Zwitterionic lipid nanoparticle compositions and methods of use thereof

Abstract

A lipid nanoparticle composition comprising: (i) at least one zwitterionic polymer-containing lipid, the lipid moiety of which is covalently attached to a zwitterionic polymer; (ii) at least one non-cationic lipid selected from charged and uncharged lipids, but which is not bound to a polymer; (iii) at least one cationic or ionizable lipid; and (iv) at least one therapeutic agent, and optionally (v) cholesterol or a derivative thereof. Also described herein is a method of delivering a therapeutic agent to a subject, the method comprising administering to a subject the lipid nanoparticle composition described above.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Application No. 63 / 129,343, filed December 22, 2020, which is incorporated by reference in its entirety.

[0002] government support This invention was made with government support under Grant Nos. 2002940 and 2103295 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] The present invention relates generally to zwitterionic lipid nanoparticle (LNP) formulations for the encapsulation and delivery of therapeutic agents, such as nucleic acids, and more specifically to the use of such LNP formulations, particularly non-PEGylated versions thereof, for the delivery of therapeutic agents for treating various diseases or disorders, such as by gene therapy, or for vaccinating a subject. [Background technology]

[0004] In vivo delivery of nucleic acids such as DNA, siRNA and mRNA has the potential to revolutionize vaccination, enzyme replacement therapy and the treatment of genetic diseases. Compared with viral vectors, lipid nanoparticle delivery platforms are considered to have fewer safety issues. Since the first LNP-siRNA drug was approved by the FDA in 2018, more and more lipid nanoparticles have been developed and moved into clinical trials. LNPs typically consist of four components: ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids. Among them, PEG-lipids are commonly used to stabilize and protect the LNP structure.

[0005] Although initially PEG was presumed to be immunologically inert, it is now known that conjugation to lipids or proteins represents a new source of immune responses due to a hapten effect, eliciting anti-PEG antibodies (Abs). In some cases, an increasing number of clinical reports have highlighted that anti-PEG Abs, rather than anti-protein Abs, are the main cause of clinical problems. In a typical example with the PEGylated uricase preparation Pegloticase (Krystexxa), more than 40% of patients with refractory chronic gout who received this drug suffered from high levels of anti-PEG Ab responses, resulting in non-responsiveness to this treatment.

[0006] Anti-PEG antibodies have impaired the efficacy of some commercially available PEG-conjugated proteins. In addition to induced anti-PEG antibodies, pre-existing anti-PEG antibodies are also important for all PEGylated drugs. In clinical trials in patients with acute coronary syndrome, severe allergic reactions occurred after the first dose of pegnivacogin, a PEGylated RNA aptamer. Doxil®, a PEGylated liposomal formulation of doxorubicin, has also been reported to cause immediate hypersensitivity reactions in some patients upon first injection. Thus, there is an unmet need to provide new lipid compositions that can replace problematic components in conventional LNPs and provide biopharmaceuticals with improved functionality. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention is most concerned with novel lipid nanoparticle (LNP) compositions for delivering therapeutic agents such as nucleic acids to subjects.The LNP compositions described herein advantageously have low immunogenicity, long circulation ability, and targeting ability.The LNP compositions are also functionalized with targeting agents for binding to specific cells or cellular components. [Means for solving the problem]

[0008] In a first aspect, the present disclosure relates to an LNP composition comprising at least the following components: (i) at least one zwitterionic polymer-containing lipid, the lipid moiety of which is covalently attached to a zwitterionic polymer; (ii) at least one non-cationic lipid selected from charged lipids and uncharged lipids not bound to a polymer; (iii) at least one cationic or ionizable lipid having a secondary, tertiary, or quaternary amino group; and (iv) at least one therapeutic agent. In a first set of embodiments, the lipid moiety of component (i) is a diacylglycerol (diacylglyceride). In a second set of embodiments, component (i) does not include a polyalkylene oxide (e.g., PEG) segment. In a third set of embodiments, the zwitterionic polymer of component (i) is a betaine polymer, or more specifically, a carboxybetaine polymer. In a fourth set of embodiments, the non-cationic lipid of component (ii) includes a zwitterionic moiety. In a fifth set of embodiments, component (ii) does not include a polyalkylene oxide segment. In a sixth set of embodiments, the non-cationic lipid of component (ii) is a phospholipid, such as a phosphatidylserine (PS) lipid. In a seventh set of embodiments, the cationic or ionizable lipid has a secondary, tertiary or quaternary group. In an eighth set of embodiments, the cationic or ionizable lipid does not include a polyalkylene oxide (e.g., PEG) segment. In a ninth set of embodiments, the lipid nanoparticle composition further comprises: (v) cholesterol or a derivative thereof. In a tenth set of embodiments, the therapeutic agent is a nucleic acid molecule, such as RNA, more specifically mRNA, more specifically viral mRNA, more specifically linear or circular mRNA. Of note, any of the first to tenth embodiments can be combined to result in the LNP composition of the present invention.

[0009] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a subject by administering to the subject any of the lipid nanoparticle compositions described above, including any of the first to tenth embodiments described above. In a first set of embodiments, the lipid nanoparticle composition is delivered to cells of the subject. In a second set of embodiments, the therapeutic agent is a nucleic acid molecule, the administration of which results in gene therapy of the subject. In a third set of embodiments, the therapeutic agent is a nucleic acid molecule, the administration of which results in vaccination of the subject.

[0010] In another aspect, the present disclosure relates to lipid compositions comprising a lipid moiety attached to a secondary, tertiary, or quaternary amine group with a functional group that is negatively charged under physiological conditions. [ka] and During the ceremony, R1 / R2=H or an alkyl group. The alkyl can be saturated or unsaturated, branched or unbranched, containing all carbon and hydrogen or heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F. L is a covalent linker group between N and A. The linker can be all carbon and hydrogen or can contain heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br and F. The structure of L includes, but is not limited to: -CH 2 -, -CH 2 CH(OH)-, -CH 2 CHClCH 2 -, -CH 2 OCH 2 -, -CH 2 SCH 2 -, -CH 2 SSCH 2 -, -CH 2 COOCH 2 As exemplified by - A-(X)n is a functional group that is negatively charged under physiological conditions. The structure may be, but is not limited to: (i) A(X)n is a carboxylic acid group (A=-COOH and n=0) or

[0011] (ii) A(X)n is a phosphate group, where A= [ka] and n=1. X=H or an alkyl group, saturated or unsaturated, branched or unbranched, all carbon or containing heteroatoms, such as, but not limited to, N, O, F, Si, P, S, Cl, Br, F, or

[0012] (iii) A(X)n is a sulfonic acid group, where A= [ka] and n=0,

[0013] (iv) A(X)n is a sulfonamide group, where A= [ka] and n=1, and X=H or an alkyl group that is saturated or unsaturated, branched or unbranched, all carbon and hydrogen, or containing heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F.

[0014] Exemplary structures of these ionizable lipids are: [ka] where n=1 to 10. [Brief description of the drawings]

[0015] [Figure 1] Chemical structure and 1H NMR spectrum of DMG-PCB (or PCB lipid), a zwitterionic polymer-containing lipid of the present invention. [Diagram 2]In vitro characterization of PCB lipid-containing LNPs (or PCB-LNPs). (a) TEM images of representative PCB-LNP formulations. (b) Luciferase expression of different PCB-LNPs in HepG2 cell line. [Diagram 3] In vivo mRNA expression of LNPs containing PCB lipids (or PCB-LNPs). Images were analyzed with Aura Image Software. All PCB-LNPs showed comparable transfection efficiency in the liver to their PEG counterparts. [Figure 4] Synthesis of ZW-A-CBn (n = 1, 2, 3). [Diagram 5] 1H NMR (CDCl3) spectrum of compound ZW-A-CB1. [Figure 6] 1H NMR (CDCl3) spectrum of compound ZW-A-CB2. [Figure 7] 1H NMR (CDCl3) spectrum of compound ZW-A-CB3. [Figure 8] Synthesis of ZW-B-CBn (n = 1, 2, 3). [Figure 9] H NMR spectrum of 3-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)propanoic acid (6). [Figure 10] H NMR spectrum of N-(8-(nonyloxy)-8-oxooctyl)-N-(8-(octadecan-9-yloxy)-8-oxooctyl)glycine (8). [Figure 11] H NMR spectrum of 4-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)butanoic acid (10). [Figure 12] Synthesis of ZW-A-sulfamide-3 [Figure 13] 1H NMR (CDCl3) spectrum of compound ZW-A-sulfamide-3. [Figure 14]A. Formulation of PCB-LNPs with different molar ratios of ZW-B-CB2. B. In vitro transfection was performed in HepG2 cells for group A and group B. Each system was performed in triplicate. [Figure 15] a) Injection scheme carried out in Example 8. b) Composition of the formulations tested in Example 8. c) EPO (erythropoietin) serum concentration analysis from serum taken at the indicated time points after the third injection of the formulations: CB1 (left), CB2 (middle), and MC3 (right). Normalized data is shown here to represent the fold change between the two cohorts. [Figure 16] In vivo luciferase expression of PCB-mLNPs (i.e., PCB-LNPs containing ZW-B-CB2 lipids) (left) and PS5-PCB-mLNPs (i.e., PCB-LNPs containing ZW-B-CB2 and PS lipids) (right) in C57B6 / L mice (0.2 mg / kg). Images were taken 6 hours after intravenous injection. [Figure 17] In vitro expression of Fluc-mRNA delivered by LNP. pH value of aqueous buffer solution: pH=5 (a) or pH=3 (b). Formulation: 50:38.5:10:1.5 (ionizable lipid + zwitterionic ionizable lipid):cholesterol:helper lipid (DOPE or DSPC):PEG-lipid. Horizontal stripes: MC3 50% + zwitterionic lipid 0%; vertical stripes: MC3 25% + ZW-B-CB1 25%; checkerboard: MC3 25% + ZW-B-CB2 25%; grid: MC3 25% + ZW-B-CB3 25%. [Figure 18] In vitro expression of Fluc-mRNA delivered by LNP. pH values ​​of aqueous buffer solutions: pH=5 (left) or pH=3 (right). Typical formulation: 50:38.5:1.5 (ionizable lipid + zwitterionic ionizable lipid):cholesterol:PEG-lipid. Horizontal stripes: MC3 50% + zwitterionic lipid 0%; vertical stripes: MC3 25% + ZW-B-CB1 25%; checkerboard: MC3 25% + ZW-B-CB2 25%; grid: MC3 25% + ZW-B-CB3 25%. [Figure 19]Luciferase expression of PS5-LNPs and PS0-LNPs (PEG formulation) in different cells: (a) HepG2, (b) Raw264.7, (c) primary mouse spleen cells. Experiments were performed in triplicate. (d) IVIS images and bioluminescence signal analysis of organs isolated from mice treated with PS0-LNPs and PS5-LNPs, respectively, carrying mRNA encoding Fluc. Organs shown in the figure are (from top to bottom): lung, superficial cervical lymph node (SCLN, attached to salivary gland), liver, kidney, and spleen. [Figure 20] Structures of some exemplary zwitterionic polymer-containing lipids and non-cationic lipids, with or without PC moieties, in LNP formulations. [Figure 21] Structures of some exemplary lipid compositions in which different types of lipid moieties (e.g., zwitterionic polymer-modified lipids, cationic lipids, non-cationic lipids, and cholesterol or its derivatives) are chemically linked. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In one aspect, the present disclosure relates to lipid nanoparticle (LNP) compositions. Some LNPs in the art are described, for example, in X. Hou et al., Nature Reviews Materials, 6, 1078-1094, 2021, the contents of which are incorporated herein by reference. The term "lipid nanoparticle" refers to a nanoparticle at least partially composed of lipid molecules. As further described below, the lipid molecules include one or more zwitterionic polymer-containing lipids described herein, as well as one or more non-cationic lipids, cationic or ionizable lipids, and / or cholesterol.

