Glycan-containing drug delivery systems

Abstract

A lipid nanoparticle (LNP) composition containing: (i) at least one glycan-containing lipid containing a glycan-containing moiety (S) linked to a lipid (L); (ii) at least one non-cationic lipid selected from charged and uncharged lipids; (iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and (iv) at least one therapeutic substance, such as a nucleic acid. Pharmaceutical compositions containing the LNP composition are also described. Methods of using the LNP composition by delivering a therapeutic substance to a subject are also described. Methods for targeted delivery of a therapeutic agent to a secondary lymphoid organ (SLO), brain, bone marrow, or specific cells (e.g., immune cells, precursor cells, or stem cells) of a subject are also described.

Description

GLYCAN-CONTAINING DRUG DELIVERY SYSTEMSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 529,512, filed on July 28, 2023, which is herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to methods of enhanced selective delivery of therapeutic agents, including nuclei acids, such as mRNA, using lipid nanoparticle (LNP) formulations.BACKGROUND

[0003] There are continuing efforts to selectively deliver any of a variety of therapeutic agents (e.g., nucleic acids and spike proteins) to specific targets in living organisms. In vivo delivery of nucleic acids, in particular, such as DNA, siRNA and mRNA, holds the potential to revolutionize vaccination, enzyme replacement therapies, and the treatment of genetic disorders. Compared to conventional protein therapeutics, mRNA therapeutics, in particular, have shown several advantages, including rapid and scalable production. The most prominent example is Modema and Pfizer / BioNTech mRNA vaccines, which have been used worldwide to combat the coronavirus disease 2019 (COVID-19) pandemics. Beyond vaccines against infectious diseases, mRNA therapeutics have shown promising applications in other fields, such as enzyme replacement therapies and gene correction against genetic disorders.

[0004] Successful implementation of mRNA therapeutics has been facilitated by the development of lipid nanoparticles (LNPs) that protect mRNA from degradation / clearance and assists in cellular uptake and mRNA endosome escape. A typical LNP formulation contains ionizable cationic lipids, helper lipids, cholesterol, and polyethylene glycol (PEG)- lipids, forming a particle around lOOnm in diameter. Compared to virus vectors, lipid nanoparticle delivery platforms tend to have less safety issues. However, LNP stability during transport along with selective delivery, particularly to secondary lymphoid organs (SLOs), continues to be a challenge. Thus, there would be a significant benefit in a method that could employ LNPs for the selective delivery of therapeutics while maintaining a highlevel of stability of the LNPs during transport. However, such a method has thus far remained elusive.SUMMARY

[0005] The present disclosure is foremost directed to LNP compositions that can provide an exceptionally selective and stable delivery of a range of therapeutics, such as nucleic acids. The LNP compositions described herein advantageously possess high selectivity, exceptional stability, low immunogenicity, and long circulation capabilities. The LNP compositions achieve this, at least in part, by the presence of at least one glycan-containing lipid containing a glycan-containing moiety (S) linked to a lipid (L) in the composition. In particular embodiments, the therapeutic substance being delivered is a nucleic acid. The nucleic acid may be, for example, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), long-noncoding RNA (IncRNA), antisense oligonucleotide (ASO), CRISPR -related RNA, Cas nuclease mRNA, guide RNA, or single-guide RNA. The LNP compositions can also be functionalized with a targeting agent (e.g., ligand) to bind to specific cells or cellular components to enhance the nucleic acid delivery. In some embodiments, the targeting ligand is or includes a phosphoserine (PS) moiety.

[0006] More specifically, the LNP composition includes: (i) at least one glycan-containing (i.e., saccharide-containing) lipid containing a glycan / saccharide-containing moiety (S) linked to a lipid (L); (ii) at least one non-cationic lipid selected from charged and uncharged lipids; (iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and (iv) at least one therapeutic substance. In some embodiments, the glycan-containing lipid of component (i) has the general structure S-L, wherein S is a glycan-containing moiety selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and L is a lipid, wherein S is covalently bonded to L.

[0007] In another aspect, the present disclosure is directed to methods of delivering a therapeutic substance to a subject by administering to the subject a lipid nanoparticle composition as described above. In embodiments, the lipid nanoparticle composition is delivered to cells of the subject. In further or separate embodiments, the therapeutic substance is a nucleic acid molecule, and administration thereof results in gene therapy ofthe subject. In further or separate embodiments, the therapeutic substance is a nucleic acid molecule, and administration thereof results in vaccination of the subject. In further or separate embodiments, the targeted delivery of the therapeutic agent provides cancer immunotherapy, autoimmune disease immunotherapy, vaccination, and / or gene editing.

[0008] In another aspect, the present disclosure is directed to methods of delivering a therapeutic substance to a secondary lymphoid organ (SLO) in a subject by administering an effective amount of the LNP composition, such as any of those described above, to an SLO of the subject. In the method, the therapeutic agent in the lipid nanoparticle is targeted to a secondary lymphoid organ (SLO) of the subject. In further or separate embodiments, the therapeutic agent is or includes a nucleic acid molecule. In further or separate embodiments, the SLO is selected from spleen and lymph nodes.

[0009] In another aspect, the present disclosure is directed to methods of targeted delivery of a therapeutic agent to the brain of a subject by administering an effective amount of a LNP composition, such as any of those described above, to the brain of the subject. In the method, the therapeutic agent in the lipid nanoparticle is targeted to the brain of the subject, such as by including a suitable targeting agent bound to the therapeutic agent or other component of the lipid nanoparticle. In further or separate embodiments, the therapeutic agent is or includes a psychiatric medication, which may function to enhance cognition or prevent or slow the onset or progression of a neurodegenerative disease.

[0010] In another aspect, the present disclosure is directed to methods of delivering a therapeutic substance to the bone marrow of a subject by administering an effective amount of a LNP composition, such as any of those described above, to the bone marrow of the subject. In the method, the therapeutic agent in the lipid nanoparticle is targeted to the bone marrow of the subject, such as by including a suitable targeting agent bound to the therapeutic agent or other component of the lipid nanoparticle.

[0011] In another aspect, the present disclosure is directed to methods of selectively delivering a therapeutic substance to specific cells (e.g., immune cells, precursor cells, or stem cells) in a subject by administering an effective amount of any of the LNP compositions described above to specific cells of the subject. To achieve, this, the LNP may be functionalized with a targeting agent in any one or more components of the LNP. In some embodiments, the therapeutic agent is or includes a nucleic acid molecule. In further or separate embodiments, the targeted delivery of the therapeutic agent provides cancerimmunotherapy, autoimmune disease immunotherapy, vaccination and / or gene editing. In further or separate embodiments, the specific cells are selected from lymphoid cells, myeloid cells, and stem cells. In further or separate embodiments, the specific cells are immune cells selected from natural killer cells, natural killer B / T cells, dendritic cells, macrophages, monocytes, B cells, T cells, neutrophils, and microglia cells. In further or separate embodiments, the specific cells are selected from liver sinusoidal endothelial cells, astrocytes, neurons, precursor cells, and pluripotent stem cells. In further or separate embodiments, the LNP is used to deliver nucleic acids encoding chimeric antigen receptor or an exogenous T cell receptor into immune cells.

[0012] In further or separate embodiments, the LNP is injected directly in vivo and delivered into the host cells in vivo. In further or separate embodiments, the LNP is transfected into the host cells ex vivo and the resulting cells are then infused in vivo. In further or separate embodiments, the LNP is administered to a mammal intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In further or separate embodiments, the LNP is used for multiple in vitro and in vivo applications, including, but not limited to, protein replacement therapy, cancer immunotherapy, cell therapy, cancer vaccines, infectious disease vaccines, gene therapy, autoimmune disease treatment and cancer diagnosis. In further or separate embodiments, the LNP is used for in vitro and in vivo production of extracellular vesicles. In further or separate embodiments, the LNP is used for clustered regularly interspaced short palindromic repeats-Cas endonuclease (CRISPR-Cas) gene editing, prime editing, based editing, and RNA editing in vitro and in vivo. In further or separate embodiments, the LNP is used to vaccinate a subject against coronavirus (e.g., SARS-CoV-2). In further or separate embodiments, the LNP is delivered along with a checkpoint inhibitor (e.g., anti- Programmed death-ligand 1 (anti-PD-Ll)) antibody, anti- cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4)) to treat cancer. In further or separate embodiments, the LNP is delivered along with cytokines (e.g., Interleukin- 12, Interleukin-35, and Interferon alpha) to treat a disease. In yet other embodiments, the LNP composition may alternatively include: (i) a viral or non- viral gene delivery system containing a glycan-containing (or saccharide-containing) lipid or LNP, (ii) extracellular vesicles (EVs) and / or Adeno- Associated Virus (AAV) in which the glycan-containing (or saccharide-containing) lipid or LNP is incorporated, and / or (iii) LNPs containing any glycan-containing or saccharide-containing lipid.

[0013] The methods described herein can be useful in a range of biotherapeutics, such as vaccines, for treating or preventing infectious diseases, cancers, and improving a subject’s health or well-being. In some embodiments, by temporary depletion of phagocytes, more LNPs can accumulate in desirable tissues and targeted cells, thus resulting in enhanced mRNA delivery or maintained efficacy with lowered dose. Without the competitive uptake by macrophages, the LNP can mainly accumulate in targeted tissues and cells. This methodology is beneficial in protein replacement or gene editing therapies for rare diseases and resulting enhanced mRNA delivery or maintained efficacy with lowered dose.

[0014] The present disclosure solves the problems noted above by providing LNP formulations that have been modified to be highly stable and more selective in targeting SLOs, such as spleen and lymph nodes. It has herein been found that, by including glycan / saccharide-containing lipids into the LNP, the glycan / saccharide-modified LNP can selectively target not only SLOs, but immune cells, including but not limited to natural killer cells (NK cells) and dendritic cells. These LNPs described herein can be used for such applications as vaccines and CAR-NK cells for cancer immunotherapies. The LNPs described herein may also be used to selectively target the brain or bone marrow of a subject. The targeted delivery of the therapeutic agent may provide, for example, cancer immunotherapy, autoimmune disease immunotherapy, vaccination and / or gene editing.

[0015] The end result of the present methods is an enhanced selective delivery of a therapeutic agent into the subject. The subject is typically a mammal, more particularly a human subject. In some embodiments, the therapeutic agent is or includes a nucleic acid, and the nucleic acid encodes a protein that is expressed in the subject. In other embodiments, the nucleic acid is a component of a gene editing machinery. In other embodiments, the nucleic acid is delivered into one or more of liver, brain, spleen, lymph nodes, kidneys, and lungs. In some embodiments, the enhanced delivery of the nucleic acid results in improved protein expression of protein encoded by the delivered nucleic acid. In other embodiments, the enhanced delivery of nucleic acid results in improved gene editing efficiency by the delivered nucleic acid. In other embodiments, the method provides an enhanced nucleic acid delivery into one or more of the liver, brain, spleen, lymph nodes, kidneys, and lungs.

[0016] In some embodiments, the lipid moiety in component (i) is a diacylglyceride. In some embodiments, component (i) excludes a polyalkylene oxide segment. In otherembodiments, component (i) includes a polyalkylene oxide segment. In some embodiments, component (i) includes a zwitterionic moiety (or more specifically, a zwitterionic polymer) attached to the glycan or lipid portion. In some embodiments, the zwitterionic polymer in component (i), if present, is a poly(carboxybetaine) (PCB), a poly (sulfobetaine), a poly (phosphobetaine), poly (phosphatidylcholine), glutamic acid- lysine (EK)-containing polypeptide, a poly(trimethylamine N-oxide) polymer, or a poly (zwitterionic phosphatidyl serine). In some embodiments, the zwitterionic polymer in component (i) is a betaine polymer, such as a poly(carboxybetaine), poly(sulfobetaine), or poly(phosphobetaine) polymer.

[0017] In some embodiments, the non-cationic lipid in component (ii) contains a zwitterionic moiety, wherein the zwitterionic moiety may be selected from phosphobetaine, phosphatidylcholine, carboxybetaine, sulfobetaine, trimethylamine N-oxide, glutamic acid- lysine (EK)-containing peptide, or zwitterionic phosphatidyl serine moiety. In some embodiments, the zwitterionic lipid is selected from 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, l-stearoyl-2-oleoyl phosphatidyethanolamine (SOPE), 1,2-dioleoyl-sn glycero-3-phophoethanolamine (transDOPE), and 1,2-disteroyl-sn-glycero- 3 -phosphocholine (DSPC).

