereof.

[0076] The helper lipid may be 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof. The included PEG lipid may be, e.g., DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, wherein PE is selected from 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and1 .3-Dipalmitoyl-glycero-2-phosphoethanolamine (DSPE).

[0077] The functionalized lipid included with the kit may be, e.g., DSPE- PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000-DBCO, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE-PEG2000- carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG-succinyl, andDGPE-PEG2000-azide, DQPE-PEG2000-amine, DGPE-PEG2000-carboxylic acid, and DGPE-PEG2000-HALO-TAG, and combinations thereof. In some embodiments, the kit includes instructions.

[0078] In some embodiments, the kit may include a single ionizable lipid, a single cationic lipid, a single cholesterol or cholesterol derivative, a single helper lipid, a single PEG lipid and a single functionalized lipid. In other embodiments, the kit may include more than one of any of the components. In still other embodiments, the kit may further include one or more nucleic acids.EXAMPLES

[0079] A folate-modified targeting lipid nanoparticle is used to deliver siRNA, as well as mRNA. The targeting lipid nanoparticle selectively targets certain cells, enhancing the cell uptake of nucleic acid payloads, and results in more efficient gene transfection.

[0080] 1. Formulation preparation

[0081] The targeting lipid nanoparticle was prepared by microfluidic method. Lipid (10 mg) was weighted and dissolved with ethanol at 5mg / mL. The microfluidic micromixer (NanoFabTx™ Microfluidic - Nano Device kit, Catalog # 911593, Sigma-Aldrich) was connected into two syringes which were controlled by syringe pumps. Nucleic acids, such as luciferase mRNA or EGFP siRNA were diluted with RNase-free Acetate buffer (pH=4.0). The concentration was calculated based on N / P ratio. For mRNA (e.g., luciferase mRNA), the N / P ratio was set as 5 while that for siRNA (e.g., eGFP siRNA) was around 10. The flow rate of ethanol phase (lipid mixture dispersions) was 50 pL / min. Meanwhile, the flow rate of aqueous phase (mRNA or siRNA dispersed in RNase-free Acetate buffer, pH=4.0) was 250 pL / min. The obtained lipid dispersion was dialyzed against cold RNase-free PBS buffer overnight and stored at 4 °C for further use.

[0082] 2. Targetability

[0083] To evaluate if the targeting lipid nanoparticles could selectively improve the cell uptake of target cell lines, the targeting lipid nanoparticles were loaded with fluorescent probes. Dil is a lipophilic dye which only shows fluorescence in lipid bilayer and represented the presence of lipid nanoparticles. To prepare Dil-loaded lipid nanoparticles, Dil in ethanol was firstly mixed with lipid dispersions to achieve a final Dil-to-lipid molar ratio of 8%. With microfluidic mixer, the aqueous phase (PBS buffer) was mixed with ethanol-lipid solutions at flow rate ratio of 5 to obtain about 100 nm lipid nanoparticles. The obtained lipid nanoparticles were dialyzed again sterile PBS buffer overnight and stored at 4 °C for further use.

[0084] Approximately 200,000 receptor-positive cells (e.g., HeLa cells) or receptor-negative cells (e.g., HEK 293 cells) in fetal bovine serum (FBS)- supplemented medium were seeded into a 96-well microplate and incubated overnight. The cells were washed with medium once and incubated with Dil- loaded lipid nanoparticles for 48 h. Subsequently, the cells were washed with sterile PBS four times. The fluorescence of obtained cells was measured by microplate reader (Varioskan LUX, Thermo Fisher Scientific) with excitation wavelength of 550 nm and emission wavelength of 585 nm.

[0085] Targetability of lipid nanoparticles is illustrated in FIG. 1. The lipid nanoparticle was loaded with hydrophobic dyes which only show fluorescence within lipid bilayers. Folate receptor overexpressed HeLa cell line was used as target cell model. In contrast, the folate receptor less-expressed cell line- HEK 293 cell was used as a control. In HeLa cells, the amount of dye-loaded folate lipid nanoparticle was more than 5 times higher than that of dye-loaded plain lipid nanoparticle, while in HEK 293 cells, the amount of dye-loaded folate- plain lipid nanoparticle was similar to that of dye-loaded plain lipid nanoparticle. This result suggested the folate-lipid nanoparticle could selectively deliver cargos to target cells.