[0017] In different embodiments, the LNPs have a size, for example, exactly, approximately, at least, or up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 microns, or within a range bounded by any two of the foregoing values.

[0018] The first component of the LNP is at least one zwitterionic polymer-containing lipid. In the zwitterionic polymer-containing lipid, the lipid moiety is covalently attached (i.e., linked) to the zwitterionic polymer. Zwitterionic polymers are generally derived from zwitterionic monomers and monomers that can be converted to zwitterionic monomers, i.e., precursors of zwitterionic monomers. Zwitterionic monomers are electronically neutral monomers that contain an equal number of positive and negative charges (e.g., one of each). In some embodiments, the zwitterionic polymer comprises multiple repeat units, each repeat unit comprising one positive and one negative charge moiety. The zwitterionic polymer typically comprises at least 2, 5, or 10 or more units, up to 100, 200, 300, 400, 500, or 1000 or less units.

[0019] As used herein, the term "zwitterionic polymer" refers to a polymer made by polymerizing a polymerizable zwitterionic monomer, which provides a zwitterionic polymer having 100 mol % zwitterionic moieties (i.e., each repeat unit of the zwitterionic polymer is a zwitterionic moiety; or refers to a polymer made by copolymerizing a polymerizable zwitterionic monomer and a polymerizable comonomer, which provides a zwitterionic polymer having less than 100 mol % zwitterionic moieties (e.g., when the polymerizable zwitterionic monomer and polymerizable comonomer are present in equal proportions in the polymerization mixture, the product is a zwitterionic polymer having 50 mol % zwitterionic moieties).

[0020] The term "zwitterionic polymer" also refers to a polymer having substantially equal numbers of negative (anionic) and positive (cationic) charges produced by copolymerizing polymerizable negatively charged monomers and polymerizable positively charged monomers, each present in substantially equal proportions in the polymerization mixture. The product of such a copolymerization is a zwitterionic polymer having 100 mol % zwitterionic moieties, each zwitterionic moiety being defined as a pair of repeat units, one with a negative charge and one with a positive charge. Such zwitterionic polymers are called mixed charge copolymers. The term "zwitterionic polymer" also refers to a polymer made by copolymerizing a polymerizable negatively charged monomer, a polymerizable positively charged monomer, and a polymerizable comonomer, each present in substantially equal proportions in the polymerization mixture, which provides a zwitterionic polymer having less than 100 mol% zwitterionic moieties (e.g., if a combination of polymerizable negatively charged monomer and polymerizable positively charged monomer and polymerizable comonomer are present in equal proportions in the polymerization mixture (i.e., 50% combination of polymerizable negatively charged monomer and polymerizable positively charged monomer and 50% polymerizable comonomer), the product is a zwitterionic polymer having 50 mol% zwitterionic moieties)."

[0021] The lipid moiety is composed of a polyol moiety (e.g., diol, glycerol, phosphatidylglycerol, phosphatidylethanolamine, or phosphatidylserine) that is esterified with one or two fatty acid molecules to result in a monoacyl or diacyl lipid, where the term "acyl" refers to the RC(=O) group, where R is a straight or branched hydrocarbon (fatty) chain containing at least 8, and typically up to 30, carbon atoms, where the hydrocarbon chain may be saturated or contain one or more carbon-carbon double bonds. The lipid moiety may be, for example, a diacyldiol (e.g., diacylethylene glycol), diacylglycerol (diacylglyceride), diacylphosphatidylglycerol, diacylphosphatidylethanolamine, or diacylphosphatidylserine moiety. The lipid may be any of the lipids described in any one of Examples 1-14 provided herein. The lipid may also be any of the lipids described in WO2011 / 057227, which is incorporated herein by reference. The fatty acyl moiety may be derived from any of the known fatty acids. Some examples of fatty acyl moieties include oleoyl, palmitoyl, lauryl, myristoyl, stearoyl, linoleoyl, and arachidonyl. The zwitterionic polymer is attached to a lipid moiety, such as any of the lipid moieties listed above, typically via a carbon on a polyol. The zwitterionic polymer may include zwitterionic groups in the side chain or backbone (or a combination thereof) of the polymer.

[0022] In one embodiment, the polymer is a homopolymer made from zwitterionic monomers and has the formula: [ka] where B is a polymer backbone, such as a polyester, polyether, polyurethane, polyamide, or polyhydrocarbon (e.g., polyethylene or polypropylene) backbone, and P is a zwitterionic moiety. In certain embodiments, the backbone (B) has any of the following structures: [ka] where R is selected from the group consisting of hydrogen and substituted or unsubstituted alkyl; E is substituted or unsubstituted alkylene, -(CH 2 ) p C(O)O-, and -(CH 2 ) p C(O)NR 2 -; p is typically an integer from 0 to 12; R 2 is selected from hydrogen and substituted or unsubstituted alkyl; L is a straight or branched chain alkylene group optionally containing one or more oxygen atoms. The subscript x is typically at least 2, 5, or 10 or more and not more than 100, 200, 300, 400, 500, or 1000 units.

[0023] In certain embodiments, P has the following structure: [ka] wherein R 3 , R 4 , and R 6 is independently selected from the group consisting of hydrogen and substituted or unsubstituted alkyl groups; R 5 or R 5 is selected from the group consisting of substituted or unsubstituted alkylene groups, phenylene groups, and polyether groups, m is an integer from 1 to 7; and x is an integer from 2 to 500.

[0024] In some embodiments, the zwitterionic polymer is a betaine polymer. In other embodiments, the zwitterionic polymer is a poly(phosphatidylcholine) polymer, a poly(trimethylamine N-oxide) polymer, a poly(zwitterionic phosphatidylserine) polymer, or a glutamic acid-lysine (EK)-containing polypeptide. In some embodiments, the zwitterionic phosphatidylserine comprises one adjacent positively charged moiety to balance the negative charge of phosphoserine. In some embodiments, the zwitterionic phosphatidylserine comprises a compound as described in "De novo design of functional zwitterionic biomimetic material for immunomodulation," Science Advances, May 29, 2020, Vol. 6, No. 22, (DOI: 10.1126 / sciadv.aba0754), which is incorporated herein by reference in its entirety.

[0025] Some examples of betaine polymers include poly(carboxybetaine), poly(sulfobetaine), and poly(phosphobetaine) polymers. Suitable poly(carboxybetaine)s can be prepared from one or more monomers selected from, for example, carboxybetaine acrylates, carboxybetaine acrylamides, carboxybetaine vinyl compounds, carboxybetaine epoxides, and mixtures thereof. In one embodiment, the monomer is carboxybetaine methacrylate. Representative monomers for making carboxybetaine polymers useful in the present invention include carboxybetaine methacrylates, such as 2-carboxy-N,N-dimethyl-N-(2'-methacryloyloxyethyl)ethanaminium inner salts; carboxybetaine acrylates; carboxybetaine acrylamides; carboxybetaine vinyl compounds; carboxybetaine epoxides; and other carboxybetaine compounds with hydroxyl, isocyanate, amino, or carboxylic acid groups. In certain embodiments, the polymer is poly(carboxybetaine methacrylate) (poly(CBMA)).

[0026] Zwitterionic polymers can be produced by any suitable polymerization method, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and free radical polymerization. Any suitable radical initiator for polymerizing such monomers can be used, including those known in the art. In some embodiments, to produce zwitterionic polymer-containing lipids or other polymer-containing lipids, zwitterionic or other monomers or precursors thereof are attached to lipids and the monomers are polymerized while attached to lipids. Alternatively, already produced polymers can be attached to lipids by means known in the art.

[0027] In other embodiments, the zwitterionic polymer is a homopolymer having a positive charge on the polymer backbone and pendant carboxylic acid groups and has the formula: [ka] wherein R is selected from the group consisting of hydrogen and substituted or unsubstituted alkyl; 1 and L 2 are independently a straight or branched chain alkylene group optionally containing one or more oxygen atoms; and x is an integer from 2 to 500.

[0028] In other embodiments, the zwitterionic polymer is a mixed-charge copolymer and has the general formula: [ka] wherein B 1 and B. 2 As mentioned above, X 1 , X 2 and X 3 R is selected from hydrogen and substituted or unsubstituted alkyl; E is substituted or unsubstituted alkylene, -(CH 2 ) p C(O)O-, and -(CH 2 ) p C(O)NR 2 -, p is an integer from 0 to 12; R 2 is selected from hydrogen and substituted or unsubstituted alkyl; L is a straight or branched chain alkylene group optionally containing one or more oxygen atoms; P 1 is a positively charged group; P 2 is a negatively charged group such as a carboxylic acid group; m is an integer from 1 to 500; and n is an integer from 1 to 500. In some embodiments, P 1 is a nitrogen atom in an aromatic ring or NR 5 R 6 where R 5 and R 6 is independently a substituted or unsubstituted alkyl group.