[0018] In some embodiments, the non-cationic lipid in component (ii) is a phospholipid, or more particularly, a phosphatidyl serine lipid. In some embodiments, the cationic or ionizable lipid in component (iii) possesses a secondary, tertiary, or quaternary amino group, any of which may or may not be attached to a lipid moiety. In other embodiments, the cationic or ionizable lipid in component (iii) possesses a secondary, tertiary, or quaternary group along with a functional group which is negatively charged under physiological conditions. In some embodiments, component (ii) excludes a polyalkylene oxide segment. In other embodiments, component (ii) includes a polyalkylene oxide segment. In some embodiments, the cationic or ionizable lipid in component (iii) excludes a poly alkylene oxide segment. In yet other embodiments, the lipid nanoparticlecomposition further comprises: (v) a steroid or sterol, such as cholesterol or a derivative thereof.BRIEF DESCRIPTION OF THE FIGURES

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary' fee.

[0020] FIGS. 1 A-1B. FIG. 1 A shows symbols used for denoting some of the glycan- containing lipids, and in particular, some exemplary S-L conjugates containing no sialic acid or 2,3-linked or 2,8-linked sialic acid, wherein the wavy lines represent a lipid. FIG. IB shows the structures of exemplary types of sialic acids, all of which can be employed as S in component (i).

[0021] FIGS. 2A-2C. Bar graphs showing the in vitro expression of Fluc-mRNA delivered by LNPs in different cell lines: THP1 monocytes (FIG. 2A); RAW264.7 macrophages (FIG. 2B); and HELA epithelial cells (FIG. 2C).

[0022] FIG. 3. Whole body bioluminescent images of C57BL / 6 male mice after intravenous injection of different LNPs encapsulating Fluc-mRNA. At 6-hr post-injection, mice were imaged upon the intraperitoneal injection of luciferin.

[0023] FIGS. 4A-4B. Different LNPs encapsulating Fluc-mRNA were injected intravenously into C57BL / 6 mice, and organs were extracted at 6 hours after injection. FIG. 4A shows bioluminescence imaging of organs extracted from C57BL / 6 mice. Organs shown here include (from top to bottom) heart, lung, superficial cervical lymph nodes (with adjacent fat), liver, kidneys and spleen. FIG. 4B is a bar chart comparing the SLOs / liver bioluminescence intensities obtained from the organ imaging for these different LNPs.SLO: secondary lymphoid organs.

[0024] FIGS. 5A-5B. Different LNPs encapsulating Cy5-labeled mRNA were injected intravenously into C57BL / 6 mice. The mice were sacrificed 3 hours after injections. Cells were extracted from the spleen and lymph nodes and analyzed under flow cytometer. FIG. 5A includes pie charts showing the comparisons between percentages of LNPs uptake by B T cells and other non-B T cells. FIG. 5B includes bar charts showing the comparisons ofLNPs uptake by CD 169+ macrophage in spleen and lymph nodes. SP = spleen; LN = Lymph Nodes.

[0025] FIG. 6. Bar chart comparing percentages of NK1. 1 cells expressing tdTomato signal are shown in the bar chart. A higher idTomato signal corresponds to a higher mRNA expression. Different LNPs encapsulating Cre-mRNA were injected intravenously into Ail4 mice, and the mice were sacrificed 48 hours after injections. Cells were extracted from the spleen and analyzed by flow cytometry.

[0026] FIGS. 7A-7D. The glycan-containing LNP formulations demonstrate enhanced antigen- specific immune responses compared to the commercial formulation SM102 (8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester). FIG. 7A shows an injection scheme for antigen-encoding mRNA in which mice were injected with LNPs encapsulating ovalbumin-encoding mRNA on days 0, 3, and 7, blood samples were drawn on days 14 and 21 for antibody testing, and spleens were extracted on day 28 for immune cell activation studies. FIG. 7B shows graphs plotting anti-ovalbumin antibodies IgG titers on day 14 (D14) and day 21 (D21 ). FIG. 7C is a bar chart showing the percentage of IFN- / + T cells in activated CD44+ CD8 T cells. FIG. 7D is a bar chart showing the percentage of 1FN- / + T cells in activated CD44+ CD4 T cells.

[0027] FIGS. 8A-8D. The glycan-containing LNP formulations demonstrate successful in- vivo CAR-NK cells engineering. FIG. 8A shows the experimental scheme for in vivo CAR- NK cells engineering. FIG. 8B shows the CAR expressions in NK cells of mice treated by glycan-containing LNP compared to mice treated by commercial formulation. In FIG. 8B, the top two images are flow cytometry graphs showing the percentages of CAR-positive NK cells in total NK cells. The bottom image in FIG. 8B is a graph quantifying CARpositive NK cells in different numbers of NK cells in the tumor killing assay. FIG. 8C is a bar chart showing the results of the specific tumor cell killing assay mediated by CAR-NK engineered in vivo. FIG. 8D show's the levels of cytotoxicity markers on CAR-NK cells after incubation with tumor cells in vitro.

[0028] FIG. 9. Depiction of an exemplar}' zwitterionic polymer containing negati vely charged sialic acid (SA) molecules on a peptide backbone containing positively charged lysine (K) units. The equal number of positive and negative charges makes the whole structure charge neutral, which corresponds to a zwitterionic structure. The zwitterionic polymer may be incorporated into component (i) or ( i i ) by attaching to a saccharide or lipid.

[0029] FIG. 10. Scheme showing chemoenzymatic synthesis of saccharide-containing lipids.

[0030] FIG. 11. Scheme showing grafl-lo and graft- from chemoenzymatic synthetic methods for producing saccharide-containing lipids.DETAILED DESCRIPTION

[0031] The present disclosure is foremost directed to lipid nanoparticle (LNP) compositions containing at least one therapeutic substance. More particularly, the LNP composition includes or exclusively contains the following components: (i) at least one glycan- containing lipid containing a gly can-containing moiety (S) linked to a lipid (L); (ii) at least one non-cationic lipid selected from charged and uncharged lipids; (iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and (iv) at least one therapeutic substance. The LNP is constructed of a self-assembled shell containing components (i), (ii), (iii) and (iv). In different embodiments, the LNP has a size of precisely, about, at least, or up to, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 nm, or a size within a range bounded by any two of the foregoing values (e.g., 10-250 nm, 10-200 nm, 50-250 nm, or 50-200 nm). As the LNP is typically spherical or spherical-like, the foregoing sizes typically refer to a diameter of the LNP. The therapeutic substance is incorporated into the LNP, either by being encapsulated within a core of the LNP or by being integrated throughout the LNP as a homogeneous or heterogeneous mixture of components (i), (ii), (iii), and (iv) and any optional additional components (e.g., a steroid or sterol, such as cholesterol, and / or targeting agent), if present.

[0032] In some embodiments, the LNP as a whole excludes a polyalkylene oxide segment, or the LNP as a whole more specifically excludes a polyethylene oxide or polypropylene oxide segment. In other embodiments, the LNP as a whole includes a polyalkylene oxide segment, or the LNP as a whole more specifically includes a polyethylene oxide or polypropylene oxide segment. In some embodiments, components (i), (ii), and / or (iii) exclude a polyalkylene oxide segment. In other embodiments, at least one of components (i), (ii), and (iii) includes a polyalkylene oxide segment.

[0033] Component (i) is or includes at least one glycan-containing lipid. The glycan- containing lipid contains a glycan-containing moiety (S) linked to a lipid (L). The term“glycan-containing lipid” of component (i) is herein meant to be synonymous with “glycolipids,” which are well known in the art. The glycan-containing lipid of component (i) can be any lipid (L) attached to any glycan-containing moiety (S). More particularly, the glycan-containing lipid can be denoted by the general structure S-L, wherein S is a glycan- containing moiety and L is a lipid, wherein S is covalently bonded to L. In some embodiments, component (i) excludes a polyalkylene oxide segment, or component (i) more specifically excludes a polyethylene oxide or polypropylene oxide segment. In other embodiments, component (i) includes a polyalkylene oxide segment, or component (i) more specifically includes a polyethylene oxide or polypropylene oxide segment. In some embodiments, component (i) includes a zwitterionic moiety, or more particularly, a zwitterionic polymer, as further described below, which may be attached to the S or L portion of the glycan-containing lipid. In some embodiments, a zwitterionic moiety is excluded from component (i).

[0034] The glycan-containing moiety (S) can be any of the known types of saccharide- containing (i.e., carbohydrate-containing) molecules, including monosaccharides, disaccharides, oligosaccharides, and polysaccharides (typically, greater than 10 or at least 15, 20, or 30 monosaccharide units). The glycan-containing moiety may also be a polyol (i.e., sugar alcohol) molecule containing precisely or at least 4, 5, or 6 hydroxy groups. Some examples of monosaccharides include glucose, fructose, galactose, mannose, ribose, and xylose. Some examples of disaccharides include sucrose, maltose, isomaltose, lactose, and trehalose. The oligosaccharides typically contain 3-10 monosaccharide units and include, for example, trisaccharides, tetrasaccharides, pentasaccharides, and higher saccharides. Some examples of oligosaccharides include fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and mannan oligosaccharides (MOS). Some examples of polyols include sorbitol, mannitol, erythritol, xylitol, galactitol, inositol, and maltitol. The glycan-containing moiety may more specifically contain or be exclusively composed of one or more glucose, fructose, galactose, iV-acetyl-D-galactosamine, sialic acid, mannose, ribose, or xylose units, or any specific combination of two or more of any of the foregoing. Typically, the glycan-containing moiety (S) is bound to the lipid (L) by at least one of its hydroxy groups (typically by one or more ether linkages between the saccharide and lipid). Thus, it is understood that any number of hydroxy groups provided above for the glycan- containing moieties corresponds to the glycan-containing moieties before attachment to the lipid.

[0035] In some embodiments, the glycan-containing moiety (S) may be selected from natural forms of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and / or polyols. In other embodiments, the glycan-containing moiety may be selected from chemically modified forms of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and / or polyols. The chemically modified forms typically result from covalently added functional groups, such as a phenyl (benzene) group, carboxyl group, amine group, amide group, or halogen atom(s). In some embodiments, the chemically modified forms of S result from chemoenzymatic reactions, graft-to, or graft-from chemical reactions. Exemplary chemoenzymatic reactions are shown in FIG. 10. Moreover, exemplary graft-to and graft-from chemoenzymatic reactions for the synthesis of saccharide-containing lipids are shown in FIG. 11.

[0036] In some embodiments, the glycan-containing moiety (S) is combined with a positively charged functional group to form a zwitterionic glycan-containing moiety, wherein the zwitterionic glycan-containing moiety may be composed of single or repeat units of a negatively charged glycan-containing moiety and a positively charged functional group. The positively charged functional group may be, for example, quaternary amine, phosphonium, or sulfonium. FIG. 9 shows an exemplary zwitterionic polymer containing negatively charged sialic acid (SA) molecules on a peptide backbone containing positively charged lysine (K) units. The equal number of positive and negative charges makes the whole structure charge-neutral, which corresponds to a zwitterionic structure. Notably, the backbone may be other than peptide, and the SA moiety can be replaced with other types of sugar molecules, including monosaccharides (e.g., mannose, galactose, glucose), oligosaccharides, and polysaccharides, and the sugar molecule can be in its natural form or modified form. For purposes of the present invention, the zwitterionic glycan-containing moiety is attached to a lipid L, such as described for component (i) or any other lipid disclosed in the present application, such as for component (ii) or (iii).

[0037] The lipid (L) may be any of the types of lipid molecules well known in the art. In some embodiments, the lipid contains one or more linear and / or branched alkyl or alkenyl groups typically containing at least 6, 7, 8, 9, or 10 carbon atoms and typically up to 12, 15, 18, 20, 24, or 30 carbon atoms. The alkenyl group contains at least one carbon-carbon double bond. In different embodiments, the alkenyl group contains one, two, three, or more carbon-carbon double bonds. The alkyl or alkenyl groups may be composed of at least (orsolely) carbon and hydrogen atoms or may (i.e., optionally) include one or more heteroatoms, such as N, O, Si, P, S, Cl, Br, and / or F. The lipid may be any of the lipids described in the Examples provided in this application. The lipid may also be any of the lipids described in International Application Pub. No. WO2011 / 057227, which is herein incorporated by reference. The lipid (L) is typically uncharged, but may, in some embodiments, be functionalized to be positively charged, negatively charged, or zwitterionic (i.e., contain a mixture of positive and negative charges counterbalanced to result in an overall neutrally charged state).

[0038] The term “lipid” refers to organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. The lipid in component (i) may be selected from any of these types of lipids. In particular embodiments, the lipid is a diacylglyceride.