[0086] 3. Characterization

[0087] The hydrodynamic diameter and the diameter distribution of obtained lipid nanoparticles were characterized with a Zetasizer (Nano-ZS90, Malvern, UK). In a sample cuvette, the lipid dispersion was diluted with PBS buffer to 0.5 mg / mL. The data were collected at room temperature with three individual scans.

[0088] Size characterization of lipid nanoparticles which did encapsulate or not encapsulate siRNA is shown in FIG. 2. The lipid nanoparticle with and without siRNA loading showed very similar (ca. 90nm) size distribution, which suggested the encapsulation of siRNA did not influence the physiochemical properties of lipid nanoparticles.

[0089] Polydispersity of lipid nanoparticles which did encapsulate or not encapsulate siRNA is illustrated in FIG. 3. The polydispersity index (PDI) of lipid nanoparticles were all around 0.2, which suggested the stability of lipid nanoparticle after siRNA encapsulation.

[0090] 4. Transfect siRNA into cells

[0091] Receptor -positive reporter cells, such as HeLa cells which overexpressed GFP were used as cell models to test if the siRNA can be delivered into cells and interfere gene expression. Approximately 50,000 GFP- expressed HeLa cells were seeded into petri dish and incubated overnight. The cells were washed with medium and incubated with siRNA-loaded lipid nanoparticles which were diluted to expected concentration with same cell medium. Lipid nanoparticles without siRNA loading or siRNA-loaded lipid nanoparticles without targeting lipids modification were used as controls to further explore the influence of targetability on gene transfection. The cells were imaged under fluorescent microscope after 24 hours.

[0092] T o quantify the siRNA transfection efficiency, the GFP-HeLa cells were seeded in a 96-well microplate instead. After the same treatment including cell washing and nanoparticles incubation, the fluorescent intensity was read with a microplate reader after different time intervals including 24 hours and 72hours. The GFP-HeLa cells which were treated with cell medium instead of nanoparticles were indicated the 100% fluorescent intensity. The silencing efficiency can be calculated by the equation of (fluorescent intensity of medium- treated cells- fluorescent intensity of nanoparticle-treated cells) / fluorescent intensity of medium-treated cells.

[0093] Fluorescent imaging data which used GFP-expressed HeLa cells as model to evaluate the siRNA transfection result is shown in FIG. 4. The expression of GFP was significantly decreased when treated with siRNA- loaded lipid nanoparticle, particularly siRNA-loaded folate-lipid nanoparticle. This result indicated the capability of lipid nanoparticle to deliver siRNA into cells.

[0094] The quantification data which also used GFP-expressed HeLa cells as model to evaluate the siRNA transfection result by microplate reader is shown in FIG. 5. The siRNA-loaded folate -lipid nanoparticles showed higher gene silencing efficacy especially at day one compared to siRNA-loaded lipid nanoparticles. And 3 days later, the silencing effects still exist. This result was consistent with the cell targetability assumption that the folate-lipid nanoparticles can selectively deliver siRNA into target cells.

[0095] 5. Cytotoxicity

[0096] Cell Counting Kit-8 assay (catalog no. 96992, Sigma-Aldrich, MO, US) was used to evaluate the cytotoxicity of lipid nanoparticle itself and nucleic acid- loaded lipid nanoparticle. Receptor -positive cells (e.g., HeLa cell) was used as a representative mammalian cell. Approximately, 40,000 cells in FBS- supplemented cell medium were seeded into a 96-well microplate and incubated overnight. The cells were washed and incubated with lipid nanoparticles which were diluted with cell medium at desired concentration. After 24 h incubation, the lipid nanoparticle dispersion was removed and washed with PBS buffer. Followed by the addition of Cell Counting Kit-8 reagents and incubation in a 37°C incubator for 1 h, the absorption at 450 nm was read with a microplate reader (Varioskan LUX, Thermo Fisher Scientific).