[0029] The positively charged units (P1-containing units) of the zwitterionic polymer can be obtained from monomers having a positively charged pendant group. Representative monomers that can be used to obtain positively charged units in the polymers of the present invention include 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl]trimethylammonium chloride, and N-acetylglucosamine.

[0030] In one embodiment, the negatively charged units of the zwitterionic polymer are derived from 2-carboxyethyl acrylate (CA) and the positively charged units are derived from 2-(dimethylamino)ethyl methacrylate (DM). In another embodiment, the negatively charged units are derived from 2-carboxyethyl acrylate (CA) and the positively charged units are derived from 2-(diethylamino)ethyl methacrylate (DE). In another embodiment, the negatively charged units are derived from 2-carboxyethyl acrylate (CA) and the positively charged units are derived from [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM). In another embodiment, the negatively charged units are derived from 2-carboxyethyl acrylate (CA) and the positively charged units are derived from 2-aminoethyl methacrylate hydrochloride (NH 2 ) is obtained.

[0031] In some embodiments, the zwitterionic polymer does not contain a polyalkylene oxide (polyalkylene glycol) segment, or the zwitterionic polymer, more specifically, does not contain a polyethylene oxide or polypropylene oxide segment. In some embodiments, all of component (i) does not contain a polyalkylene oxide segment, or component (i) does not contain a polyethylene oxide or polypropylene oxide segment, more specifically. In some embodiments, the lipid nanoparticle as a whole does not contain a polyalkylene oxide segment or molecule.

[0032] The term "lipid" refers to organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," including fats and oils, as well as waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. In one embodiment, the lipid comprises a diacylglyceride.

[0033] In one embodiment, the zwitterionic polymer is linked to a "complex lipid". Some examples of such lipids include dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine, 18-1-trans-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In another embodiment, the zwitterionic polymer is linked to a "simple lipid". In other embodiments, the zwitterionic polymer is linked to a "derivative lipid." In some embodiments, the zwitterionic polymer comprises a zwitterionic compound as disclosed in WO2011057225A2, which is incorporated herein by reference.

[0034] The second component of LNP is at least one non-cationic lipid selected from charged lipid and uncharged lipid that is not bound to polymer.The term "non-cationic lipid" as used herein refers to lipid that is not positively charged and cannot be ionized to positively charged state.However, non-cationic lipid can be neutrally charged by containing zwitterion (positive and negative charges in polymer), such as any of the zwitterionic groups and moieties described above.

[0035] In some embodiments, the non-cationic lipid comprises a zwitterionic moiety. The zwitterionic moiety can be any such moiety detailed above. The zwitterionic moiety can be, for example, a phosphobetaine, a phosphatidylcholine, a carboxybetaine, a sulfobetaine, a trimethylamine N-oxide, a glutamic acid-lysine (EK)-containing, or a zwitterionic phosphatidylserine (phosphoserine) moiety, or a combination thereof. In some embodiments, the zwitterionic lipid is a phospholipid, such as a phosphatidylcholine or a phosphatidylserine lipid.

[0036] In other embodiments, the non-cationic lipids are not zwitterionic, but are negatively charged by including a negatively charged group (e.g., phosphoserine). A metal (e.g., alkali) or ammonium counteranion may be ionically and fluidly associated with the negatively charged group.

[0037] In another set of embodiments, the non-cationic lipid is uncharged by not containing a charged group. The uncharged non-cationic lipid may be, for example, a phosphatidylglycerol lipid, a phosphatidylethanolamine lipid, or a sphingolipid. The non-cationic lipid may be, in some embodiments, a simple lipid, such as a fat, oil, and / or wax. The non-cationic lipid may be, in some embodiments, a complex lipid, such as a phospholipid or a glycolipid. The non-cationic lipid may be, in some embodiments, a derived lipid, such as a steroid, a phospholipid, a sphingolipid, and / or a sterol. In some embodiments, the non-cationic lipid is selected from diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, or cerebroside. In certain embodiments, the non-cationic lipid is selected from one or more of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), or phosphatidylinositol (PI). In other particular embodiments, the non-cationic lipid is selected from dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine, 18-1-trans-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE). The non-cationic lipid is not bound to a polymer, and therefore does not contain a polyalkylene oxide (polyalkylene glycol) segment.

[0038] In certain embodiments, the non-cationic lipid comprising ionic moiety is a phospholipid.In one embodiment, the non-cationic lipid comprising ionic moiety is a lipid conjugated with one or more carboxybetaine groups.In one embodiment, the non-cationic lipid comprising ionic moiety is a lipid conjugated with one or more sulfobetaine groups.In one embodiment, the non-cationic lipid comprising ionic moiety is a lipid conjugated with one or more trimethylamine N-oxide groups.

[0039] The third component of the LNP is at least one cationic or ionizable lipid. The cationic or ionizable lipid may or may not include lipids bound to a polymer with cationic groups. In some embodiments, the cationic or ionizable lipid is not bound to a polymer. The term "cationic lipid" as used herein refers to a lipid that is positively charged (typically by having an ammonium group). In a cationic lipid, the positively charged group is not associated with a negative charge in the cationic lipid. Thus, the cationic lipid is not a zwitterionic lipid. The term "ionizable lipid" as used herein refers to a lipid that contains one or more groups that can be ionized to provide a positive charge in the polymer. Ionizable lipids generally have secondary, tertiary, or quaternary amino groups, particularly alkylated amines, more particularly monoalkylamine or dialkylamine groups, any of which can be protonated or alkylated to provide an alkylated ammonium group. The cationic lipid may contain a trialkylamine group, which is necessarily positively charged when bound to a lipid. In certain embodiments, the cationic or ionizable lipid has dimethylamino or trimethylamino (or dimethylammonium or trimethylammonium) group.In certain embodiments, the cationic or ionizable lipid is selected from 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2DMA), and [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (DLinMC3DMA).

[0040] In some embodiments, the ionizable lipid comprises a lipid moiety attached to a secondary, tertiary, or quaternary amine group with a functional group that is negatively charged under physiological conditions. [ka] wherein R1 / R2=H or an alkyl group. The alkyl may be saturated or unsaturated, branched or unbranched, containing all carbon and hydrogen or heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F. L is a covalent linker group between N and A. The linker may be all carbon and hydrogen or may contain heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F. The structure of L includes, but is not limited to: -CH 2 -, -CH 2 CH(OH)-, -CH 2 CHClCH 2 -, -CH 2 OCH 2 -, -CH 2 SCH 2 -, -CH 2 SSCH 2 -, -CH 2 COOCH 2 As exemplified by - A-(X)n is a functional group that is negatively charged under certain pH conditions. Examples of structures include, but are not limited to, the following: (v) A(X)n is a carboxylic acid group (A=-COOH and n=0), or

[0041] (vi) A(X)n is a phosphate group, where A= [ka] and n=1. X=H or an alkyl group, saturated or unsaturated, branched or unbranched, all carbon or containing heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, F, or

[0042] (vii) A(X)n is a sulfonic acid group, where A= [ka] and n=0,

[0043] (viii) A(X)n is a sulfonamide group, where A= [ka] and n=1, and X=H or an alkyl group, including but not limited to, saturated or unsaturated, branched or unbranched, all carbon and hydrogen, or heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F.

[0044] Exemplary structures of these ionizable lipids are: [ka] where n=1 to 10.

[0045] In some embodiments, the cationic or ionizable lipids are free of polyalkylene oxide (polyalkylene glycol) segments, or the cationic or ionizable lipids are more specifically free of polyethylene oxide or polypropylene oxide segments. In some embodiments, the cationic or ionizable lipids are free of polyalkylene oxide segments, or the cationic or ionizable lipids are more specifically free of polyethylene oxide or polypropylene oxide segments. In some embodiments, the lipid nanoparticles are free of polyalkylene oxide segments.

[0046] In some embodiments, the LNP further comprises cholesterol or a derivative thereof, which is considered herein to be an optional additional component of the LNP. In certain embodiments, the cholesterol derivative is a plant sterol, such as β-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol. In certain embodiments, the cholesterol derivative is dihydrocholesterol, entcholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol.

[0036] In some embodiments, the acetylcholinesterol may be acetylcholine, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, or fecosterol, or a salt or ester thereof, such as sodium cholate.

[0047] The LPN further comprises a therapeutic substance incorporated into or encapsulated by the self-assembled shell constructed of one or more of the first, second, third, and / or fourth lipid components described above. The therapeutic substance can be any substance that has therapeutic value to a living organism, particularly a mammal, such as a human or animal subject. The therapeutic substance can be, for example, a negatively charged core molecule. The core molecule can be, for example, a nucleotide, a nucleoside, a nucleic acid base, or a nucleic acid (e.g., DNA or RNA). In certain embodiments, the therapeutic substance comprises one or more such core molecules. In some embodiments, the therapeutic substance comprises RNA, more particularly mRNA, more particularly viral mRNA. In other specific embodiments, the therapeutic substance is a spike protein of a virus, such as a coronavirus, SARS-COV2 (COVID-19), or HIV virus.

[0048] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, including delivery of nucleic acid molecules, ribonucleoproteins (RNPs), and many other therapeutic substances. Examples of nucleic acids include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), antisense oligonucleotides (ASO), short interfering RNA (siRNA), microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interfering complementary RNA (micRNA), polyvalent RNA, circular RNA (circRNA), crispr RNA (crRNA), long non-coding RNA (lncRNA), plasmid DNA, oligo DNA, and complementary DNA (cDNA). In certain embodiments, the therapeutic molecule comprises one or more of DNA, RNA, ssDNA, dsDNA, ssRNA, dsRNA, and hybrids thereof. In other embodiments, the therapeutic molecule comprises one or more of plasmid DNA or linearized DNA. In other embodiments, the therapeutic molecule comprises one or more of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), and / or long non-coding RNA (lncRNA). In other embodiments, the therapeutic molecule comprises an antisense oligonucleotide (ASO). In other embodiments, the therapeutic molecule comprises a Cas nuclease mRNA and / or a guide RNA nucleic acid. The guide RNA nucleic acid may be, for example, a single guide RNA (sgRNA). In any of the foregoing embodiments, the therapeutic molecule comprises a vaccine against SARS-Cov-2, in particular, the vaccine is an mRNA vaccine or the mRNA vaccine corresponds to the spike protein or a portion thereof.