[0039] The lipid is oftentimes constructed of a polyol portion (e.g., a diol, glycerol, phosphatidylglycerol, phosphatidylethanolamine, or phosphatidylserine portion) that has been esterified with one or more fatty acid molecules to result in a fatty acyl (e.g., monoacyl, diacyl, or triacyl) lipid, wherein the term “fatty acyl” refers to a RC(=O) group in which R is a linear or branched alkyl or alkenyl group as described above. Some examples of fatty acyl groups include oleoyl, palmitoyl, lauryl, myristoyl, stearoyl, linoleoyl, and arachidonyl. The lipid may be, for example, a diacyldiol (e.g., diacylethyleneglycol), diacylglycerol (diacylglyceride), diacylphosphatidylglycerol, diacylphosphatidyl- ethanolamine, or diacylphosphatidylserine moiety.

[0040] In some embodiments, the lipid is a glycerolipid. The glycerolipid may be, for example, a glyceride, diglyceride, or triglyceride, which may alternatively be referred to as an acylglycerol, diacylglycerol, or triacylglycerol, respectively. In other embodiments, the lipid may be a phospholipid, or more particularly, a glycerophospholipid, as well known in the art. In other embodiments, the lipid may be a sphingolipid or a derivative thereof, such as a ceramide, phosphosphingolipid, glycosphingolipid (or more particularly, a cerebroside or ganglioside), or sphingosine. In other embodiments, the lipid may be a linear or branched, saturated or unsaturated fatty acyl group not esterified with a polyol portion, butinstead, directly attached to the saccharide by an ester or amide bond. In other embodiments, the lipid may contain one or more hydrophobic rings, such as a steroid or sterol, or more particularly, cholesterol or a derivative thereof, or a phytosterol (e.g., |3- sitosterol, stigmasterol, or brassicasterol). Cholesterol derivatives are compounds that preserve the tetracyclic fused ring structure of cholesterol but contain a modification, such as one or more additional hydroxy groups, methyl groups, or the presence of one or more halogen atoms, or a change in position of a double bond, hydroxy group, or methyl group, or the absence of a double bond, hydroxy group, or methyl group, as well as esterified, etherified, oxo, and epoxidized derivatives of cholesterol. The cholesterol derivative may alternatively be as further described later on in this disclosure. When a steroid or sterol is selected as L in component (i), it is necessarily covalently attached to the glycan portion (S) of component (i). In yet other embodiments, the lipid may be a fat-soluble vitamin, such as Vitamin A, D, E, or K.

[0041] Any of a variety of lipids, as described above, can be conjugated with any of a variety of glycan-containing molecules by methods well known in the art. For example, lipids with functional head groups can be conjugated with saccharides by known chemical reactions. Moreover, after an initial synthesis of a saccharide-lipid conjugate, additional saccharides can be attached to the saccharide-lipid head by incubation with suitable enzymes and one or more saccharide monomers, as shown in FIG. 10. In some embodiments, the lipid (L) attaches to the glycan-containing moiety (S) by one or more hydroxy groups of the polyol portion of the lipid. In other embodiments, a functionalized lipid can be reacted with a suitably functionalized glycan-containing molecule to form a glycan-containing lipid, wherein the lipid and glycan-containing molecules contain functional groups reactive with each other to result in a linkage. For example, a lipid containing either an alkyne group, azido group, thiol group, or amine group can be conjugated with a saccharide molecule containing either an azido group, alkyne group, maleimide group, or acylated N-hydroxy succinimide group, respectively. As well known in the art, an alkyne group and azide group can be reacted by click chemistry to form a triazole linkage. Thus, in some embodiments, an azide or alkyne derivative of any lipid described herein can be attached to an alkyne or azide derivative (respectively) of any glycan-containing molecule described herein to attach the lipid and glycan-containing molecule by a triazole linkage. In specific embodiments, sialic acid functionalized with an alkyne can be conjugated to a lipid functionalized with azide group. Moreover, apolysialyltransferase from Neisseria meningitidis can synthesize polysialic acid on the initially produced sialic-acid-lipid conjugate in the presence of its donor substrate CMP- Neu5Ac. After this chemoenzymatic reaction, a polysialic-acid-lipid conjugate can be synthesized.

[0042] The resulting glycan-containing lipid may be, for example, a natural or modified glycolipid, or a natural or modified glycosphingolipid, or a natural or modified ganglioside, or a natural or modified sialic acid-containing lipid. In more specific embodiments, the glycan-containing lipid is a 2,3-linked sialic acid-containing lipid, wherein the 2,3-linked sialic acid-containing lipid is a natural or modified form, and may, in some embodiments, be selected from GM3 and GD1A. In other specific embodiments, the glycan-containing lipid is a 2,8-linked sialic acid-containing lipid, wherein the 2,8-linked sialic acidcontaining lipid is a natural or modified form, and may, in some embodiments, be selected from GD3 and GQ1B. In other specific embodiments, the glycan-containing lipid is a 2,6- linked sialic acid-containing lipid, wherein the 2,6-linked sialic acid-containing lipid is a natural or modified form. FIG. 1 A shows various exemplary S-L conjugates containing no sialic acid or 2,3-linked or 2,8-linked sialic acid, wherein the wavy line represents a lipid. FIG. IB shows the structures of exemplary types of sialic acids, all of which can be employed as S in component (i).

[0043] In some embodiments, any glycan-containing molecule disclosed in this application is combined with any lipid(s) disclosed in this application to result in an S-L conjugate containing a specific combination of S and L. In some embodiments, one or more glycan- containing molecules (S) disclosed in this application is excluded from the S-L conjugate. In other embodiments, one or more lipids (L) disclosed in this application is excluded from the S-L conjugate. In some embodiments, the lipid (L) in component (i) is structurally distinct from the lipid in components (ii) and (iii), while in other embodiments, the lipid (L) in component (i) is structurally overlapping with one or both types of lipids in components (ii) and / or (iii).

[0044] In some embodiments, a negatively charged glycan-containing moiety (S), such as a sialic acid or derivative thereof, is combined with (i.e., attached to) a positively charged functional group to form a zwitterionic glycan-containing moiety. The positively charged functional group may be, for example, a quaternary ammonium, quaternary phosphonium, or sulfonium group. In some embodiments, a zwitterionic glycan-containing moiety is apolypeptide containing positively charged amino acids (arginine, lysine or histidine), and optionally, one or more uncharged amino acids (e.g., proline, serine, or glycine), wherein the zwitterionic glycan-containing moiety is modified with one or more sialic acid units. In particular embodiments, a zwitterionic glycan-containing moiety is formed by binding one or more sialic acid units with one or more lysine units that contain a side chain with a quatemized amine group. More specifically, a polylysine chain containing quaternized lysine side chains can be derivatized with sialic acid units to form a zwitterionic sialic acid- lysine moiety that can function as a glycan-containing moiety (S).

[0045] In other embodiments, component (i) may be described by the following formula:wherein:Y: can be various forms of lipids with different numbers of carbon atoms and double bondsA: can be various forms of saccharides (i.e. Glc; GlcNAc; GlcN; Gal; ....); its connection to B can be in a single or multiple branchesB: can be various forms of saccharides (i.e. Glc; GlcNAc; GlcN; Gal; . . ..)X: can be various forms (natural / synthetically modified) of saccharides (especially sialic acid Neu5Ac / Neu5Gc / Neu;. . ..)

[0046] An exemplary S-L molecule for component (i) is the following:

[0047] Component (ii) is or includes at least one non-cationic lipid. The term “non-cationic lipid” in component (ii) is meant to include any of the charged and uncharged lipids that are not overall cationic (positively charged) and not capable of being ionized to a positively charged state. As further discussed below, the non-cationic lipid may be uncharged, negatively charged (anionic), or may be zwitterionic by containing a mix of positive and negative charges that are counterbalanced to result in a neutrally charged lipid. In embodiments, the non-cationic lipid is distinct from component (i) by at least not being attached to a glycan-containing group (S). The non-cationic lipid may he selected from any of the types of lipids described above for component (i). In some embodiments, the noncationic lipid in component (ii) is not zwitterionic but negatively charged by containing a negatively charged group (e.g., phosphoserine). A metal (e.g., alkali) or ammonium counteranion may be ionically and fluxionally associated with the negatively charged group. In specific embodiments, the non-cationic lipid in component (ii) is a phospholipid. In some embodiments, component (ii) excludes a polyalkylene oxide segment, or component (ii) more specifically excludes a polyethylene oxide or polypropylene oxide segment. In other embodiments, component (ii) includes a polyalkylene oxide segment, or component (ii) more specifically includes a polyethylene oxide or polypropylene oxide segment.

[0048] The non-cationic lipid in component (ii) may more particularly be, for example, a phosphatidylglycerol lipid, phosphatidylethanolamine lipid, or sphingolipid. The noncationic lipid may, in some embodiments, be a simple lipid, such as a fat, oil, and / or wax. The non-cationic lipid may, in some embodiments, be a compound lipid, such as a phospholipid or glycolipid. The non-cationic lipid may, in some embodiments, be 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 a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, or a cerebroside. In particular embodiments, the non-cationic lipid is selected from one or more of a phosphatidylethanolamine (PE), a phosphatidylglycerol (PG), a phosphatidic acid (PA), or a phosphatidylinositol (PI). In other particular embodiments, the non-cationic lipid is a dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl-phospho ethanolamine, 16-O-dimethyl-phosphoethanolamine, 18-1-trans-phosphoethanolamine, 1-stearoyl-2-oleoyl phosphatidyethanolamine (SOPE), 1 ,2-dioleoyl-sn glycero-3- phophoethanolamine (transDOPE), and l,2-disteroyl-sn-glycero-3-phosphocholine (DSPC). Since the non-cationic lipid is not attached to a polymer, the non-cationic lipid excludes a polyalkylene oxide (polyalkylene glycol) segment.

[0049] In some embodiments, the non-cationic lipid in component (ii) contains a zwitterionic moiety, thus resulting in a zwitterionic lipid. The zwitterionic lipid may be denoted by the formula L-ZM, wherein L is a lipid (such as any of the lipids described above) and ZM is a zwitterionic moiety, and L and ZM are covalently bonded to each other. The zwitterionic moiety (ZM) may be monomeric, dimeric, trimeric, oligomeric, or polymeric. The zwitterionic moiety may be, for example, a phosphobetaine, phosphatidylcholine, phosphoserine, carboxybetaine, sulfobetaine, trimethylamine N-oxide, glutamic acid-lysine (EK) -containing peptide, or zwitterionic phosphatidyl serine (phosphoserine) moiety, or a combination thereof. In some embodiments, the zwitterionic lipid is a phospholipid, such as a phosphatidylcholine or phosphatidylserine lipid. In some embodiments, the non-cationic lipid corresponds to any of the lipids (L) described above for component (i) but conjugated with one or more zwitterionic groups, such as one or more carboxybetaine groups. In other embodiments, the non-cationic lipid corresponds to any of the lipids (L) described above for component (i) but conjugated with one or more zwitterionic groups, such as one or more sulfobetaine groups. In other embodiments, the non-cationic lipid corresponds to any of the lipids (L) described above for component (i) but conjugated with one or more zwitterionic groups, such as one or more trimethylamine N- oxide groups. In some embodiments, any of the zwitterionic moieties described in this application excludes a polyalkylene oxide (polyalkylene glycol) segment, or any of the zwitterionic moieties described in this application more specifically excludes a polyethylene oxide or polypropylene oxide segment.

[0050] In other embodiments, the zwitterionic moiety (ZM) is more specifically a zwitterionic polymer, herein denoted as ZP. Thus, the non-cationic lipid in component (ii) may more specifically have the formula L-ZP, wherein L is a lipid (such as any of the lipids described above) and ZP is a zwitterionic polymer, and L and ZP are covalently bonded to each other. As used herein, the term “zwitterionic polymer” refers to a polymer containing an equal number of positive and negative charges within the polymer. In some embodiments, the zwitterionic polymer (ZP) contains a plurality of repeating units, whereineach repeating unit contains one positive and one negative charged moiety. The zwitterionic polymer typically contains at least or greater than 2, 5, or 10, and up to or less than 100, 200, 300, 400, 500, or 1000 units.