[0097] FIG. 6 shows the HeLa cells maintained high cell viability while in contact with the plain lipid nanoparticles or folate-lipid nanoparticles, which suggests the complete biocompatibility of our delivery system. In addition, the cell viability of siRNA-loaded lipid nanoparticles and siRNA-loaded folate lipid nanoparticles were also around 100%, which suggested the transfection of siRNA did not interfere the growth of target cells.

[0098] In another example, the lipid nanoparticle was used to deliver mRNA into targeted cells.

[0099] FIG. 7 shows size characterization of lipid nanoparticles which did encapsulate or not encapsulate mRNA. A luciferase mRNA strand was chosen as model. After encapsulation of mRNA, the size of lipid nanoparticles changed from 100 nm to 130nm, which suggested the successful encapsulation of mRNA.

[0100] FIG. 8 shows polydispersity of lipid nanoparticles which did encapsulate or not encapsulate mRNA. The polydispersity index (PDI) of all lipid nanoparticles were around 0.2, which suggested the good stability even after targeting modification and mRNA loading.

[0101] Transfect mRNA into targeted cells

[0102] Receptor -positive cells (e.g., HeLa cells) are used as cell models to test if mRNA can be delivered into cells and express proteins successfully. Luciferase mRNA was used as a mRNA model. Approximately 50,000 HeLa cells were seeded into a 96-well microplate reader and grown overnight. Subsequently, the cells were washed and incubated with mRNA-loaded lipid nanoparticles which were diluted with medium. After 24 hours incubation, the cells were washed and lysed, followed by the addition of luciferase substrate (Promega luciferase assay). The luminescence which indicated the expression of luciferase mRNA was read by the microplate reader. Lipid nanoparticles which were not loaded with mRNA and mRNA-loaded lipid nanoparticles which did not carry targeting moieties were used as controls.

[0103] FIG.9 shows the capability to selectively deliver mRNA into target cells (HeLa). Luciferase mRNA was used as mRNA model and evaluated by luciferase assay. Compared to lipid nanoparticles themselves, the mRNA- loaded lipid nanoparticles can significantly express luciferase, which suggested the successful transfection of mRNA into cells. In addition, the amount of luciferase expression induced by folate- lipid nanoparticles were much higher than that induced by plain lipid nanoparticles, which suggested the targetability of folate- lipid nanoparticles and its capability to selectively deliver mRNA into target cells.

[0104] FIG.10 shows the cytotoxicity of both mRNA-loaded lipid nanoparticles and mRNA-loaded folate lipid nanoparticles were >80%, which suggested the delivery system was biocompatible and did not interfere mRNA expression.

[0105] The Examples provided herein are illustrative in nature and are not meant to limit the scope of the invention as defined by the claims.

Claims

CLAIMSThe invention claimed is:1 . A targeting lipid nanoparticle for nucleic acid delivery comprising: an ionizable lipid, a cholesterol or cholesterol derivative, a helper lipid, a PEG lipid, a targeting lipid; and a nucleic acid.

2. The targeting lipid nanoparticle of claim 1 wherein the molar ratio of ionizable lipid: cholesterol: helper lipid: PEG lipid: targeting lipid is in the range from (40-60): (20-40): (10-20): (0.5-5): (0.5-5).

3. The targeting lipid nanoparticle of claim 1 or 2 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 3R>-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof.

4. The targeting lipid nanoparticle of any of claims 1 to 3, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

5. The targeting lipid nanoparticle of any of claims 1 to 4, wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine(DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

6. The targeting lipid nanoparticle of any of claims 1 to 5 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000 and combinations thereof, wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE), and is preferably DOPE-PEG2000.

7. The targeting lipid nanoparticle of any of claims 1 to 6 wherein the targeting lipid is selected from the group consisting of DSPE-PEG2000 folate, DSPE-PEG2000-biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD and DSPE-PEG-peptide, preferably DSPE-PEG2000- folate; wherein the peptide is selected from the group consisting of GALA peptide, TAT peptide, and DSPE-PEG-antibody.

8. The targeting lipid nanoparticle of any of claims 1 to 7 wherein nucleic acid is selected from the group of mRNA or siRNA, preferably, luciferase mRNA or eGFP siRNA.