[0049] In any of the foregoing embodiments, the nucleotides may encode a fusion biological moiety comprising a protection domain and a functional domain. In some embodiments, the functional domain is fused to the protection domain directly or via a linker consisting of amino acids. In further particular embodiments, the protection domain may comprise a plurality of additional amino acids independently selected from the group consisting of: a plurality of negatively charged amino acids (e.g., aspartic acid, glutamic acid, and derivatives thereof); a plurality of positively charged amino acids (e.g., lysine, histidine, arginine, and derivatives thereof); and proline, serine, threonine, asparagine, glutamine, glycine, and derivatives thereof, wherein the ratio of the number of positively charged amino acids to the number of negatively charged amino acids is about 1:0.5 to about 1:2. In some embodiments, the protection domain may be selected from other amino acid polymers (e.g., extended recombinant polypeptides (XTEN), proline-alanine-serine, and elastin-like polypeptides). In any of the foregoing embodiments, the protection domain may be selected from natural half-life extending domains (e.g., Fc fragments).

[0050] LNPs may also include lipid components with mixed functionality. In embodiments, any of the lipid components, including but not limited to the zwitterionic polymer-containing lipids, non-cationic lipids, cationic lipids, and cholesterol and / or cholesterol derivatives, may include one or more functionalities of different lipid components. In embodiments, the zwitterionic polymer-containing lipids include the functionality of the zwitterionic polymer-containing lipids and cationic lipids or ionizable lipids. In embodiments, any of the zwitterionic polymer-containing lipids, non-cationic or ionizable lipids, cationic lipids, and cholesterol and / or cholesterol derivatives may also include one or more functionalities of the zwitterionic polymer-containing lipids, non-cationic lipids, cationic lipids, and cholesterol and / or cholesterol derivatives. In embodiments, the lipid components may include the functionality of other lipid components, thereby eliminating the need for additional lipid components with that functionality. In embodiments, the zwitterionic polymer-containing lipids may include the functionality of non-cationic lipids, thereby eliminating or reducing the need for non-cationic lipids. In embodiments, the zwitterionic polymer-containing lipids can include cationic lipid functionality, thereby eliminating or reducing the need for cationic lipids. In embodiments, the zwitterionic polymer-containing lipids can include cholesterol and / or cholesterol derivative functionality, thereby eliminating or reducing the need for cholesterol and / or cholesterol derivatives.

[0051] In embodiments, any lipid component (zwitterionic polymer-modified lipid, cationic lipid, non-cationic lipid, and cholesterol or derivatives) can be chemically bound to any other lipid component. In embodiments, the zwitterionic polymer-modified lipid and cationic lipid are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid and non-cationic lipid are chemically bound to one lipid. In embodiments, the cationic lipid and non-cationic lipid are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid, cationic lipid, and non-cationic lipid are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid and cholesterol or derivatives are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid, cationic lipid, and cholesterol or derivatives are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid, non-cationic lipid, and cholesterol or derivatives are chemically bound to one lipid. In embodiments, the zwitterionic polymer-modified lipid, the non-cationic lipid, the non-cationic lipid and the cholesterol or derivative are chemically bound to one lipid, in other embodiments, each lipid component is separate and distinct and is not combined with other lipid components.

[0052] Any of the components of the lipid nanoparticles described above are chemically modified to include targeting ligands (e.g., Fab or Fc). In certain embodiments, the zwitterionic polymer-containing lipids have targeting ligands, such as peptides (e.g., RGD), lipids (e.g., phosphoserine-containing lipids), proteins (e.g., apolipoprotein E), aptamers (e.g., anti-VEGF aptamers), sugars (e.g., sialic acid), and antibodies (e.g., anti-PD1) or antibody fragments (e.g., Fab or Fc), to deliver LNPs loaded with therapeutic or diagnostic agents or both to target regions within specific organs in the body.

[0053] In another aspect, the present disclosure relates to a method of delivering a therapeutic substance to a subject by administering any one or more of the above LNP compositions to the subject. The subject is typically a mammal, more typically a human subject, but may be other types of mammals, such as pets or livestock, such as dogs, cats, cows, or sheep. In some embodiments, the method of delivering a therapeutic substance results in a method of treating the subject. As a method of treatment, the LNP composition may be administered for the purpose of, for example, protein replacement therapy, cancer immunotherapy, cancer vaccine therapy, infectious disease vaccine, gene editing, autoimmune disease treatment, and / or cancer diagnosis. In certain embodiments, the LNP composition is administered for gene therapy, including CRISPR-Cas gene editing, or for in vitro and in vivo production of extracellular vesicles, or for vaccination against coronavirus (e.g., SARS-CoV-2). In more specific embodiments, the LNPs are used for in vitro and in vivo clustered regularly interspaced palindromic repeats-Cas endonuclease (CRISPR-Cas) gene editing, including, but not limited to, delivering one or more nucleic acids encoding one or more CRISPR-associated proteins, such as, for example, Cas proteins. In some embodiments, the LNPs are administered with a checkpoint inhibitor (e.g., an anti-programmed death-ligand 1 (anti-PD-L1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4)) to treat cancer.

[0054] In some embodiments, the therapeutic method includes targeted delivery of a therapeutic agent to a secondary lymphoid organ (SLO) of a subject, where the subject is administered lipid nanoparticles comprising a phosphoserine-containing lipid and a therapeutic agent. The SLO can be, for example, the spleen and / or lymph nodes. The phosphoserine-containing lipid can be, for example, a naturally occurring PS-lipid such as 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) or L-α-phosphatidylserine (brain). In some embodiments, the targeted delivery results in cancer immunotherapy, autoimmune disease immunotherapy, or gene editing.

[0055] LNP compositions are typically administered in the form of a pharmaceutical composition comprising the LNP. In the pharmaceutical composition, the LNP can be dissolved or suspended in or mixed with a pharma- ceutically acceptable carrier, which may be liquid, semi-solid (e.g., gel or wax) or solid, as is well known in the art. The phrase "pharma-ceutically acceptable" as used herein refers to a compound, material, composition, and / or dosage form that is, within the bounds of sound medical judgment, suitable for administration to a subject. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically safe for the subject. As used herein, any carrier known in the art may be suitable, depending on the mode of administration.

[0056] Some examples of pharmaceutically acceptable liquid carriers include alcohols (e.g., ethanol), glycols (e.g., propylene glycol and polyethylene glycol), polyols (e.g., glycerol), oils (e.g., mineral or vegetable oils), paraffins, and aprotic polar solvents (e.g., dimethylsulfoxide or N-methyl-2-pyrrolidone), which may or may not contain an aqueous component (e.g., at least 10, 20, 30, 40, or 50 vol.% or more of water, up to, or below, these values).Some examples of pharmaceutically acceptable gels include long-chain polyalkylene glycols and their copolymers (e.g., poloxamers), cellulosic and alkylcellulose-based materials (e.g., as described in U.S. Pat. No. 6,432,415), and carbomers. The pharma- ceutically acceptable wax may be or include, for example, carnauba wax, white wax, beeswax, glyceryl monostearate, glyceryl oleate, and / or paraffin.

[0057] In some embodiments, the pharmaceutical composition contains only the LNP and one or more solvents or carriers. In other embodiments, the pharmaceutical composition contains one or more additional components. The additional components can be, for example, a pH buffer, a monosaccharide or polysaccharide (e.g., lactose, glucose, sucrose, trehalose, lactose, or dextran), a preservative, an electrolyte, a surfactant, or an antibacterial agent. Optionally, sweeteners, flavoring agents, or coloring agents can be included. Other suitable excipients are described in standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Edition, Mack Publishing Company, Easton, Pa., 1995.

[0058] The LNP composition can be administered to a subject by any suitable route, typically in the form of a pharmaceutical composition in which the LNPs are mixed with or suspended in a liquid or solid pharma- ceutically acceptable carrier. The LNPs can be administered intravenously, orally, intramuscularly, intradermally, subcutaneously, nasally, or by inhalation. In certain embodiments, the LNPs are administered to a subject by injection. In some embodiments, the LNPs are delivered to cells of a subject. In some embodiments, the LNPs are delivered by removing cells from a subject, administering lipid nanoparticles to the removed cells, and then reintroducing the removed cells to the subject. In some embodiments, the LNPs are directly injected in vivo and delivered to host cells in vivo. In other embodiments, the LNPs are transfected into host cells ex vivo, and the resulting cells are injected in vivo.

[0059] Examples are set forth below for illustrative purposes and to set forth the best currently known modes of the invention, however, the scope of the invention is in no way limited by the examples set forth herein. EXAMPLES

[0060] Example 1. Preparation and properties of zwitterionic polymer-modified lipids (DMG-PCB or PCB lipids) In this example, the preparation and properties of DMG-PCB, a representative zwitterionic polymer-modified lipid of the present invention, are described. The structure and 1 The 1 H NMR spectrum is shown in Figure 1.

[0061] DMG-N-BOC. Triethanolamine (1.6 mL, 9.36 mmol, 1.2 equiv.) was added to a solution of tert-butyl N-(2,3-dihydroxypropyl)carbamate (1.5 g, 7.8 mmol, 1 equiv.) in dry DCM (10 mL) under nitrogen atmosphere and the mixture was cooled to 0° C. in an ice bath. To this solution, stearoyl chloride (2.83 g, 9.36 mmol, 1.2 equiv.) was dissolved in 20 mL of DCM and added dropwise over 30 min. The reaction was stirred at room temperature overnight. The reaction mixture was extracted with saturated NaHCO3 (aqueous [aq]). The organic layer was separated, washed with brine, dried over MgSO4, filtered, and evaporated under vacuum. The residue was further purified by flash chromatography to give DMG-N-BOC (1.42 g, 40%).