[0051] The zwitterionic polymer (ZP) can be prepared by any of the suitable methods well known in the art. In some embodiments, the zwitterionic polymer (ZP) is prepared by polymerizing one or more zwitterionic monomers that are polymerizable. Zwitterionic monomers are electronically neutral monomers that include equal numbers of positive and negative charges (e.g., one of each). In other embodiments, the zwitterionic polymer is prepared by polymerizing one or more uncharged monomers that can later (after polymerization) be converted to charged groups. In this way, the uncharged monomers function as precursors of zwitterionic monomers. The zwitterionic polymer may be composed of 100 mole percent zwitterionic moieties, i.e., each repeating unit of the zwitterionic polymer is a zwitterionic moiety; or the zwitterionic polymer may be a copolymer of a zwitterionic monomer and non-zwitterionic comonomer, which corresponds to a zwitterionic polymer having less than 100 mole percent zwitterionic moieties (e.g., when the polymerizable zwitterionic monomer and the polymerizable comonomer are present in equal proportions in the polymerization mixture, the product is a zwitterionic polymer having 50 mole percent zwitterionic moieties).

[0052] In some embodiments, the zwitterionic polymer is prepared by a known polymerization method, such as atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT) polymerization, and free radical polymerization. Any suitable radical initiators for polymerizing such monomers including those well known in the art, may be used. In some embodiments, to prepare the zwitterionic polymer-containing lipid or other polymer-containing lipid, a zwitterionic or other monomer or precursor thereof is attached to a lipid, and the monomer is polymerized while attached to the lipid. Alternatively, an already produced polymer may be attached to a lipid by means well known in the art.

[0053] The zwitterionic polymer (ZP) can be attached to the lipid moiety by means well known in the art. In typical embodiments, any one or more of the lipids in components (i), (ii), and / or (iii) are conjugated to a zwitterionic polymer via a carbon residing in a polyol portion or polar head group of the lipid. Methods of attaching a lipid to a zwitterionic polymer are provided in detail in, for example, U.S. Patent 8,617,592, the contents of whichare herein incorporated by reference. For example, a zwitterionic polymer (e.g., PCB) may be functionalized with a succinimide group by methods well known in the art, followed by reaction with an amino-functionalized lipid (e.g., DSPE) to attach the lipid to the zwitterionic polymer. The zwitterionic polymer may contain the zwitterionic groups in side chains or the backbone (or combination thereof) of the zwitterionic polymer. Notably, for any of the zwitterionic polymer (ZP) formulas provided in this application, it is understood that the ZP structure necessarily includes an attachment point to the lipid (L) to result in the L-ZP structure in component (ii).

[0054] In one embodiment, the zwitterionic polymer (ZP) is a homopolymer prepared from zwitterionic monomers and has the formula:wherein BB is a polymer backbone, L’ is an optional linker, and Z is a zwitterionic moiety. The backbone (BB) can be any polymeric backbones known in the art, including linear, branched, and cyclic backbone structures, e.g., a polyester, polyether, polyurethane, polyamide, or polyhydrocarbon (e.g., polyethylene or polypropylene) backbone. The linker (L’) can be any of the linkers commonly included in pendant groups of polymers, e.g., linear or branched alkylene linkers or cyclic linkers, any of which may contain precisely or at least 1, 2, 3, 4, 5, or 6 carbon atoms and optionally containing one or more heteroatoms (typically selected from oxygen and nitrogen atoms). The subscript x is typically at least or greater than 2, 5, 10, 20, 50, or 100, and up to or less than 100, 200, 300, 400, 500, 1000, 2000, or 5000 units.

[0055] In particular embodiments, the backbone (BB) of the zwitterionic polymer may have any of the following structures:wherein R is selected from hydrogen atom and substituted or unsubstituted alkyl; and E is selected from substituted or unsubstituted alkylene, -(CH2)PC(O)O-, and -(CH2)PC(O)NR2-, wherein p is typically an integer from 0 to 12 and R2is selected from hydrogen and substituted or unsubstituted alkyl.

[0056] In particular embodiments, for any of the formulas for the zwitterionic polymer (ZP) provided herein, Z is selected from any of the following structures:wherein R3, R4, and R6are independently selected from the group consisting of hydrogen and substituted or unsubstituted alkyl group, R5or Rs is selected from the group consisting of substituted or unsubstituted alkylene, phenylene, and polyether groups, and m is an integer from 1 to 7; and x is an integer from 2 to 500. Any of the above formulas may also function as a ZM group in the L-ZM structure described earlier above.

[0057] In one set of embodiments, the zwitterionic polymer (ZP) has the following structure:

[0058] In Formula (1) above, the variable Rais H or an alkyl group containing 1-3 carbon atoms. In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. Some examples of alkyl groups containing 1 -3 carbon atoms include methyl, ethyl, n-propyl, and isopropyl. In some embodiments, Rais H or methyl. The variable X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms. In some embodiments, Rbis H or methyl. The variable Z is a zwitterionic moiety. The subscript n is typically an integer of at least 2, 5, or 10 (e.g., at least or greater than 10, 50, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 50,000, or 100,000 units, or a number of units within a range bounded by any two of the foregoing values). The subscript m is an integer of at least 1, such as a value of precisely or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a value within a range bounded by any two of the foregoing values (e.g., 1-12, 1-10, 1-6, 1-4, 1-3, 2-4, or 2-3).

[0059] In some embodiments, for any of the formulas for the zwitterionic polymer (ZP) provided herein, the group Z represents a single zwitterionic group containing a positive and negative charge in the same Z group. The group Z may be selected from, for example, carboxybetaine, sulfobetaine, phosphobetaine, and trialkylamine-jV-oxide zwitterionic moieties. The corresponding polymer according to Formula (1) is poly (carboxy betaine), poly (sulfobetaine), poly(phosphobetaine), poly(phosphatidylcholine), glutamic acid-lysine (EK)-containing polypeptide, poly(trialkylamine-N-oxide), and poly(zwitterionic phosphatidyl serine), as well known in the art. A poly(ethylene glycol) (PEG) may or may not also be attached to the lipid, which may be a lipid in component (i) and / or (ii). In other embodiments, a certain number of the Z groups are positively charged and an equal number of Z groups are negatively charged to result in Z zwitterionic pairs (i.e., Z+Z" zwitterionic pairs). Thus, the non-cationic lipid L-ZP of Formula (ii) may contain any one of the above exemplary ZPs linked to L.

[0060] Notably, although Formulas (I), (1), and sub-formulas thereof may appear to depict zwitterionic homopolymers (100 mol% zwitterionic moieties), Formulas (I), (1), and subformulas thereof include the possibility that one or more non- zwitterionic or uncharged monomer units is situated (inserted) between zwitterionic monomeric units depicted in any of these formulas, thereby resulting in a copolymer. If zwitterionic monomeric units (such as any of those described above, i.e., where n = 1) are labeled as A units, and non-zwitterionic or uncharged monomeric units are labeled as B units, the copolymer can have any of the known copolymer arrangements, including alternating (e.g., A-B-A-B), block (e.g., A-A-A-A-B-B-B-B), or random (e.g., A-B-B-A-B-A-A-B-A-B-B). The zwitterionic polymer may also include more than one type of non-zwitterionic or uncharged monomer unit, such as in the structures A-B-C-A-B-C (repeating), A-A-A-B-B-B-C-C-C (block), or A-C-B-B-C-A-B-C-A-C-A-B-C-A-B-C-B (random), wherein B and C represent non- zwitterionic or uncharged monomeric units.

[0061] In some embodiments of Formula (1), Z contains a positively charged group directly bound to a negatively charged group in the zwitterionic polymer. The resulting zwitterionic polymer may have the following structure:

[0062] In Formula (la), Ra, X, n, and m are as defined above under Formula (1). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables Ci and C2 are independently selected as positively charged and negatively charged moieties to form a zwitterionic moiety C1-C2. Some examples of positively charged moieties include ammonium (-NRV-) and phosphonium (-PRV-) moieties. Some examples of negatively charged moieties include terminal oxide (-O’), carboxylate (-C(O)O-), phosphate (-OPO3 ), phosphonate (-PO3 ), sulfate (-OSO3 ), and sulfonate (-SO3 ). In some embodiments, Ci is positively charged and C2 is negatively charged. For example, Ci may be an ammonium moiety and C2 may be oxide, which together results in an ammonium X-oxide (-NRV-O ) zwitterionic group. As the ammonium moiety is also attached to a carbon atom of the polymer, the ammonium oxide zwitterionic group is also herein referred to as a trialkylamine-X-oxide group. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In other separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0063] In specific embodiments of Formula (la), Ci is an ammonium moiety and C2 is oxide, which together results in an ammonium X-oxide (-NRV-0 ) zwitterionic group. The resulting polymer is a poly(trialkylammonium oxide), i.e., pTMAO, and may have the following structure:

[0064] In Formula (la-1), Ra, X, n, and m are as defined above under Formula (1). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables R1and R2are independently selected from Ra. In some embodiments, R1and R2are both alkyl, or more particularly, both methyl. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0065] In other embodiments of Formula (1), Z contains a positively charged group indirectly bound to a negatively charged group via a linker in the zwitterionic polymer. The resulting zwitterionic polymer may have the following structure:

[0066] In Formula (lb), Ra, X, n, and m are as defined above under Formula (1). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The subscript p is an integer of at least 1 , such as a value of precisely or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a value within a range bounded by any two of the foregoing values (e.g., 1-12, 1-10, 1-6, 1-4, 1-3, 2-4, or 2-3). The variables Ci and C2are independently selected as positively charged and negatively charged moieties to form a zwitterionic spaced pair. Some examples of positively charged moieties include ammonium (-NRa2+-) and phosphonium (-PRV-) moieties. Some examples of negatively charged moieties include terminal oxide (-O’), carboxylate (-C(O)O-), phosphate (-OPO3 ), phosphonate (-PO3 ), sulfate (-OSO3 ), and sulfonate (-SO3 ). In some embodiments, Ci is positively charged and C2 is negatively charged. For example, Ci may be an ammonium or phosphonium moiety and C2 may be carboxylate, sulfate, sulfonate, phosphate, or phosphonate moiety. In other embodiments, Ci is negatively charged and C2 is positively charged. For example, Ci may be a phosphate, phosphonate, sulfate, or sulfonate moiety and C2 may be an ammonium or phosphonium moiety. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In other separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0067] In specific embodiments of Formula (lb), Ci is an ammonium moiety and C2 is a negatively charged moiety, such as a carboxylate, sulfate, sulfonate, phosphate, or phosphonate moiety, which together results in a spaced zwitterionic group. The resulting zwitterionic polymer may have the following structure:

[0068] In Formula (lb-1), Ra, X, n, m, and p are as defined above under Formulas (1) and (lb). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables R1and R2are independently selected from Ra. In some embodiments, R1and R2are both alkyl, or more particularly, both methyl. C2 is a negatively charged moiety, such as a carboxylate, phosphate, phosphonate, sulfate, or sulfonate moiety. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specificembodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl. In embodiments where C2 is a carboxylate moiety, the polymer of Formula (lb-1) can generally be referred to as a poly(carboxybetaine). In embodiments where C2 is a sulfonate moiety, the polymer of Formula (lb-1) can generally be referred to as a poly (sulfobetaine). In embodiments where C2 is a phosphate moiety, the polymer of Formula (lb-1) can generally be referred to as a poly (phosphobetaine).

[0069] In specific embodiments of Formula (lb-1), C2 is a sulfonate or carboxylate moiety. The resulting zwitterionic polymer may have any of the following structures:

[0070] In Formulas (lb-2) and (lb-3), Ra, X, n, m, and p are as defined above under Formulas (1) and (lb). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables R1and R2are independently selected from Ra. In some embodiments, R1and R2are both alkyl, or more particularly, both methyl. In separate or further specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0071] In other specific embodiments of Formula (lb), Ci is a phosphonium moiety and C2 is a negatively charged moiety, such as a carboxylate, sulfate, sulfonate, phosphate, or phosphonate moiety, which together results in a spaced zwitterionic group. The resulting zwitterionic polymer may have the following structure:

[0072] In Formula (lb-4), Ra, X, n, m, and p are as defined above under Formulas (1) and (lb). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables R1and R2are independently selected from Ra. In some embodiments, R1and R2are both alkyl, or more particularly, both methyl. C2 is a negatively charged moiety, such as a carboxylate, phosphate, phosphonate, sulfate, or sulfonate moiety. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0073] In specific embodiments of Formula (lb-4), C2 is a sulfonate or carboxylate moiety. The resulting zwitterionic polymer may have any of the following structures:

[0074] In Formulas (lb-5) and (lb-6), Ra, X, n, m, and p are as defined above under Formulas (1) and (lb). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound tothe lipid at a terminal position of the backbone. The variables R1and R2are independently selected from Ra. In some embodiments, R1and R2are both alkyl, or more particularly, both methyl. In separate or further specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0075] In other specific embodiments of Formula (lb), Ci is a phosphate moiety and C2 is a positively charged moiety, such as an ammonium or phosphonium moiety, which together results in a spaced zwitterionic group. The resulting zwitterionic polymer may have the following structure:

[0076] In Formula (lb-7), Ra, X, n, and m are as defined above under Formula (1). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The subscript p is an integer of at least 1 , such as a value of precisely or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a value within a range bounded by any two of the foregoing values (e.g., 1-12, 1-10, 1-6, 1-4, 1-3, 2-4, or 2-3). The variable C2+is a positively charged moiety that forms a zwitterionic spaced pair with the phosphate moiety in Formula (lb-7). Some examples of positively charged moieties include ammonium (- NRa2+-) and phosphonium (-PRa2+-) moieties. In specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In other separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0077] In specific embodiments of Formula (lb-7), C2+is an ammonium moiety. The resulting zwitterionic polymer may have the following structure:(lb-8)

[0078] In Formula (lb-8), Ra, X, n, m, and p are as defined above under Formulas (1) and (lb). In some embodiments, the variable Rarepresents a bond to or linkage to the lipid (L). In some embodiments, the zwitterionic polymer may be bound to the lipid at a terminal position of the backbone. The variables R3, R4, and R5are independently selected from Ra. In some embodiments, at least one or two of R3, R4, and R5are alkyl (or more specifically, methyl and / or ethyl) or all of R3, R4, and R5are alkyl (or more specifically, methyl and / or ethyl). In separate or further specific embodiments, m has a value of 1, 2, 3, or 4. In separate or further specific embodiments, p has a value of 1, 2, 3, or 4, or a value of 2, 3, or 4. In separate or further specific embodiments, Rais H or methyl. In separate or further specific embodiments, X is O or NRb, wherein Rbis H or an alkyl group containing 1-3 carbon atoms, or Rbis H or methyl.