9. A targeting lipid nanoparticle for nucleic acid delivery comprising: an ionizable lipid, a cationic lipid, a helper lipid, a cholesterol, a PEG lipid, a targeting lipid; anda nucleic acid.

10. The targeting lipid nanoparticle of claim 9 wherein the molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: targeting lipid is in the range from (30-50): (5-20): (10-25): (25-40): (0.5-5): (0.5-5).

11. The targeting lipid nanoparticle of claim 9 or 10 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 3fi-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3- dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 -aminium (ALC-0315) and combinations thereof, and is preferably DODMA.

12. The targeting lipid nanoparticle of claim 9 to 11 wherein the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3- trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3- trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB) and combinations thereof, and is preferably DOTMA.

13. The targeting lipid nanoparticle of any of claims 9 to 12, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

14. The targeting lipid nanoparticle of any of claims 9 to 13, wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

15. The targeting lipid nanoparticle of any of claims 9 to 14 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG-PEG1000,DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000 and combinations thereof, preferably DMG-PEG2000, wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE).

16. The targeting lipid nanoparticle of any of claims 9 to 15 wherein the targeting lipid is selected from lipid is selected from the group consisting of DSPE-PEG2000 folate, DSPE-PEG2000-Biotin, DSPE-PEG2000-DBCG, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE-PEG-antibody, and is preferably DSPE-PEG2000-folate.

17. The targeting lipid nanoparticle of any of claims 9 to 16 wherein nucleic acid is selected from the group of mRNA or siRNA, and is preferably luciferase mRNA or eGFP siRNA.

18. A kit for preparing a targeting lipid nanoparticle for nucleic acid delivery, the kit comprising: an ionizable lipid, a cationic lipid a helper lipid, a cholesterol, a PEG lipid, a targeting lipid.

19. The kit of claim 18 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 3fS-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof, and is preferably DODMA.

20. The kit of claim 18 or 19 wherein the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB) and combinations thereof, and is preferably DOTMA.21 . The kit of any of claims 18 to 20, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

22. The targeting lipid nanoparticle of any of claims 18 to 21 , wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

23. The kit of any of claims 18 to 22 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE- PEG500, PE-PEG1000, PE-PEG2000; preferably DMG-PEG2000; wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE).

24. The kit of any of claims 18 to 23 wherein the targeting lipid is selected from lipid is selected from the group consisting of DSPE-PEG2000 folate, DSPE-PEG2000-biotin, DSPE-PEG2000-DBCO, DSPE-PEG2000-mannose, DSPE-PEG2000-RGD, DSPE-PEG-peptide, and DSPE-PEG-antibody, and is preferably DSPE-PEG2000-folate.

25. The kit of any of claims 18-24 further comprising instructions.

26. A targeting lipid nanoparticle for nucleic acid delivery comprising:an ionizable lipid, a cholesterol, a helper lipid, a PEG lipid, a functionalized lipid; and a nucleic acid.

27. The targeting lipid nanoparticle of claim 26 wherein the molar ratio of ionizable lipid: cholesterol: helper lipid: PEG lipid: functionalized lipid is in the range from (40-60): (20-40): (10-20): (0.5-5): (0.5-5).

28. The targeting lipid nanoparticle of claim 26 or 27 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 3fi-[N-(N’,N’-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3- dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 -aminium (ALC-0315) and combinations thereof.

29. The targeting lipid nanoparticle of any of claims 26 to 28, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

30. The targeting lipid nanoparticle of any of claims 26 to 29, wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

31. The targeting lipid nanoparticle of any of claims 26 to 30 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000, PE-PEG2000, preferably PE-PEG2000; wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE).

32. The targeting lipid nanoparticle of any of claims 26 to 31 wherein the functionalized lipid is selected from lipid is selected from the group consisting of DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000- DBCO, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE-PEG2000- carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG-succinyl, and DGPE-PEG2000-azide, DOPE-PEG2000-amine, DOPE-PEG2000-carboxylic acid and DOPE-PEG2000-HALO-TAG.