[0062] DMG-N. DMG-N-BOC (100 mg) was dissolved in 5 mL of DCM and 5 mL of TFA. The reaction was stirred at room temperature for 3 h. The solvent was then removed in vacuo to give DMG-N as a white powder.

[0063] CTA-DMG lipid. A solution of DMG-N (100 mg, 0.16 mmol), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester (92.34 mg, 0.2 mmol) and TEA (22.3 μL, 0.16 mmol) in DCM (5 mL) was allowed to stir at room temperature overnight. The reaction mixture was concentrated under vacuum and purified by flash chromatography to obtain CTA-DMG lipid.

[0064] DMG-PCB. In a typical reversible addition-fragmentation chain transfer (RAFT) polymerization reaction to obtain a 10 kDa polymer, CTA-DMG lipid (100 mg, 0.142 mmol), and CBAAM-1-tBu (1036.32 mg, 2.84 mmol) 2,2'-azobis(2-methylpropionitrile) (AIBN, 4.66 mg, 0.028 mmol) were dissolved in anhydrous dimethylformamide (DMF, 5 mL) in a 25 mL round-bottom flask equipped with a stir bar. The flask was sealed with a rubber septum and purged with nitrogen for 30 min. The reaction was stirred overnight in a 65 °C oil bath. The resulting polymer was precipitated three times in ethyl ester, centrifuged to collect the pellet, and dried under vacuum overnight to obtain a pale yellow powder. To remove the trithiocarbonate polymer (258.6 mg), the resulting polymer (258.6 mg, 0.05 mmol), EPHP (89.2 mg, 0.5 mmol) and AIBN (3.3 mg, 0.02 mmol) were dissolved in 3 mL of DMF. The solution was purged with nitrogen for 30 min and stirred at 100 °C for 2 h. The product was precipitated in diethyl ether, centrifuged and dried under vacuum to obtain a white solid powder. The final product DMG-PCB was obtained by deprotecting the tert-butyl groups with trifluoroacetic acid (TFA, 5 mL per 100 mg of polymer) at room temperature for 4 h, followed by precipitation in ethyl ether and centrifugation three times. The pellet was dried under vacuum overnight, dissolved in RNase-free water and dialyzed for 2 days. The molecular weight (approximately 12 kDa) was 1 Determined from H NMR (DO).

[0065] Example 2. Preparation and in vitro characterization of LNPs containing zwitterionic polymer-modified lipids (DMG-PCB or PCB lipids) In this example, DMG-PEG from two commercially available LNP formulations is replaced with DMG-PCB from Example 1 to form LNPs containing PCB lipids (or PCB-LNPs). The in vitro transfection efficiency of PCB-LNPs is shown in Figure 2b. The morphology of PCB-LNPs was observed by transmission electron microscopy (TEM), as shown in Figure 2a. The preparation of PCB-LNPs containing mRNA therapeutics is described below.

[0066] DLin-MC3-DMA (MC3) was purchased from Organixinc Inc. 1,1'-((2-(4-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200) was purchased from Cordenpharma Inc. Other components include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids, USA), cholesterol (Sigma, USA), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K, Avanti Polar Lipids, USA). DMG-PCBs containing different polymer molecular weights (4 kDa or 7 kDa) were synthesized from Example 1.

[0067] Inclusion of mRNA into PCB-LNPs was produced by mixing the lipid components with mRNA in a microfluidic mixer NanoAssemblr Ignite (Precision NanoSystems Inc.). Two different ionizable lipid-based LNP formulations were investigated. For MC3-based LNPs, the lipid mixture molar ratio was MC3:DSPC:cholesterol:X = 50:40:38.5:1.5, where X is DMG-PEG2K for MC3-PEG, DMG-PCB4K for MC3-PCB4, and DMG-PCB7K for MC3-PCB7. For C12-200-based LNPs, the lipid mixture molar ratio was C12-200:DOPE:cholesterol:Y = 35:16:46.5:2.5, where Y is DMG-PEG2K for C12-PEG and DMG-PCB4K for C12-PCB. Briefly, the four lipid components were dissolved in ethanol, and separately, mRNA encoding firefly luciferase (Fluc) was dissolved in 50 mM citrate buffer (pH = 3). The aqueous and ethanol phases were then mixed in a microfluidic device at a flow rate ratio of 3:1. To remove residual organic solvents, the resulting LNPs were washed with PBS through a 100 kDa centrifugal filter (MilliporeSigma, USA).

[0068] LNPs were transfected into HepG2 (ATCC no. HB-8065) ​​and luciferase expression was analyzed 6 h after transfection. Briefly, cells were seeded at 60–70% confluence and incubated at 37 °C and 5% CO2 in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin (Pen-Strep). During transfection, RNA was added per well at the doses shown in Figure 2b. Six hours after transfection, the culture medium containing LNPs was carefully removed and cells were gently rinsed once with PBS. Luciferase expression was measured using a luciferase assay (Promega Cat#: E1501) according to the manufacturer's protocol. All PCB-LNPs showed comparable in vitro transfection efficiency to the corresponding PEG-LNPs, indicating that DMG-PCBs can be used in LNPs to deliver mRNA into cells.

[0069] Example 3. In vivo delivery of LNPs containing zwitterionic polymer-modified lipids (DMG-PCB or PCB lipids) The PCB-LNP formulation from Example 2 was delivered to mice to test the biodistribution of protein expression following systemic delivery.

[0070] Briefly, 6-8 week-old C57BL / 6 mice were administered LNPs carrying Fluc-mRNA via retro-orbital injection at a dosage of 0.4 mg / kg. Bioluminescence imaging was performed 6 h after transfection. Mice were injected with 150 mg / kg of D-luciferin diluted in PBS. All images were analyzed using Aura Image Software. As shown in Figure 3, all PCB-LNPs promoted high mRNA expression in the liver. Efficient in vivo expression of mRNA was corroborated in two different LNP systems: one with MC3 as the ionizable lipid; the other with C12-200 as the ionizable lipid. Thus, the incorporation of this superhydrophilic polymer-conjugated lipid into LNPs resulted in efficient in vivo transfection, demonstrating the feasibility of DMG-PCB as an alternative to DMG-PEG.

[0071] Example 4. Synthesis and characterization of ZW-A-CBn (n=1, 2, 3). In this example, ZW-A-CBn (n=1, 2, 3) was synthesized, 1 The synthesis of ZW-A-CBn- is shown in Figure 4 and described below.

[0072] Compound S1 was synthesized according to the conditions of Jayaraman and Hope (Angew. Chem. Int. Ed. 2012, 51, 8529-8533). Mg turnings were freshly activated with 0.1 M HCl, washed with water and acetone, and dried under high vacuum. Mg (680 mg) was added to a three-necked round-bottom flask equipped with a magnetic stirrer, an addition funnel, and a reflux condenser. The flask was degassed and filled with 2.3 mL of anhydrous ether. Linoleyl bromide (7.50 g) was dissolved in 14 mL of ether, and then 2 mL of the ether solution was added to the flask at room temperature in one go. The reaction mixture was stirred for 2 min, after which a small crystal of iodine was added. The dark brown color due to iodine disappeared rapidly and the reaction mixture began to reflux. The remainder of the linoleyl solution in ether was added dropwise over 5–10 min while maintaining the reaction under gentle reflux. After the addition, the reaction mixture was kept in a water bath at 35° C. for 3 h and then cooled in an ice bath. Ethyl formate (0.8 mL, 0.734 g) was added dropwise at 0° C., and the reaction was then allowed to warm to room temperature. After 6 h, NH 4 The reaction was quenched by adding Cl solution. The solution was extracted with ether (x3) and 2 SO 4 The crude mixture was dried over 10 mL of ethyl acetate and concentrated to give a yellow oil. The major by-product was determined to be the formate ester of S1, so the crude mixture was dissolved in 10 mL of a saturated solution of KOH in ethanol and stirred overnight to hydrolyze the by-product. The reaction mixture was then diluted with DCM and diluted with NH 4 Wash with Cl, concentrate, and add Na 2 SO 4 and purified by flash chromatography on silica gel (1% to 15% ethyl acetate in hexanes) to give the desired product as a pale yellow oil. Yield: 3.56 g.

[0073] Compound S2: In a two-neck round-bottom flask under nitrogen was added S1 (2.00 g), 4-[(tert-butoxycarbonyl)(methyl)amino]butanoic acid (0.87 g), DMAP (0.045 g), and 25 mL of DCM. The solution was cooled to 0° C., and then a 5 mL solution of DCC in DCM was added dropwise. The solution was filtered, concentrated, and purified by silica gel flash chromatography using 0-10% ethyl acetate in hexanes. 2.658 g of a colorless oil was obtained as product.

[0074] Compound S3: To a 50 mL round bottom flask was added S2 (1.758 g) and 5 mL of a 50% v / v solution of TFA in DCM. The reaction was stirred at room temperature for 1.5 h and then concentrated. The crude was dissolved in DCM and added Na 2 CO 3 Wash with solution of Na 2 SO 4 The yellow oil was purified by flash chromatography on silica gel (2-15% MeOH in DCM) to give 1.02 g of a slightly yellow oil.

[0075] Compound ZW-A-CB1: In a 2 mL autosampler vial, compound S3 (114 mg), tert-butyl bromoacetate (35.4 mg), K 2 CO 3 (80 mg), KI (4.3 mg), and a mixture of anhydrous acetonitrile (0.3 mL) and THF (0.1 mL) were added. The mixture was stirred at room temperature for 15 h, then diluted with DCM and filtered. The filtrate was concentrated and purified by silica gel flash column chromatography (5% to 15% MeCN in DCM) to give compound 1' as a colorless oil. Yield: 61 mg. Compound 1' (61 mg) was dissolved in a 50 v / v% solution of TFA in DCM and stirred at room temperature for 2 h. The reaction mixture was concentrated and then purified via silica gel flash chromatography using 5 to 20% MeOH in DCM to give compound ZW-A-CB1 as a slightly yellow oil. Yield: 53 mg. 1 H NMR (CDCl3) is shown in Figure 5.