[0079] Some specific examples of zwitterionic polymers within the above formulas include:

[0080] The zwitterionic polymer of component (ii) may alternatively be within the scope of Formula (I) but not within the scope of Formula (1) or sub-formulas thereof. Some examples of zwitterionic polymers not within the scope of Formula (1) or sub-formulas thereof but which may be included in the lipid nanoparticle composition include the following, wherein q can have any of the values given above for m (e.g., at least 1):

[0081] In some embodiments, the zwitterionic polymer (ZP) in component (ii) is a betaine polymer. In other embodiments, the zwitterionic polymer is a poly(phosphatidylcholine) polymer, poly(trimethylamine N-oxide) polymer, poly(zwitterionic phosphatidylserine) polymer, or glutamic acid-lysine (EK)-containing polypeptide. In some embodiments, zwitterionic phosphatidyl serine comprises one neighboring positive charged moiety to balance the negative charge of the phosphoserine. In some embodiments, zwitterionic phosphatidyl serine comprises a compound as described in “De novo design of functionalzwitterionic biomimetic material for immunomodulation” Science Advances, 29 May 2020, Vol. 6, Issue 22, (DOI: 10.1126 / sciadv.aba0754) which is hereby incorporated by reference in its entirety.

[0082] 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 invention include carboxybetaine methacrylates, such as 2-carboxy-N,N-dimethyl-N-(2’- methacryloyloxyethyl) ethanaminium inner salt; carboxybetaine acrylates; carboxybetaine acrylamides; carboxy betaine vinyl compounds; carboxybetaine epoxides; and other carboxybetaine compounds with hydroxyl, isocyanates, amino, or carboxylic acid groups. In a particular embodiment, the polymer is a poly(carboxybetaine methacrylate) (poly(CBMA)).

[0083] In another embodiment, the zwitterionic polymer (ZP) is a homopolymer that has a positive charge in the polymer backbone and a pendant carboxylic acid group and has the formula:wherein R is selected from the group consisting of hydrogen and substituted or unsubstituted alkyl; Li and L2 are independently a straight or branched alkylene group optionally including one or more oxygen atoms; and x is an integer from 2 to 500.

[0084] Alternatively, the zwitterionic polymer (ZP) may be a mixed-charge zwitterionic polymer. The mixed-charge zwitterionic polymer has an equal number of negative (anionic) charges and positive (cationic) charges in separate units of the polymer. The mixed-charge zwitterionic polymer may be prepared by copolymerizing a polymerizablenegatively charged monomer and a polymerizable positively charged monomer, each present in substantially equal proportions in the polymerization mixture. The product of such a copolymerization is a zwitterionic polymer having 100 mole percent zwitterionic moieties, where each zwitterionic moiety is defined as a pair of repeating units: a repeating unit having a negative charge and a repeating unit having a positive charge. The mixed- charge zwitterionic polymer may also be copolymer produced from a polymerizable negatively charged monomer, a polymerizable positively charged monomer, and a polymerizable comonomer, which provides a zwitterionic polymer having less than 100 mole percent zwitterionic moieties. The mixed charge copolymer may contain, for example, 50% of a combination of negatively charged monomer and positively charged monomer and 50% of a polymerizable comonomer, which corresponds to a mixed-charge copolymer product having 50 mole percent zwitterionic moieties.

[0085] The mixed-charged zwitterionic polymer may be conveniently represented by the following general formula:wherein BBi and BB2 are independently selected from polymer backbones, such as Xi, X2, and X3, as described earlier above; Li and L2 are linkers, such as linear or branched alkylene groups optionally including one or more oxygen atoms; Zi is a positively charged group, such as an ammonium, phosphonium, or sulfonium group; Z2 is a negatively charged group, such as a carboxylate or borate group; x is an integer from 1 to 500; and y is an integer from 1 to 500.

[0086] The positively charged unit (Zi containing unit) of the mixed-charge zwitterionic polymer can be derived from a monomer having a positively charged pendant group. Representative monomers that can be used to derive the positively charged unit in the polymers of the present invention include 2-(dimethylamino)ethyl methacrylate, 2- (diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethylammonium chloride, and N-acetylglucosamine. In some embodiments, Zi is a heteroaromatic ring containing a positively charged nitrogen atom linked to Li, or Zi can be denoted as NR5R6,wherein R5and R6are independently substituted or unsubstituted alkyl group, and wherein R5and R6are optionally interconnected to form a heteroaromatic ring.

[0087] In one embodiment, the negatively charged unit of the mixed-charge zwitterionic polymer is derived from 2-carboxyethyl acrylate (CA), and the positively charged unit is derived from 2-(dimethylamino)ethyl methacrylate (DM). In another embodiment, the negatively charged unit is derived from 2-carboxyethyl acrylate (CA), and the positively charged unit is derived from 2-(diethylamino)ethyl methacrylate (DE). In another embodiment, the negatively charged unit is derived from 2-carboxyethyl acrylate (CA), and the positively charged unit is derived from [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM). In another embodiment, the negatively charged unit is derived from 2- carboxyethyl acrylate (CA), and the positively charged unit is derived from 2-aminoethyl methacrylate hydrochloride (NH2).

[0088] Component (iii) is or includes at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group. In some embodiments, the cationic or ionizable lipid contains a lipid moiety attached to a polymer that contains a cationic group or ionizable group. In some embodiments, the cationic or ionizable lipid contain a lipid moiety not attached to a polymer that contains a cationic group or ionizable group, or the lipid moiety is not attached to a polymer altogether. The term “cationic lipid,” as used herein, refers to a positively charged lipid (typically, by possessing an ammonium group). The cationic lipid may contain a trialkylamine group, which is necessarily positively charged when bound to the lipid. The term “ionizable lipid,” as used herein, refers to lipids that contain one or more groups capable of being ionized to result in a positive charge in the polymer. The ionizable lipid generally possesses a secondary or tertiary amino group, which may be an alkylated secondary or tertiary amine. More particularly, the ionizable lipid is a monoalkylamine or dialkylamine group, any of which can be protonated or alkylated to result in an alkylated ammonium group. In some embodiments, component (iii) is chemically distinct from component (i) such as by not including a glycan.

[0089] In some embodiments of the cationic lipid of component (iii), the positively charged group or ionizable group is not associated with a negative charge within the cationic lipid. Thus, the cationic lipid in this embodiment is not a zwitterionic lipid or a precursor to a zwitterionic lipid. In other embodiments of the cationic lipid of component (iii), the positively charged group or ionizable group is present along with a negatively chargedfunctional group within the cationic lipid. Thus, the cationic lipid in this embodiment is a zwitterionic lipid or a precursor to a zwitterionic lipid. In some embodiments, the cationic or ionizable lipid in component (iii) possesses a secondary, tertiary, or quaternary group along with a functional group which is negatively charged under physiological conditions.

[0090] In particular embodiments, the cationic or ionizable lipid possesses a dimethylamino or trimethylamino (or dimethylammonium or trimethylammonium) group. In particular embodiments, the cationic or ionizable lipids are selected from l,2-dioleoyl-3- dimethylammonium-propane (DODAP), 1 ,2-diIinoIeyloxy-N,N-dimethyI-3-aminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2DMA), and [(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 -tetraen- 19-yl] 4-(dimethylamino)butanoate (DLinMC3DMA).

[0091] In some embodiments, the ionizable lipid contains a lipid moiety attached to a secondary, tertiary or quaternary amine group along with a functional group that is negatively charged under physiological conditions. This lipid moiety can have the following formula:R1N - L' — AR2wherein:R1and R2are independently selected from H and alkyl groups and wherein the alkyl group may be saturated, unsaturated, branched, and / or unbranched, and may optionally include one or more heteroatoms selected from N, O, Si, P, S, Cl, Br, and F, wherein at least one of R1and R2corresponds to a lipid group, which may correspond to any of the lipids described in detail earlier above;L’ is a covalent linker group between N and A, wherein the covalent linker group is a linear or branched alkyl group containing 1-20 carbon atoms and may optionally include one or more heteroatoms selected from N, O, Si, P, S, Cl, Br, and F. Some examples of L include -CH2-, -CH2CH(OH)-, -CH2CHC1CH2-, -CH2OCH2-, -CH2SCH2-, -CH2SSCH2-, and -CH2COOCH2-; andA is a functional group that is negatively charged or capable of being deprotonated to form a negatively charged group at a pH of 5-8. Some examples of A include the following:(i) carboxylic acid group (A = -COOH or -COO ); or(ii) phosphate, i.e., where A =I— o-p-o-i -o-p-o-OH or SHand deprotonated (anionic) forms thereof;(iii) sulfonic acid group, i.e., where A =OiiO and deprotonated (anionic) forms thereof; and(iv) sulfonamide group, i.e., where A =

[0092] In some embodiments, the cationic or ionizable lipid is denoted by the following alternative formula:R1zN — L — A - (X)„R2.9, wherein n is 0 or 1; R , R , and L’ are as defined above, A- (X)n is a functional group that is negatively charged under certain pH conditions. X is an H or alkyl group that is saturated or unsaturated, linear or branched, and optionally containing heteroatoms such as one or more of N, O, Si, P, S, Cl, Br, and F.

[0093] Some examples of A-(X)ninclude the following:(i) A(X)n is a carboxylic acid group (A = -COOH and n=0) or(ii) A(X)n is a phosphate, where A =and n - 1. X = H or an alkyl group that is saturated or unsaturated, branched or not branched, all-carbon or containing heteroatoms such as but not limited to N, O, Si, P, S, Cl, Br, and F or(iii) A(X)nis a sulfonic acid group, where A =and n - 0(iv) A(X)n is a sulfonamide group, where A =

[0094] Some exemplary structures of cationic or ionizable lipids include:wherein n is typically a value in the range of 1-10.

[0095] In some embodiments, the cationic or ionizable lipid excludes a polyalkylene oxide (polyalkylene glycol) segment. In some embodiments, the cationic or ionizable lipid more specifically excludes a polyethylene oxide or polypropylene oxide segment. In some embodiments, the lipid nanoparticle excludes a polyalkylene oxide segment.

[0096] In some embodiments, the LNP further includes a steroid or sterol, or more particularly, cholesterol or a derivative thereof, which is considered herein to be an optional further component of the LNP. In some embodiments, the cholesterol derivative is a phytosterol, e.g., P-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol. In other embodiments, the cholesterol derivative is dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'- hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3p[N — (N'N'- dimethylaminoethyl)carbamoyl cholesterol (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, 25 -hydroxy cholesterol, 7-dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, 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, e.g., sodium cholate.