33. The targeting lipid nanoparticle of any of claims 26 to 32 wherein nucleic acid is selected from the group of mRNA or siRNA, and is preferably luciferase mRNA or eGFP siRNA.

34. A targeting lipid nanoparticle for nucleic acid delivery comprising: an ionizable lipid, a cationic lipid, a helper lipid, a cholesterol, a PEG lipid, a functionalized lipid; and a nucleic acid.

35. The targeting lipid nanoparticle of claim 34 wherein the molar ratio of ionizable lipid: cationic lipid: helper lipid: cholesterol: PEG lipid: functionalized lipid is in the range from (30-50): (5-20): (10-25): (25-40): (0.5-5): (0.5-5).

36. The targeting lipid nanoparticle of claim 34 or 35 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 3fS-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3- dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1 -aminium (ALC-0315) and combinations thereof, and is preferably DODMA.

37. The targeting lipid nanoparticle of claim 34 to 36 wherein the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3- trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3- trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB), and combinations thereof, and is preferably DOTMA.

38. The targeting lipid nanoparticle of any of claims 34 to 37, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

39. The targeting lipid nanoparticle of any of claims 34 to 38, wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

40. The targeting lipid nanoparticle of any of claims 34 to 39 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG- PEG1000, DMG-PEG2000, PE-PEG500, PE-PEG1000 and PE-PEG2000, preferably DMG-PEG2000; wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE).

41. The targeting lipid nanoparticle of any of claims 34 to 40 wherein the targeting lipid is selected from lipid is selected from the group consisting of DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000- DBCO, DSPE-PEG2000-azide, DSPE-PEG2000-maleimide, DSPE-PEG2000- carboxyl NHS, DSPE-PEG2000-benzylguanine, DSPE-PEG-succinyl, DOPE- PEG2000-azide, DGPE-PEG2000-amine, DOPE-PEG2000-carboxylic acid, and DGPE-PEG2000-HALO-TAG.

42. The targeting lipid nanoparticle of any of claims 34 to 41 wherein nucleic acid is selected from the group of mRNA or siRNA, and is preferably luciferase mRNA or eGFP siRNA.

43. A kit for preparing a targeting lipid nanoparticle for nucleic acid delivery, the kit comprising: an ionizable lipid, a cationic lipid, a helper lipid, a cholesterol, a PEG lipid, and a targeting lipid.

44. The kit of claim 43 wherein the ionizable lipid is selected from the group consisting of 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 3fS-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol), 1 ,2-dipalmitoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-1-aminium (ALC-0315) and combinations thereof, and is preferably DODMA.

45. The kit of claim 43 or 44 wherein the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3-trimethylammonium propane(chloride salt) (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dimethyldioctadecylammonium (bromide salt) (DDAB), and combinations thereof, and is preferably DOTMA.

46. The kit of any of claims 43 to 45, wherein the cholesterol or cholesterol derivative is selected from the group consisting of cholesterol, cholesterol hemisuccinate, cholesterol NHS succinate and combinations thereof.

47. The targeting lipid nanoparticle of any of claims 43 to 46, wherein helper lipid is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and combinations thereof.

48. The kit of any of claims 43 to 47 wherein the PEG lipid is selected from the group consisting of DMG-PEG500, DMG-PEG1000, DMG-PEG2000, PE- PEG500, PE-PEG1000, PE-PEG2000, preferably DMG-PEG2000; wherein PE is selected from the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,3-dipalmitoyl-glycero-2- phosphoethanolamine (DSPE).

49. The kit of any of claims 43 to 48 wherein the targeting lipid is selected from lipid is selected from the group consisting of DSPE-PEG2000-carboxylic acid, DSPE-PEG2000-amine, DSPE-PEG2000-DBCO, DSPE-PEG2000- azide, DSPE-PEG2000-maleimide, DSPE-PEG2000-carboxyl NHS, DSPE- PEG2000-benzylguanine, DSPE-PEG-succinyl, DOPE-PEG2000-azide, DOPE-PEG2000-amine, DOPE-PEG2000-carboxylic acid and DOPE- PEG2000-HALO-TAG.

50. The kit of claim 43 further comprising instructions.

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