[0076] Compound ZW-A-CB2: A 2 mL autosampler vial was charged with compound S3 (102 mg), acrylic acid (38.4 mg), N,N-diisopropylethylamine (2.4 mg) and 0.3 mL of ethanol. The mixture was stirred at 37 °C for 30 h, then diluted with DCM and washed with water and brine. The extract was purified by HPLC with NaCl. 2 SO 4 The mixture was dried over 100 ml, concentrated and purified by silica gel flash column chromatography (1% to 20% MeOH in DCM / ethyl acetate 1:1) to give compound ZW-A-CB2 as a pale yellow oil. Yield: 86 mg. 1 H NMR (CDCl3) is shown in Figure 6.

[0077] Compound ZW-A-CB3: In a 2 mL autosampler vial, compound S3 (99.4 mg), t-butyl 4-bromobutyrate (46.8 mg), K 2 CO 3 (89.6 mg), KI (8 mg), and a mixture of anhydrous acetonitrile (0.3 mL) and THF (0.1 mL) were added. The mixture was stirred at 40° C. for 18 h, then diluted with DCM and filtered. The filtrate was concentrated and purified by silica gel flash column chromatography (1%-15% MeCN in DCM) to give compound 3' as a colorless oil. Yield: 95 mg. Compound 3' (90 mg) was dissolved in a 50 v / v% solution of TFA in DCM and stirred at room temperature for 3 h. The reaction mixture was concentrated and purified by silica gel flash chromatography using 1-15% MeOH in DCM to give compound ZW-A-CB3 as a gummy liquid. Yield: 54 mg. 1 H NMR (CDCl3) is shown in Figure 7.

[0078] Commercially available reagents were used without further purification unless otherwise noted. All reactions were carried out at room temperature unless otherwise noted. 2or under argon atmosphere. Column chromatography was performed on a Biotage Isolera system using SiliCycle SiliaSep HP flash cartridges. NMR spectra were recorded on Bruker or Varian 400 or 500 MHz spectrometers. All 1 1 H NMR experiments are reported in δ units, parts per million (ppm), measured relative to the signal of residual chloroform (7.26 ppm) in deuterated solvents.

[0079] Example 5 Synthesis and Characterization of ZW-B-CBn (n = 1-3) In this example, ZW-B-CBn (n=1, 2, 3) was synthesized, 1 The synthesis method of ZW-B-CBn is shown in Figure 8 and described below.

[0080] Nonan-2-yl 8-bromooctanoate (1). A 100 mL round-bottom flask was charged with 8-bromoacetic acid (2.00 g), nonan-1-ol (2.59 g), and 20 mL of dichloromethane. The flask was heated with N 2 The mixture was purged with N and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.25 g) and 4-dimethylaminopyridine (0.22 g) in 20 mL of dichloromethane were added dropwise. 2 The mixture was stirred under reduced pressure for 18 hours. Water was added and the crude product was extracted with dichloromethane, washed with brine and dried over sodium sulfate. The product was purified by column chromatography on silica gel using dichloromethane / hexane as eluent. The yield was 2.36 g (75%).

[0081] Heptadecan-9-yl 8-((tert-butoxycarbonyl)amino)octanoate (2). A 100 mL round-bottom flask was charged with 8-((tert-butoxycarbonyl)amino)octanoic acid (1.04 g) and heptadecan-9-ol (1.13 g) along with 20 mL of dichloromethane. The flask was filled with N 2The mixture was purged with N and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.01 g) and 4-dimethylaminopyridine (0.05 g) in 20 mL of dichloromethane were added dropwise. 2 The mixture was stirred under reduced pressure for 20 hours. Water was added and the crude product was extracted with dichloromethane, washed with brine and dried over sodium sulfate. The product was purified by column chromatography on silica gel using ethyl acetate / hexane as eluent. The yield was 1.42 g (72%).

[0082] Heptadecan-9-yl 8-aminooctanoate (3). To a 20 mL vial was added heptadecan-9-yl 8-((tert-butoxycarbonyl)amino)octanoate (0.13 g), 2 mL dichloromethane and 2 mL trifluoroacetic acid. The mixture was stirred for 3 h. After removing the volatiles in vacuo, the crude product was dissolved in ethyl acetate, washed with 1N NaOH and brine, and dried over sodium sulfate. The yield was 93.2 mg (93%).

[0083] Nonyl 8-((8-(octadecan-9-yloxy)-8-oxooctyl)amino)octanoate (4). A flame-dried 50 mL Schlenk reactor was charged with activated 4 Å molecular sieves (500 mg), cesium hydroxide monohydrate (188 mg), and 6 mL of anhydrous dimethylformamide. The reaction was stirred for 2 hours at 37° C. for 1 hour at 37° C. for 2 hours at 37° C. 2 The mixture was stirred under N2 for 10 min. Heptadecan-9-yl 8-aminooctanoate (500 mg) was added and the mixture was stirred under N2 for an additional 30 min. Nonan-2-yl 8-bromooctanoate (483 mg) was then added dropwise to the suspension and the reaction was cooled to 10° C. 2 The mixture was stirred under reduced pressure for 24 hours. The mixture was filtered, poured into 1N NaOH, extracted with dichloromethane, washed with brine, and dried over sodium sulfate. The product was purified by column chromatography on silica gel using methanol / chloroform as eluent. The yield was 705 mg (84%).

[0084] Heptadecan-9-yl 8-((3-(tert-butoxy)-3-oxopropyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octanoate (5). To a 1.5 mL vial was added nonyl 8-((8-(octadecan-9-yloxy)-8-oxooctyl)amino)octanoate (50 mg) and t-butyl acrylate (10.6 mg) in 0.5 mL of methanol. The reaction was stirred for 2 h at 37° C. for 1 h at 37° C. for 1 h at 37° C. 2 The mixture was stirred under reduced pressure for 20 h. The volatiles were removed in vacuo. The yield was 58 mg (98%).

[0085] 3-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)propanoic acid (6). To a 1.5 mL vial was added heptadecan-9-yl 8-((3-(tert-butoxy)-3-oxopropyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octanoate (50 mg), 0.5 mL dichloromethane and 0.5 mL trifluoroacetic acid. The mixture was stirred for 3 h. The volatiles were removed in vacuo and the product was purified by column chromatography on silica gel using methanol / dichloromethane as eluent. The yield was 45 mg (94%). 3-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)propanoic acid (6) 1 The H NMR spectrum is shown in Figure 9. This compound is designated ZW-B-CB2.

[0086] Heptadecan-9-yl 8-((2-(tert-butoxy)-2-oxoethyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octanoate (7). To a flame-dried 1.5 mL vial was added nonyl 8-((8-(octadecan-9-yloxy)-8-oxooctyl)amino)octanoate (100 mg), t-butyl bromoacetate (117 mg), N,N-diisopropylethylamine (97 mg) and 1 mL of anhydrous dimethylformamide. The reaction was diluted with N 2The mixture was stirred under 50° C. for 24 hours. The mixture was poured into 1N NaOH, extracted with dichloromethane, washed with brine and dried over sodium sulfate. The product was purified by column chromatography on silica gel using methanol / dichloromethane / ammonia as eluent. The yield was 92 mg (79%).

[0087] N-(8-(nonyloxy)-8-oxooctyl)-N-(8-(octadecan-9-yloxy)-8-oxooctyl)glycine (8). To a 1.5 mL vial was added heptadecan-9-yl 8-((2-(tert-butoxy)-2-oxoethyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octannoate (50 mg), 0.5 mL dichloromethane and 0.5 mL trifluoroacetic acid. The mixture was stirred for 3 h. The volatiles were removed in vacuo and the product was purified by column chromatography on silica gel using methanol / dichloromethane as eluent. The yield was 42 mg (88%). The H NMR spectrum of N-(8-(nonyloxy)-8-oxooctyl)-N-(8-(octadecan-9-yloxy)-8-oxooctyl)glycine (8) is shown in FIG. 10. This compound is designated ZW-B-CB1.

[0088] Heptadecan-9-yl 8-((4-(tert-butoxy)-4-oxobutyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octanoate (9). To a flame-dried 1.5 mL vial was added nonyl 8-((8-(octadecan-9-yloxy)-8-oxooctyl)amino)octanoate (100 mg), t-butyl 4-bromobutyrate (134 mg), N,N-diisopropylethylamine (97 mg) and 1 mL of anhydrous dimethylformamide. The reaction was diluted with N 2 The mixture was stirred under 50° C. for 24 hours. The mixture was poured into 1N NaOH, extracted with dichloromethane, washed with brine and dried over sodium sulfate. The product was purified by column chromatography on silica gel using methanol / dichloromethane / ammonia as eluent. The yield was 98 mg (81%).

[0089] 4-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)butanoic acid (10). To a 1.5 mL vial was added heptadecan-9-yl 8-((4-(tert-butoxy)-4-oxobutyl)(8-(nonan-2-yloxy)-8-oxooctyl)amino)octanoate (50 mg), 0.5 mL dichloromethane and 0.5 mL trifluoroacetic acid. The mixture was stirred for 3 h. The volatiles were removed in vacuo and the product was purified by column chromatography on silica gel using methanol / dichloromethane as eluent. The yield was 44 mg (93%). 4-((8-(nonyloxy)-8-oxooctyl)(8-(octadecan-9-yloxy)-8-oxooctyl)amino)butanoic acid (10) 1 The H NMR spectrum is shown in Figure 11. This compound is designated ZW-B-CB3.

[0090] Example 6. Synthesis and characterization of ZW-A-sulfoamide-3 In this example, ZW-A-sulfamide-3 was synthesized. 1 The synthesis of ZW-A-sulfamide-3 is shown in Figure 12 and described below.

[0091] S4: To a 50 mL round bottom flask was added compound S1 (600 mg), bromoacetic acid (178 mg), DMAP (6 mg) and 5 mL of DCM. The mixture was cooled in an ice bath and 5 mL of DCC (178 mg) solution was added dropwise. The reaction was allowed to warm to room temperature and stirred for 12 h before being filtered, concentrated and purified by silica gel flash chromatography (5% to 30% DCM in hexanes). Compound S4 was obtained as a colorless oil. Yield: 0.59 g.