[0097] In some embodiments, each of components (i), (ii), and (iii) are chemically distinct from each other. In other embodiments, two or three of components (i), (ii), and (iii) are chemically combined by forming a single molecule that has a structure within the scope of the two or three components. The term “chemically combined” indicates that the two or three components are covalently bonded to each other. As a first example, the glycan- containing lipid and cationic lipid may be chemically combined into a single lipid that has a structure that includes the scope of both the glycan-containing lipid and cationic lipid as defined above. As a second example, the glycan-containing lipid and non-cationic lipid may be chemically combined into a single lipid that has a structure that includes the scope of both the glycan-containing lipid and non-cationic lipid as defined above. As a third example, the glycan-containing lipid, cationic lipid, and non-cationic lipid may be chemically combined into a single lipid that has a structure that includes the scope of the glycan-containing lipid, cationic lipid, and non-cationic lipid as defined above. Notably, cationic and non-cationic lipids can be combined since they differ not only in the charge domain but also in their linker and / or lipid structures. Thus, a lipid may be composed of a portion of the cationic lipid and non-cationic lipid according to the description of these lipids above. For example, a lipid tail structure of a cationic lipid may be combined with a hydrophilic head group of a non-cationic lipid. As a fourth example, the glycan-containing lipid and a steroid or sterol (e.g., cholesterol or a derivative thereof) may be chemically combined into a single lipid that has a structure that includes the scope of both the glycan- containing lipid and steroid or sterol (e.g., cholesterol or a derivative thereof) as defined above. As a fifth example, the glycan-containing lipid, cationic lipid, and a steroid or sterol (e.g., cholesterol or a derivative thereof) may be chemically combined into a single lipid that has a structure that includes the scope of the glycan-containing lipid, cationic lipid, andsteroid or sterol (e.g., cholesterol or a derivative thereof) as defined above. As a sixth example, the glycan-containing lipid, non-cationic lipid, and a steroid or sterol (e.g., cholesterol or a derivative thereof) may be chemically combined into a single lipid that has a structure that includes the scope of the glycan-containing lipid, non-cationic lipid, and steroid or sterol (e.g., cholesterol or a derivative thereof) as defined above. As a seventh example, the glycan-containing lipid, non-cationic lipid, cationic lipid, and a steroid or sterol (e.g., cholesterol or a derivative thereof) may be chemically combined into a single lipid that has a structure that includes the scope of the glycan-containing lipid, non-cationic lipid, cationic lipid, and steroid or sterol (e.g., cholesterol or a derivative thereof) as defined above.

[0098] The LNP further includes a therapeutic substance, herein denoted as component (iv). The therapeutic substance is incorporated into or encapsulated by the self- assembled shell constructed of one or more of the first, second, and / or third lipid components described above. The therapeutic substance can be any substance having therapeutic value for a living organism, particularly a mammal, such as a human or animal subject. The therapeutic substance may be selected from, for example, nucleic acids (including nucleobases, nucleotides, oligonucleotides, and polynucleotides), peptides (including amino acids, dipeptides, tripeptides, oligopeptides, and proteins), and pharmaceuticals (e.g., anti-cancer drugs, antibiotics, anti-inflammatory drugs, and psychiatric drugs).

[0099] In particular embodiments, the therapeutic substance is a nucleic acid molecule, or more particularly, a negatively charged nucleic molecule. The nucleic acid molecule may be, for example, a nucleotide, nucleoside, nucleobase, oligonucleotide, or a polynucleotide (e.g., DNA or RNA). In particular embodiments, the therapeutic substance contains one or more such nucleic acid molecules. Examples of RNA molecules that can be included as the therapeutic substance include messenger RNA (mRNA), viral RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), short (or small) interfering RNA (siRNA), microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interf ering complementary RNA (micRNA), multivalent RNA, circular RNA (circRNA), crispr RNA (crRNA), long-noncoding RNA (IncRNA), CRISPR-related RNA, Cas nuclease mRNA, guide RNA, or single-guide RNA, and combinations thereof. Examples of DNA molecules that can be included as the therapeutic substance include plasmid DNA, oligo DNA, and complementary DNA (cDNA).

[0100] In particular embodiments, the therapeutic molecule is selected from one or more of DNA, RNA, ssDNA, dsDNA, ssRNA, dsRNA, and hybrids thereof. In other embodiments, the therapeutic molecule is selected from one or more of plasmid DNA or linearized DNA. In other embodiments, the therapeutic molecule is selected from one or more of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), and / or long-noncoding RNA (IncRNA). In other embodiments, the therapeutic molecule is an antisense oligonucleotide (ASO). In other embodiments, the therapeutic molecule is Cas nuclease mRNA and / or 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 may be or include a vaccine against SARS-Cov-2, particularly wherein the vaccine is an mRNA vaccine or wherein the mRNA vaccine corresponds to a spike protein or portion thereof. In particular embodiments, the therapeutic substance contains RNA, or more particularly, mRNA, or more particularly viral mRNA. In other particular embodiments, the therapeutic substance is or includes a nucleic acid encoded in a spike protein of a virus, such as a coronavirus, SARS-COV2 (COVID- 19), or HIV virus.

[0101] In other particular embodiments, the nucleic acid is a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and / or hybrids thereof, or wherein the DNA is plasmid DNA or linearized DNA. In some embodiments, the RNA is a combination of: a) Cas nuclease mRNA, and b) a guide RNA nucleic acid that is or is a single-guide RNA (sgRNA).

[0102] In any of the nucleic acids disclosed in this application, the nucleotide (e.g., DNA or RNA) may encode fusion biological moieties containing protective domains and functional domains. In some embodiments, the functional domains are fused directly or via a linker (e.g., containing amino acids) to the protective domains. In some embodiments, the functional domains are fused to the protective domains directly or via a linker consisting of amino acids. In further particular embodiments, the protective domain may comprise: 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 a plurality of additional amino acids independently selected from the group consisting of proline, serine, threonine, asparagine, glutamine, glycine, and derivatives thereof, wherein the ratio of the number of positively chargedamino acids to the number of positively charged amino acids is from about 1 :0.5 to about 1:2. In some embodiments, the protective domain can be selected from other amino acid polymers, such as extended recombinant polypeptide (XTEN), proline- alanine- serine, and / or elastin-like polypeptides. In any of the foregoing embodiments, the protective domain can be selected from natural half-life extension domains (e.g., Fc fragment).

[0103] In some embodiments, the LNP composition is chemically modified with a targeting ligand. Any one or more of the components of the lipid nanoparticle described above may be chemically modified to contain a targeting ligand. In some embodiments, the targeting ligand is in the glycan-containing lipid, i.e., component (i). In other embodiments, the targeting ligand is in the non-cationic lipid, i.e., component (ii). In other embodiments, the targeting ligand is in the cationic lipid, i.e., component (iii). In other embodiments, the targeting ligand is in the therapeutic substance, i.e., component (iv). The targeting agent may also be included, in two, three, or all of components (i)-(iv). The targeting ligand may be, for example, an antibody (or Fab or Fc). In some embodiments, the glycan-containing moiety (S) is chemically conjugated or fused to an extracellular vesicle (EV). In other embodiments, the glycan-containing moiety (S) is chemically conjugated to an adeno- associated virus (AAV). In particular embodiments, the LNP is modified to possess a targeting ligand to deliver LNPs loaded with a therapeutic or diagnostic agent or both of them to a targeted area within a specific organ in the body. The targeting agent may be, for example, a peptide (e.g., RGD), a lipid (e.g., phosphoserine-containing lipid), a protein (e.g., apolipoprotein E), an aptamer (e.g., anti-VEGF aptamer), a sugar (e.g., sialic acid) and an antibody (e.g., anti-PDl) or antibody fragment (e.g., Fab or Fc).

[0104] In some embodiments, any component (i) disclosed in this application is combined with any component (ii) disclosed in this application. Moreover, any component (iii) disclosed in this application may be combined with any combination of components (i) and (ii). Still further, any component (iv) disclosed in this application may be combined with any combination of components (i), (ii), and (iii). Still further, any combination of components (i), (ii), (iii), and (iv) can be used for any method disclosed in this application.

[0105] In other aspects, the present disclosure is directed to methods of delivering a therapeutic substance to a subject. In the method, an LNP formulation containing a therapeutic substance is administered to a subject. The therapeutic substance may be any of those described above. Moreover, the LNP being delivered in the method can include anycombination of components (i), (ii), (iii), and (iv). In some embodiments, the LNP composition may be delivered to cells of the subject, wherein the LNP composition may or may not be targeted to specific cells of the subject. In embodiments, the therapeutic substance is a nucleic acid molecule, and administration of the LNP results in gene therapy or vaccination of the subject. In some embodiments, the method is for targeted delivery of a therapeutic agent to a secondary lymphoid organ (SLO) in a subject, wherein the SLO may be the spleen or lymph nodes. The therapeutic agent may be or include a nucleic acid molecule, such as any of those described earlier above. In other embodiments, the method is for targeted delivery of a therapeutic agent to the brain of a subject or bone marrow of a subject. The targeted delivery of the therapeutic agent may provide, for example, cancer immunotherapy, autoimmune disease immunotherapy, vaccination, and / or gene editing.

[0106] In some embodiments, the method delivers any of the LNP formulations described above for targeted delivery of a therapeutic agent to specific cells (e.g., immune cells, precursor cells, or stem cells) in a subject, wherein the therapeutic agent may be, for example, a nucleic acid, such as any of those described above. In the method, the therapeutic agent in the lipid nanoparticle is targeted to specific cells of the subject. The targeted delivery of the therapeutic agent may provide, for example, cancer immunotherapy, autoimmune disease immunotherapy, vaccination and / or gene editing. The specific cells may be selected from, for example, lymphoid cells, myeloid cells, and stem cells. In some embodiments, the specific cells are immune cells selected from natural killer cells, natural killer B / T cells, dendritic cells, macrophages, monocytes, B cells, T cells, neutrophils, and microglia cells. In other embodiments, the specific cells are selected from liver sinusoidal endothelial cells, astrocytes, neurons, precursor cells, and pluripotent stem cells. In some embodiments, the LNP is used to deliver nucleic acids encoding chimeric antigen receptor or an exogenous T cell receptor into immune cells.

[0107] In any of the above described targeted delivery methods, the LNP may be used in different ways, such as, for example: injected directly in vivo and delivered into the host cells in vivo; or the LNP is transfected into the host cells ex vivo and the resulting cells are then infused in vivo; or the LNP is administered to a mammal intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation; or the LNP is used for multiple in vitro and in vivo applications, including, but not limited to, protein replacement therapy, cancer immunotherapy, cell therapy, cancer vaccines, infectiousdisease vaccines, gene therapy, autoimmune disease treatment and cancer diagnosis; or the LNP is used for in vitro and in vivo production of extracellular vesicles; or the LNP is used for clustered regularly interspaced short palindromic repeats-Cas endonuclease (CRISPR- Cas) gene editing, prime editing, based editing, and RNA editing in vitro and in vivo; or the LNP is used for vaccines against coronavirus (e.g., SARS-CoV-2); or the LNP is delivered along with checkpoint inhibitor (e.g., anti- Programmed death-ligand 1 (anti-PD-Ll) antibody, anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4)) to treat cancer; or the LNP is delivered along with cytokines (e.g., Interleukin- 12, Interleukin-35, and Interferon alpha) to treat a disease.

[0108] In other aspects, the present disclosure is also directed to a method of delivering a therapeutic substance (e.g., a nucleic acid) to a subject in which monocytes and / or macrophages have been depleted. The lipid nanoparticles and compositions of the present invention may be used for a variety of purposes, including the delivery of nucleic acid molecules, rihonucleoprotein (RNP) and numerous other therapeutic substances. In the method, a lipid nanoparticle (LNP) composition loaded with the therapeutic substance is administered to a subject in which monocytes and / or macrophages have been depleted. In some embodiments, the method may be practiced by first depleting monocytes and / or macrophages in the subject followed by administering the LNP loaded with the therapeutic substance (e.g., a nucleic acid) to the subject, thereby delivering the therapeutic substance into the subject. The subject is typically a mammal, more typically a human subject, but may also be another type of mammal, such as a pet or farm animal, such as a primate, dog, cat, cow, sheep, mouse, rat, rabbit, horse, or pig.

[0109] In some embodiments, the method of delivering the therapeutic substance results in a method of treating the subject. As a method of treatment, the LNP composition, which is loaded with a therapeutic substance (e.g., nucleic acid), can be administered for the purpose of, for example, protein replacement therapy, cancer immunotherapy, cancer vaccine therapy, infectious disease vaccines, gene editing, autoimmune disease treatment, and / or cancer diagnosis. In particular embodiments, the LNP composition is administered for gene therapy comprising 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 particular embodiments, the LNP is used for clustered regularly interspaced short palindromic repeats-Cas endonuclease (CRISPR-Cas) gene editing in vitro and in vivo, forexample including but not limited to delivering one or more nucleic acids that encode for one or more CRISPR associated proteins such as Cas protein. In some embodiments, the LNP is administered along with a checkpoint inhibitor (e.g., anti- Programmed death-ligand 1 (anti-PD-Ll) antibody or anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) to treat cancer.