[0092] S5: In a 25 mL round-bottom flask, add 4-hydroxybenzenesulfonamide (60.8 mg), K 2 CO 3(43.4 mg), and 0.5 mL of DMF were added. The mixture was stirred for 5 min, then 1 mL of a solution of S4 (121 mg) in DMF was added. The reaction mixture was stirred at 35° C. for 20 h or more, then diluted with 5 mL of DCM and filtered. The filtrate was concentrated and purified by silica gel flash chromatography (10% to 40% ethyl acetate in hexane) to give S5 as a colorless oil. Yield: 102 mg.

[0093] ZW-A-Sulfamide-3: To a 20 mL vial was added S5 (100 mg), 3-carboxy-N,N,N-trimethylpropan-1-aminium chloride (48 mg), N'-ethylcarbodiimide hydrochloride (75 mg), DMAP (32 mg), triethylamine (55 μL) and 2 mL of DMF. The reaction mixture was stirred at room temperature overnight and then purified by silica gel flash chromatography (5% to 50% MeOH in DCM) to give the title compound as a pale yellow solid. Yield: 55 mg. The 1H NMR (CDCl3) of compound ZW-A-Sulfamide-3 is shown in Figure 13.

[0094] Example 7. Preparation and in vitro characterization of ZW-B-CB2-containing PCB-LNPs (non-PEG formulation) In this example, ZW-B-CB2 synthesized from Example 5 was incorporated into PCB-LNP described in Example 2 at different ratios to replace MC3. Other components included DSPC, cholesterol and DMG-PCB4K. The molar ratio of each component is shown in Figure 14.

[0095] Lipids were dissolved in ethanol and mRNA was dissolved in 50 mM citrate buffer (pH 3). Inclusion of mRNA into LNPs was prepared by mixing the two phases in a ratio of 1:3 (ethanol:water, v / v%) under vigorous stirring. The formulation was dialyzed against PBS (pH 7.4) in a dialysis cassette for at least 10 hours, concentrated by passing through a 0.22 μm filter, and stored at 4°C until use.

[0096] To evaluate the transfection efficiency of the formulations, in vitro transfection of LNPs encapsulating mRNA encoding Fluc was performed. Briefly, LNPs were transfected into HepG2 (ATCC number HB-8065) ​​and luciferase expression was analyzed 6 hours after transfection. Cell culture conditions were described in Example 2. During transfection, RNA was added in triplicate at 100 ng per well. Six hours after transfection, the culture medium containing LNPs was carefully removed and the cells were gently rinsed once with PBS. Luciferase expression was measured using a luciferase assay (Promega Cat#: E1501) according to the manufacturer's protocol.

[0097] Example 8. Multiple systemic delivery of ZW-B-CBn (n=1-3)-containing PCB-LNPs (non-PEG formulation) in vivo Accelerated blood clearance (ABC) effect has been reported when LNPs are injected multiple times and rapid clearance of LNPs is observed with subsequent injections. The ABC effect is largely caused by antibodies generated against the lipid components of LNPs, which have been reported to be immunogenic. In this example, ZW-B-CBn-containing PCB-LNPs from Example 7 were used to test the ABC effect of LNPs. MC3-based PEG-LNPs from Example 2 were used as a control. The composition of each formulation is shown in Figure 15b.

[0098] For in vivo pharmacokinetic (PK) studies, 3-4 week old male C57BL / 6J mice were divided into three groups and intravenously administered mRNA-LNPs at a dose of 5 μg of mRNA. Two different injection cohorts were performed. (Figure 15a) In cohort 1, only one dose of hEPO-mRNA (human erythropoietin mRNA) was administered. In cohort 2, Fluc-mRNA (false luciferase mRNA) was administered for the first two injections and hEPO-mRNA was administered for the last injection. (Figure 15a) Cohort 2 was designed to eliminate the anti-hEPO immune response induced by the first two injections, whereby the PK profile of hEPO in the third injection shows an ABC effect mainly due to the LNP component. To determine the PK profile of hEPO, mouse serum was collected 6 h and 24 h after injection and analyzed using ELISA (DuoSet, hEPO ELISA kit, R&D).

[0099] As shown in Figure 15c, LNPs containing PCB and ZW-B-CB1 / ZW-B-CB2 showed less ABC effect compared to the MC3 formulation in injection cohort 2. This result indicates that PCB-LNPs with ZW-B-CBn induce a reduced ABC effect and are therefore less immunogenic.

[0100] Example 9. In vivo systemic delivery of PCB-LNPs (non-PEG formulation) containing ZW-B-CB2 and targeting phosphoserine (PS) lipids In this example, a negatively charged phospholipid, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), was incorporated into the PCB-LNP formulation described in Example 7. Briefly, the molar ratio of MC3:ZW-B-CB2:DSPC:cholesterol:DMG-PCB4k:DOPS is equal to 20:30:10:38.5:1.5:5. The formulation containing DOPS is named PS-PCB-mLNP (i.e., PCB-LNP containing ZW-B-CB2 and PS lipids), and the one without DOPS is named PCB-mLNP (i.e., PCB-LNP containing ZW-B-CB2 lipids) as a control. The lipid mixture and the aqueous mRNA solution were rapidly mixed in a microfluidic channel as described in Example 2, followed by washing with PBS to produce the final product. DOPS was used as a non-cationic lipid for targeting to secondary lymphoid organs (SLOs).

[0101] To test protein expression in vivo, PS-PCB-mLNPs with mRNA encoding Fluc were delivered to mice by intravenous injection. Bioluminescence images were taken 6 h after administration using IVIS as described above. As shown in Figure 16, the IVIS images indicate that mRNA expression shifted from the liver toward the spleen and superficial lymph nodes. In the control group, in PCB-mLNPs without the targeting lipid DOPS, Fluc expression mostly occurred in the liver. Thus, incorporation of DOPS allowed more protein expression in the spleen and lymph nodes, indicating a targeting effect to these SLOs. This result indicates that PS-PCB-mLNPs are a promising platform for in vivo mRNA delivery to SLOs for applications including immunotherapy and vaccines.

[0102] Example 10. Preparation and in vitro characterization of ZW-B-CBn (n = 1 to 3)-containing PEG LNPs (PEG formulations) The ionizable lipid, which is the main component of LNPs (or designated as PEG LNPs to distinguish them from PCB LNPs in which the PEG lipids are replaced by PCB lipids), has been reported to be immunogenic. To reduce immunogenicity, the ionizable lipids were partially replaced with ZW-B-CBn obtained in Example 6 to create a formulation. Briefly, three conditions were tested in which different helper lipids were varied: 1) DSPC, 2) DOPE, and 3) no helper lipid. In conditions 1 and 2, the LNPs were composed of the following lipid: MC3+ZW-B-CBn, DSPC or DOPE, cholesterol, and DMG-PEG2k in a molar ratio of 50:10:38.5:1.5. In condition 3, the LNPs were composed of the following lipid: MC3+ZW-B-CBn, cholesterol, and DMG-PEG2k in a molar ratio of 50:38.5:1.5. The results of in vitro transfection are shown in Figures 17 and 18. The production and in vitro transfection of LNPs is described below.

[0103] Lipids were dissolved in ethanol at the indicated molar ratios, and mRNA was dissolved in 20 mM sodium acetate buffer (pH 5) or 20 mM citrate buffer (pH 3). To generate LNPs, the ethanol and aqueous solutions were rapidly mixed in a ratio of 1:3 (ethanol:aqueous) with vigorous stirring. The formulations were dialyzed against PBS (pH 7.4) in dialysis cassettes for at least 10 h, concentrated through a 0.22 μm filter, and stored at 4 °C until use. All formulations were tested for particle size, and RNA encapsulation.

[0104] To evaluate the transfection efficiency of the formulation, in vitro transfection of LNPs encapsulating mRNA encoding Fluc was performed. Briefly, LNPs were transfected into HepG2 (ATCC number HB-8065) ​​and luciferase expression was analyzed 6 hours after transfection. Cell culture conditions were described in Example 2. At the time of transfection, RNA was added in triplicate at 100 ng per well. Six hours after transfection, the culture medium containing LNPs was carefully removed and the cells were gently rinsed once with PBS. Luciferase expression was measured using a luciferase assay (Promega Cat#: E1501) according to the manufacturer's protocol.

[0105] Example 11. Preparation and in vitro characterization of PEG LNPs (PEG formulations) containing PS lipids as targeting lipids In this example, a negatively charged phospholipid, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), was incorporated into the MC3-based formulation to generate PS5-LNPs. Briefly, PS5-LNPs were fabricated using the microfluidics described in Example 2, which contained MC3, DSPC, cholesterol, DMG-PEG2k, and DOPS in a molar ratio of 50:10:38.5:1.5:5. DOPS-free LNPs were used as controls (PS0-LNPs).

[0106] Both PS5-LNPs and PS0-LNPs were encapsulated with mRNA encoding firefly luciferase (Fluc) and transfected into HepG2 (ATCC number HB-8065) ​​and Raw264.7 (ATCC number TIB-71) to evaluate the transfection efficiency in vitro. HepG2 cells were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin (Pen-Strep) at 37°C and 5% CO. 2Similarly, Raw264.7 was maintained under the same culture conditions in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1x Pen-Strep. To transfect cells with LNPs, different amounts of RNA (indicated by plot) were added to cells seeded in 96-well plates at 60–70% confluence. Six hours after transfection, the culture medium containing PS-LNPs was carefully removed, and cells were gently rinsed once with PBS. Cells were lysed by adding 30 μL of Passive Lysis Buffer (Promega Cat#: E1941), and 100 μL of D-luciferin substrate (Promega Cat#: E1501) was added to each well. Immediately after adding the substrate, luminescence intensity was measured in white opaque plates using a Cytation5 microplate reader (Biotek, USA). As shown in Figure 19, PS5-LNPs have transfection efficiency in both the hepatic cell line HepG2 (Figure 19a) and the monocyte / macrophage cell line RAW264.7 (Figure 19b). Furthermore, compared with PS0-LNPs, PS5-LNPs show higher mRNA expression levels in monocytes / macrophages but much lower in hepatocytes. In addition, PS5-LNPs also showed high transfection efficiency in primary mouse splenocytes (Figure 19c). The difference in protein expression in these cells indicates that PS5-LNPs may selectively deliver mRNA to immune cells but not hepatocytes.