[0110] In some embodiments, the method of treatment includes targeted delivery of a therapeutic agent to a secondary lymphoid organ (SLO) in a subject, wherein the subject is administered any of the LNP compositions described above containing any suitable therapeutic agent described above. The SLO may be, for example, spleen and / or lymph nodes. To achieve this, the therapeutic agent or other component of the lipid nanoparticle is capable of targeting a secondary lymphoid organ (SLO) of the subject. The targeting agent may be a glycan-containing lipid, as described above, such as GM3, GD1A, GD3, GQ1B, GT1B, or other glycan-containing lipid. In some embodiments, the lipid nanoparticle is modified to include a further or alternative targeting agent that targets SLO cells. The further or alternative targeting agent may include antibodies targeting to specific cell types. For example, an NK1.1 antibody can be conjugated to the lipid to target NK cells. The lipid nanoparticle can alternatively or in addition include lipids containing active targeting ligands to immune cells. For example, a phosphoserine-containing lipid can be added to lipid nanoparticle for macrophage targeting. In some embodiments, the therapeutic agent is or includes a nucleic acid molecule. Some types of nucleic acids that may be selectively delivered to an SLO include any of those described earlier above, e.g., mRNA, DNA, siRNA, circular RNA, self-amplify RNA, long-noncoding RNA, tRNA, and the like. The targeting to SLOs can provide specific gene editing in immune cells in SLOs, improve vaccinations, and facilitate the engineering of specific immune cells, such as engineering of immune cells with CAR for therapeutic purposes. In some embodiments, a phosphoserine- containing lipid is included in the LNP. The phosphoserine-containing lipid may be, for example, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or a naturally-occurring PS- lipid, such as L-a-phosphatidylserine (brain). In some embodiments, the targeted delivery results in cancer immunotherapy, autoimmune disease immunotherapy, or gene editing.

[0111] In some embodiments, the method of treatment includes targeted delivery of a therapeutic agent to the brain of a subject, wherein the subject is administered any of the LNP compositions described above containing any suitable therapeutic agent describedabove. To achieve this, the therapeutic agent or other component of the lipid nanoparticle is capable of targeting brain cells of the subject. In some embodiments, the lipid nanoparticle is modified to include a targeting agent that targets brain cells. The targeting agent may be, for example, a glycan that can bind to receptors on the cells in the brain such as glia cells and neurons. The targeting glycan may mediate important biological processes in the brain. In some embodiments, the therapeutic agent is or includes a nucleic acid molecule. Some types of nucleic acids that may be selectively delivered to a brain include any of those described earlier above, e.g., mRNA, DNA, siRNA, circular RNA, self-amplify RNA, long- noncoding RNA, tRNA, and the like. Targeting to the brain can provide specific gene editing and cell engineering in neurons and glia cells, which can be used to treat a neurodegenerative disease, such as Alzheimer’s disease, Parkinson’s disease, forms of dementia, and the like.

[0112] In some embodiments, the method of treatment includes targeted delivery of a therapeutic agent to the bone marrow of a subject, wherein the subject is administered any of the LNP compositions described above containing any suitable therapeutic agent described above. To achieve this, the therapeutic agent or other component of the lipid nanoparticle is capable of targeting the bone marrow of the subject. In some embodiments, the lipid nanoparticle is modified to include a targeting agent that targets bone marrow. The targeting agent may be, for example, a glycan that can bind to receptors on the cells in the bone marrow, such as stem cells and blood cells. For example, C-type lectin receptors on blood cells in the bone marrow can recognize glycans which consequently mediate cellular immune responses. Some glycan-containing lipid nanoparticles can be used to transfect these blood cells with lectin receptors and activate immune responses for specific antigens encoded by mRNA. In some embodiments, the therapeutic agent is or includes a nucleic acid molecule. Some types of nucleic acids that may be selectively delivered to a bone marrow include any of those described earlier above, e.g., mRNA, DNA, siRNA, circular RNA, self-amplify RNA, long-noncoding RNA, tRNA, and the like. Targeting to bone marrow can provide specific gene editing and cell engineering in stem cells and blood cells, which can be applicable for vaccinations, genetic modifications, and the like.

[0113] The LNP composition is typically administered in the form of a pharmaceutical composition containing the LNP. In the pharmaceutical composition, the LNP may be dissolved or suspended in, or admixed with, a pharmaceutically acceptable carrier, whichmay be a liquid, semi-solid (e.g., gel or wax), or solid, as well known in the art. The phrase “pharmaceutically acceptable” refers herein to those compounds, materials, compositions, and / or dosage forms which are, within the scope 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 to the subject. Any of the carriers known in the art can be suitable herein depending on the mode of administration.

[0114] Some examples of pharmaceutically acceptable liquid carriers include alcohols (e.g., ethanol), glycols (e.g., propylene glycol and polyethylene glycols), polyols (e.g., glycerol), oils (e.g., mineral oil or a plant oil), paraffins, and aprotic polar solvents acceptable for introduction into a mammal (e.g., dimethyl sulfoxide or N-methyl-2- pyrrolidone) any of which may or may not include an aqueous component (e.g., at least, above, up to, or less than 10, 20, 30, 40, or 50 vol% water). Some examples of pharmaceutically acceptable gels include long-chain polyalkylene glycols and copolymers thereof (e.g., poloxamers), cellulosic and alkyl cellulosic substances (as described in, for example, U.S. Patent 6,432,415), and carbomers. The pharmaceutically acceptable wax may be or contain, for example, carnauba wax, white wax, bees wax, glycerol monostearate, glycerol oleate, and / or paraffins.

[0115] In some embodiments, the pharmaceutical composition contains solely the LNP and one or more solvents or the carrier. In other embodiments, the pharmaceutical composition includes one or more additional components. The additional component may be, for example, a pH buffering agent, mono- or polysaccharide (e.g., lactose, glucose, sucrose, trehalose, lactose, or dextran), preservative, electrolyte, surfactant, or antimicrobial. If desired, a sweetening, flavoring, or coloring agent may be included. Other suitable excipients can be found in standard pharmaceutical texts, e.g. in “Remington's Pharmaceutical Sciences”, The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., 1995.

[0116] The LNP composition, typically in the form of a pharmaceutical composition in which the loaded LNP is admixed with or suspended in a liquid or solid pharmaceutically acceptable carrier, can be administered to the subject by any suitable route. The LNP may be administered intravenously, orally, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In particular embodiments, the LNP is administered byinjection into the subject. In some embodiments, the LNP is delivered to cells of the subject. In some embodiments, the LNP is delivered by removing cells from the subject, administering the lipid nanoparticle to the removed cells, and then reintroducing the removed cells to the subject. In some embodiments, the LNP is injected directly in vivo and delivered into the host cells in vivo. In other embodiments, the LNP is transfected into the host cells ex vivo and the resulting cells are then infused in vivo.

[0117] As noted earlier above, by temporarily depleting monocytes and / or macrophages, more LNPs are advantageously permitted to accumulate in desirable tissues and or targeted cells, thereby resulting in enhanced nucleic acid (e.g., mRNA) delivery or maintained efficacy with lowered dose. In embodiments of the method, the subject is pre-treated by depleting monocytes and / or macrophages in the subject, i.e., before the subject is administered or otherwise treated with the loaded LNP composition described above. In some embodiments, the depletion of monocytes and / or macrophages is achieved by administering to the subject small molecule drugs having this ability. Some examples of small molecule drugs include clodronate, zoledronate, etidronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, and risedronate. In other embodiments, the depletion of monocytes and / or macrophages is achieved by administering to the subject liposome compositions loaded with small molecule drugs having this ability. Some examples of such liposome compositions include liposome clodronate (LipoD) and liposome zoledronate. In other embodiments, the depletion of monocytes and / or macrophages is achieved by administering to the subject antibodies configured to deplete monocytes and / or macrophages. Some examples of antibodies having this ability include anti-CDl 15, anti-CCR2, anti-Ly6C, anti-Gr-1. The depletion of monocytes and / or macrophages may be achieved within hours (e.g., 1, 2, 3, 4, 5, 6, 12, 18, or 24 hours, or range therein), within days (e.g., 1, 2, 3, 4, 5, 6, or 7 days, or range therein), or within weeks (e.g., 1, 2, 3, or 4 weeks), and the LNP may be administered to the subject after any one of the periods of time exemplified above for achieving depletion of monocytes and / or macrophages.

[0118] In some embodiments, the nucleic acid encodes a protein that is expressed in the subject. In some embodiments, the nucleic acid is a component of a gene editing machinery. In some embodiments, the nucleic acid is delivered into one or more of liver, brain, spleen, lymph nodes, kidneys, and lungs. The nucleic acid may be delivered into anybiological tissue of the subject, including extracellular fluid (e.g., blood or blood plasma, i.e., systemically) and / or cells of the subject, wherein the cells may or may not be within organs or blood of the subject. The enhanced delivery of nucleic acid into the subject, as provided by the method described above, may result in an improved expression of protein encoded by the delivered nucleic acid. Alternatively or in addition, the enhanced delivery of nucleic acid into the subject may result in an improved gene editing efficiency by the delivered nucleic acid. Alternatively or in addition, the enhanced delivery of nucleic acid into the subject may enhance nucleic acid delivery into one or more of liver, brain, spleen, lymph nodes, kidneys, and lungs. The term “enhanced” or “enhancement,” as used herein, indicates the improvement in delivery of the nucleic acid achieved by the present method (i.e., wherein monocytes and / or macrophages have been depleted in the subject) compared to the delivery of the nucleic acid to a subject in which monocytes and / or macrophages have not been depleted. In embodiments, the enhancement may be quantified as an at least or greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 120%, 150%, 180%, or 200% enhancement. In some embodiments, the nucleic acid encodes a protein, and the protein expression is enhanced by any of the values provided above. In other embodiments, the nucleic acid (e.g., mRNA) is a component of a gene editing machinery, and the gene editing is enhanced by any of the values provided above. In some embodiments, the nucleic acid is delivered into one or more of liver, brain, spleen, lymph nodes, kidneys, and lungs, and the delivery is enhanced by any of the values provided above.

[0119] Examples have been set forth below for the purpose of illustration and to describe the best mode of the invention at the present time. However, the scope of this invention is not to be in any way limited by the examples set forth herein.Examples

[0120] Example 1. Preparation and Characterization of LNP-mRNA Formulations by Microfluidic Mixing

[0121] The LNPs were formulated according to the lipid formulations in Table 1 below. The firefly luciferase-encoding mRNA (Fluc-mRNA) was dissolved in 10 mM citrate buffer (pH 5). The lipid solutions were mixed with the acidic mRNA phase rapidly in a microfluidic device to generate LNPs. Successful encapsulation of the LNPs wasconfirmed by a size measurement and polydispersity index (PDI) using dynamic light scattering (DLS). The LNPs were studied further only if they had a size smaller than 200 nm, above which generally leads to phagocytosis and quick removal from the blood stream. Finally, the RNA content and encapsulation efficiency were evaluated. Briefly, the LNPs were diluted in a lx TE buffer. Then, the LNPs were lysed in a IX TE buffer containing 2% Triton X-100 (TE-TX) and incubated at 37 C for 30 min. The fluorescent dye was added to the intact sample to detect the free RNA molecules outside the particle and added to the lysed sample to detect the total amount of RNA including those entrapped in the particles. Quantification of RNA was conducted using a standard curve with known RNA concentration in TE buffer and TE-TX buffer, respectively. The encapsulation efficiency (EE) was given as EE% = (1 - free mRNA / total RNA)xl00%. The anticipated encapsulation efficiencies was greater than 70%.

[0122] The novel LNPs with glycan-containing lipids ranging from 2.5 to 10 are within the normal range of functional LNPs. The size numbers are below 200 nm in diameter. The PDI numbers are below 0.7. The mRNA encapsulation rate was found to be within the range of 70%-100%.Table 1. The formulation, diameter, polydispersity index, and mRNA encapsulation rate for each LNP.

[0124] Example 2. In Vitro Transfection

[0125] In this example, an in vitro test was performed to evaluate the transfection efficiency of LNPs. Different gangliosides were incorporated into lipid nanoparticles (LNP) with different molar ratios as SM102: cholesterol: DSPC: DMG-PEG2k: gangliosides = 50: 38.5: 10: 1.5: X, where, for example, X=2.5 (GM3,1), 5 (GM3.2), 10 (GM3, 3) or 20 (GM3,4), representing the amount of GM3 included in the LNP. SMI 02- LNP was used as a control without ganglioside incorporation. LNPs encapsulating Fluc- mRNA were added to a 100 pL / well of culture medium at a concentration of 100 ng mRNA per well. After 24 hours of transfection, the bioluminescence signal was detected using a luciferase assay system in a black 96- well plate following the manufacturer’s protocol.