[0107] The findings from the in vitro studies were also supported in vivo. Both PS5-LNPs and PS0-LNPs were loaded with mRNA encoding firefly luciferase (Fluc) and delivered to C57BL / 6 mice by retro-orbital injection (0.2 mg / kg). Six hours after administration, mice were euthanized and various organs were isolated to analyze mRNA expression levels. PS5-LNPs showed 40% of the total bioluminescence signal in the cervical lymph nodes and spleen. In contrast, control group PS0-LNPs showed over 90% of the bioluminescence signal in the liver, but almost none in the lymph nodes and spleen (Figure 19d). This result clearly concludes the biodistribution shift toward SLO after incorporation of DOPS. It is noteworthy that while many other reports have shown that negatively charged lipids reduce the transfection efficiency of particles, the addition of PS targets lymph nodes and spleen without sacrificing overall transfection efficiency.

[0108] Example 12 - Prophetic Example. Fabrication and Characterization of Reduced Component PCB-LNPs A library of PCB-LNPs with decreasing amounts of cholesterol is created. As shown in Table 1, the ratio of cholesterol ranges from 0 to 38.5 mol%, with a corresponding range of DMG-PCB from 1.5 to 40 mol%. The amounts of ionizable lipids (50 mol%) and helper lipids (10 mol%) are not varied as described in Example 2.

[0109] [Table 1]

[0110] Each formulation is manufactured according to the same procedure as described in Example 2. Each formulation is characterized for size, zeta potential, and encapsulation efficiency. For efficacy testing, each formulation is transfected in vitro to evaluate transfection efficiency. Specifically, mRNA encoding firefly luciferase (Fluc) is encapsulated in each formulation. HEK293T (ATCC, CRL-11268) are seeded in 96-well plates at 4×104 cells / well in 100 μL of culture medium (DMEM supplemented with 10% FBS) and incubated at 37° C., 5% CO 2 The plates were incubated overnight at 4°C for 6 h. Then, 5 μL of Fluc mRNA-loaded LNPs were added to the medium and incubated for 6 h. Each preparation was in quintuplicate, and PBS was added as a negative control. After transfection, the culture medium was carefully removed and the expression of Fluc was evaluated using a luciferase assay system.

[0111] Furthermore, the best-performing formulations are delivered to mice to evaluate their expression and biodistribution in vivo. Specifically, Fluc mRNA-loaded LNPs (1 mg / kg of mRNA) are injected into 6-7 week-old male C57BL / 6J mice. Six hours after transfection, mice are administered D-luciferin (30 mg / mL in PBS) by intraperitoneal injection. Ten minutes later, mice are sacrificed and eight organs (liver, spleen, kidney, lung, and spleen) are harvested. Organ luminescence is analyzed using an optical imaging system and quantified using appropriate software, measuring the radiance of each organ in photons / second.

[0112] Example 13 - Prophetic Example. Preparation and Characterization of LNPs with and without PC Moieties In this example, to address immunogenicity from the PC portion, a formulation using a molecule that does not contain a PC portion is generated. (Figure 20) In one formulation that does not contain a PC portion, the four components of the LNP include an ionizable lipid, DMG-CB1, cholesterol, and DMG-PCB. DMG-CB1 is generated from Example 1, where CB is bound to DMG-N and replaces the helper lipid DSPC of the previous formulation. In the formulation with a PC portion, the four components of the LNP include an ionizable lipid, DSPC, cholesterol, and DSPE-PCB. DSPE-PCB is generated according to reference (Z. Cao, L. Zhang, and S. Jiang, Superhydrophilic Zwitterionic Polymers Stabilize Liposome, Langmuir, 28, 11625, 2012).

[0113] Each formulation is manufactured according to the same procedure as described in Example 2. Each formulation is characterized for size, zeta potential, and encapsulation efficiency. For efficacy testing, each formulation is transfected in vitro to evaluate the transfection efficiency. Specifically, mRNA encoding firefly luciferase (Fluc) is encapsulated in each formulation. HEK293T (ATCC, CRL-11268) are seeded in 96-well plates at 2×104 cells / well in 100 μL of culture medium (DMEM supplemented with 10% FBS) and incubated at 37° C., 5% CO 2 The plates were incubated overnight at 4°C for 6 h. Then, 5 μL of Fluc mRNA-loaded LNPs were added to the medium and incubated for 6 h. Each preparation was in quintuplicate, and PBS was added as a negative control. After transfection, the culture medium was carefully removed and the expression of Fluc was evaluated using a luciferase assay system.

[0114] Furthermore, the best formulations are delivered to mice to evaluate their expression and biodistribution in vivo. Specifically, Fluc mRNA-loaded LNPs (1 mg / kg of mRNA) are injected into 6-7 week-old male C57BL / 6J mice. Six hours after transfection, mice are administered D-luciferin (30 mg / mL in PBS) by intraperitoneal injection. Ten minutes later, mice are sacrificed and eight organs (liver, spleen, kidney, lung, and heart) are harvested. Organ luminescence is analyzed using an optical imaging system and quantified using appropriate software, measuring the radiance of each organ in photons / second.

[0115] Example 14 - Prophetic Example. Combination of lipid components As also shown in FIG. 21, any of the lipid components (zwitterionic polymer-modified lipids, cationic lipids, non-cationic lipids, and cholesterol or its derivatives) can be chemically combined with any other components to achieve the compositions and methods described above.

[0116] While what are considered to be preferred embodiments of the present invention have been shown and described, those skilled in the art could make various changes and modifications which remain within the scope of the invention as defined by the appended claims.

Claims

1. 1. A lipid nanoparticle composition comprising: (i) at least one zwitterionic polymer-containing lipid, wherein a lipid moiety is covalently attached to the zwitterionic polymer; (ii) at least one non-cationic lipid selected from charged lipids and uncharged lipids, wherein the non-cationic lipid is not bound to a polymer; (iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and (iv) at least one therapeutic agent A lipid nanoparticle composition comprising:

2. 2. The composition of claim 1, wherein the lipid moiety of component (i) is a diacylglyceride.

3. 2. The composition of claim 1, wherein the cationic or ionizable lipid of component (i) and / or component (ii) and / or component (iii) does not contain a polyalkylene oxide segment.

4. 2. The composition of claim 1, wherein the zwitterionic polymer of component (i) is selected from the group consisting of poly(carboxybetaine) (PCB), poly(sulfobetaine), poly(phosphobetaine), poly(phosphatidylcholine), glutamic acid-lysine (EK)-containing polypeptides, poly(trimethylamine N-oxide) polymers, and poly(zwitterionic phosphatidylserine).

5. 2. The composition of claim 1, wherein the zwitterionic polymer of component (i) is a betaine polymer, optionally wherein the betaine polymer is a poly(carboxybetaine), poly(sulfobetaine) or poly(phosphobetaine) polymer.

6. The non-cationic lipid of component (ii) comprises a zwitterionic moiety, and optionally the zwitterionic moiety is a phosphobetaine, a phosphatidylcholine, a carboxybetaine, 2. The composition of claim 1, wherein the phosphatidylserine moiety is selected from the group consisting of a sulfobetaine, a trimethylamine N-oxide, a glutamic acid-lysine (EK)-containing peptide, or a zwitterionic phosphatidylserine moiety.

7. 2. The composition of claim 1, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine, 18-1-trans-phosphoethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dioleoyl-snglycero-3-phosphoethanolamine (transDOPE).

8. 2. The composition of claim 1, wherein the non-cationic lipid of component (ii) is a phospholipid, and optionally, the phospholipid is a phosphatidylserine lipid.

9. The composition of claim 1, wherein the cationic or ionizable lipid of component (iii) optionally has a secondary, tertiary, or quaternary group, together with a functional group that is negatively charged under physiological conditions.

10. The cationic or ionizable lipid comprises: 【Chemistry 1】 [In the formula, R １ / R ２ =H or an alkyl group, which can be saturated, unsaturated, branched, and / or unbranched, and can further contain one or more heteroatoms, including, but not limited to, N, O, F, Si, P, S, Cl, Br, and F; L comprises a covalent linker group between N and A, said covalent linker group being -CH ２ --, --CH ２ CH(OH)-,-CH ２ CHClCH ２ --, --CH ２ OCH ２ --, --CH ２ SCH ２ --, --CH ２ S.S.C.H. ２ --, --CH ２ COOCH ２ may contain one or more of -, C, H, and may further contain one or more heteroatoms, including but not limited to N, O, F, Si, P, S, Cl, Br, and F; A-(X)n is a functional group that is negatively charged under certain pH conditions and can include: (i) A(X)n is a carboxylic acid group (A=-COOH and n=0), or (ii) A(X)n is a phosphate group, where A= 【Chemistry 2】 and n=1 and X=H or an alkyl group, saturated or unsaturated, branched or unbranched, all carbon or containing heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F; (iii) A(X)n is a sulfonic acid group, where A= 【Chemistry 3】 and n=0, (iv) A(X)n is a sulfonamide group, where A= 【Chemistry 4】 and n=1 and X=H or an alkyl group that is saturated or unsaturated, branched or unbranched, all carbon and hydrogen, or that contains heteroatoms such as, but not limited to, N, O, F, Si, P, S, Cl, Br, and F. The composition of claim 9 comprising:

11. The functional group is: 【Chemistry 5】 is selected from the group consisting of The composition of claim 9, wherein n=1 to 10.

12. The composition of claim 1 , wherein the lipid nanoparticle composition further comprises: (v) cholesterol or a derivative thereof.

13. The composition of claim 1 , wherein the therapeutic agent of component (iv) is a nucleic acid molecule.

14. 14. The composition of claim 13, wherein the nucleic acid molecule is RNA, optionally wherein the RNA is selected from the group consisting of messenger RNA (mRNA) such as viral mRNA, small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), antisense oligonucleotide (ASO), CRISPR-associated RNA, Cas nuclease mRNA, guide RNA, single guide RNA, and combinations thereof.

15. The composition of claim 1 , wherein the therapeutic agent is a viral spike protein.

16. A lipid nanoparticle composition for use as a medicament, said composition being as defined in claims 1 to 15.

17. A lipid composition comprising a lipid moiety bound to a secondary, tertiary, or quaternary amine group with a functional group that is negatively charged under physiological conditions, and optionally comprising a lipid moiety described in claim 10 or 11.

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