[0126] FIGS. 2A-2C show the in vitro expression of Fluc-mRNA delivered by LNPs in different cell lines: THP1 monocytes (FIG. 2A); RAW264.7 macrophages (FIG. 2B); and HELA epithelial cells (FIG. 2C). As shown in FIGS. 2A-2C, a higher RLU number refers to a higher transfection efficiency and mRNA expression. Due to the glycan moieties on the LNPs, the novel LNPs have different transfection levels compared to the control SM102-LNP. Moreover, changing the ratios of the glycan-containing lipids in the formulations also impacts the transfection. The novel LNPs without sialic acids on the surface (LacCer, GlcCer, GalCer) tend to have highest transfection in HELA (epithelial cells) while the ones with sialic acids on the surface (GM3, GD3, GM1, GDI A, GQ1B, GT1B) barely transfect any HELA cells. However, the novel LNPs with sialic acids have higher transfection in immune cell lines, including THP1 (monocytes) and especially RAW264.7 (macrophages). Also, a glycan-containing lipid composing 2.5%-10% of the total lipids is optimal to form an LNP.

[0127] Example 3. Whole Body Imaging

[0128] In this example, in vivo delivery of LNPs encapsulating Fluc-mRNA was performed to evaluate the mRNA expression level in different organs. C57BL / 6 male mice with a weight of 20-25g were administered the selected novel LNPs and SM102-LNP encapsulating the Fluc-mRNA via retro-orbital injections. The dosage was 0.1 mg mRNA / kg. After 6 hours, shaved mice were injected with 150 mg / kg of D-luciferin substrate diluted in PBS. The whole-body images were taken 10 minutes after the substrate injectionunder the IVIS Lumina imaging system. The bioluminescent images were shown as surface radiance (p / sec / cm2 / sr) at a same scale.

[0129] As shown in the whole body images in FIG. 3, the majority of luminescence signal is in the liver of mice administered with LNPs without sialic acids (GlcCer, LacCer, GalCer, and SM102). However, incorporation of glycan-containing sialic acids (GD3, GDI A, GQ1B) shifts the expression significantly towards the spleen and lymph nodes. Notably, the GM1-LNP, which does not have the sialic acid at the outermost of the lipid, has highest signal in the liver but not the spleen. In summary, the novel LNPs with sialic acids at the outermost of the lipid shown specific targeting to secondary lymphoid organs (SLOs).

[0130] Example 4. Targeting to Secondary Lymphoid Organs

[0131] In this example, in vivo delivery of LNPs encapsulating Fluc-mRNA was performed to evaluate the mRNA expression level in different organs. C57BL / 6 male mice with a weight of 20-25g were administered with the selected novel LNPs and SM102-LNP encapsulating the Fluc-mRNA via retro-orbital injections. The dosage was 0.1 mg mRNA / kg. After 6 hours, shaved mice were injected with 150 mg / kg of D-luciferin substrate diluted in PBS. The organs were extracted from mice 10 minutes after the substrate injection and imaged under the IVIS Lumina imaging system. The bioluminescence signal was then analyzed.

[0132] As shown in FIG. 4A, the majority of the luminescence signal was in the liver for SM102-LNP. For LNPs modified with sialic acids (GD1A, GM3), spleen and lymph nodes showed significantly higher signals than the liver. The total flux of bioluminescent signal of were analyzed to quantitively evaluate the targeting effects to the SLOs. As shown in FIG. 4B, the ratio of the total flux in SLOs versus liver was approximately 6-fold higher in the novel LNPs with sialic acids than that in the SM102-LNP. Therefore, the novel LNPs with sialic acids have specific targeting to secondary lymphoid organs (SLOs).

[0133] Example 5. Targeting to Non-B and T cells

[0134] In this example, an in vivo study of the distribution and accumulation of mRNA / LNP was performed. The novel LNPs and SM102-LNP encapsulating Cy5 -labeled mRNAwere injected into the mice intravenously at a dosage of 0.5 mg / kg. After 3 hours, the spleen and lymph nodes were extracted from the mice. Cells extracted from the spleen and lymph nodes were labeled and identified by surface marker-specific antibodies. Samples were then analyzed. The percentages of Cy5 signal in different cell types were quantified using commercial software.

[0135] As shown in FIG. 5 A, the pie charts show the percentages of lymphocytes and myeloid cells in Cy5+ cells in both spleen and lymph nodes. Compared to SM102-LNP, both GM3 and GDI A have higher accumulation in myeloid cells. Moreover, as shown in FIG. 5B, the LNPs have higher accumulations in CD 169 macrophages in both spleen and lymph nodes. These macrophages highly express sialoadhesins (CD169), which can specifically recognize sialic acid and mediate the targeted delivery of the novel LNPs, on their surfaces.

[0136] Example 6. Targeting NK Cells

[0137] In this example, the in vivo mRNA expression level in different cell types was studied. The Cre recombinase (Cre)-loxP system was implemented to show the single-cell resolution of mRNA expression. Ail4 / Cre mice were injected with Cre mRNA encapsulated by the novel LNPs and SM102-LNP. In this mouse model, a strong fluorescent signal from tdTomato correlates with efficient cell uptake and translation of the mRNA encoding Cre. After 2 days post-injection, the main organs including the liver, spleens, and lymph nodes were extracted to obtain single-cell suspensions. Cells were stained with surface-marker specific antibodies and processed under a flow cytometer to identify their populations and the tdTomato signal.

[0138] As shown in FIG. 6, over 70% of NK1.1 cells efficiently express tdTomato in groups of GM3- and GDlA-LNPs, whereas the percentage is only 20% in the control group of SMI 02. Given the fact that the population of NK cells is relatively rare, the result here indicates GM3 as well as GDlA-LNPs as promising drug carriers for in vivo NK cells- based therapies, such as CAR-NK. Moreover, these two LNPs show higher transfection efficiencies in monocytes and plasmacytoid dendritic cells. Accordingly, the novel LNPs have specific targeting to immune cells such as monocytes, dendritic cells, and especially NK1.1 cells.

[0139] Example 7. Inducing Antigen-Specific Responses

[0140] In this example, the ability of glycan-containing LNPs to induce antigen-specific responses was studied. Since glycan-containing LNPs can target antigen-presenting cells (APCs) and modulate immune responses, they can deliver antigen-encoding mRNA to APCs more efficiently than commercial formulations, resulting in stronger antigen-specific responses.

[0141] Mice were injected with LNPs encapsulating mRNA encoding the ovalbumin (OVA) antigen on days 0, 3, and 7. Blood samples were drawn on days 14 and 21 to measure anti-OVA antibodies. On day 28, spleens were collected, and cells were extracted to co-incubate with OVA antigens to assess antigen-specific T-cell responses.

[0142] As shown in FIG. 7A, the overall injection scheme is described. As shown in the graphs in FIG. 7B, anti-OVA antibody levels were significantly higher in mice treated with glycan-containing LNPs. This indicates that glycan LNP induces a stronger humoral response. Moreover, FIGS. 7C and 7D demonstrate that glycan-containing LNPs induce more OVA-specific pro-inflammatory (IFN-y+) CD4 and CD8 T cells. Therefore, the glycan-containing LNPs exhibit an excellent capability for inducing stronger antigenspecific immune responses, which makes them suitable for various applications, such as vaccines.

[0143] Example 8. Engineering CAR-NK Cells In Vivo

[0144] In this example, the ability of glycan-containing LNPs to engineer NK cells with chimeric antigen receptors (CAR) in vivo was investigated. Glycan-containing formulations have been shown to efficiently deliver mRNA to NK cells, facilitating in vivo CAR NK cell engineering by introducing CAR-encoding mRNA into NK cells.

[0145] As shown schematically in FIG. 8A, mice were intravenously injected with antiCD 19 CAR mRNA. NK cells were then extracted from the spleen and incubated with CD 19-positive tumor cells to assess specific tumor cell killing and NK cell cytotoxicity. As shown in FIG. 8B, glycan-containing LNPs resulted in higher CAR expression on NK cells. In FIG. 8B, the top two images are flow cytometry graphs showing the percentages of CAR-positive NK cells in total NK cells. The bottom image in FIG. 8B is a graphquantifying CAR-positive NK cells in different numbers of NK cells in the tumor killing assay. Additionally, as shown in FIG. 8C, these engineered CAR-NK cells demonstrate specific killing of CD19-positive tumor cells, with a cytotoxicity level of 30%. As indicated in Figure. 8D, the levels of cytotoxic markers, such as CD107a and granzyme B, are also higher in NK cells treated with glycan-containing LNPs compared to those treated with the commercial formulation. In conclusion, the glycan-containing LNPs can efficiently mediate CAR-NK cells engineering in vivo. These findings highlight the potential of glycan-containing LNPs in enhancing the efficiency and effectiveness of CAR-immune cell therapies. FIG. 8D shows the levels of cytotoxicity markers on CAR-NK cells after incubation with tumor cells in vitro.

[0146] While there have been shown and described what are at present considered the preferred embodiments of the invention, those skilled in the art may make various changes and modifications which remain within the scope of the invention defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A lipid nanoparticle (LNP) composition comprising:(i) at least one glycan-containing lipid containing a glycan-containing moiety (S) linked to a lipid (L);(ii) at least one non-cationic lipid selected from charged and uncharged lipids;(iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and(iv) at least one therapeutic substance.

2. The composition of claim 1 , wherein the therapeutic substance in component (iv) is a nucleic acid molecule.

3. The composition of claim 2, wherein the nucleic acid molecule is an RNA.

4. The composition of claim 3, wherein the RNA is mRNA.

5. The composition of claim 3, wherein the RNA is viral mRNA.

6. The composition of claim 3, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), viral RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), long- noncoding RNA (IncRNA), antisense oligonucleotide (ASO), a CRIS PR-related RNA, a Cas nuclease mRNA, a guide RNA, a single-guide RNA, and combinations thereof.

7. The composition of claim 1, wherein the therapeutic substance is a nucleic acid encoded in a spike protein of a virus.

8. The composition of claim 2, wherein the nucleic acid is a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.

9. The composition of claim 8, wherein the DNA is plasmid DNA or linearized DNA.

10. The composition of claim 8, wherein the RNA is a combination of:a. Cas nuclease mRNA, and; b. a guide RNA nucleic acid that is or is a single-guide RNA (sgRNA).

11. The composition of claim 8, wherein the DNA or RNA is encoding fusion biological moieties comprising protective domains and functional domains.

12. The composition of claim 11, wherein the functional domains are fused directly or via a linker consisting of amino acids to the protective domains.

13. The composition of claim 11 , wherein the protective domain of the fusion biological moieties is a domain or domains comprising: a) a plurality of negatively charged amino acids (e.g., aspartic acid, glutamic acid, and derivatives thereof); b) a plurality of positively charged amino acids (e.g., lysine, histidine, arginine, and derivatives thereof); and c) a plurality of additional amino acids independently selected from the group consisting of proline, serine, threonine, asparagine, glutamine, glycine, and derivatives thereof; and wherein the ratio of the number of positively charged amino acids to the number of positively charged amino acids is from about 1 :0.5 to about 1 :2.

14. The composition of claim 11, wherein the protective domain of the fusion biological moieties are selected from other amino acid polymers, such as extended recombinant polypeptide (XTEN), proline-alanine-serine, and elastin-like polypeptides.

15. The composition of claim 11, wherein the protective domain of the biological moieties of can be selected from other natural half-life extension domains (e.g., Fc fragment).

16. The composition of any one of claims 1-15, wherein the gly can-containing lipid of component (i) has the general structure S-L, wherein S is a glycan-containing moiety selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and L is a lipid, wherein S is covalently bonded to L.

17. The composition of any one of claims 1-16, wherein the gly can-containing lipid is a natural or modified glycolipid.

18. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a natural or modified glycosphingolipid.

19. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a natural or modified ganglioside.

20. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a natural or modified sialic acid containing lipid.

21. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a 2,3-linked sialic acid-containing lipid, wherein the 2,3-linked sialic acid-containing lipid is a natural or modified form.

22. The composition of claim 21, wherein the 2,3-linked sialic acid-containing lipid is selected from GM3 and GDI A.

23. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a 2,8-linked sialic acid-containing lipid, wherein the 2,8-linked sialic acid-containing lipid is a natural or modified form.

24. The composition of claim 23, wherein the 2,8-linked sialic acid-containing lipid is selected from GD3 and GQ1B.

25. The composition of any one of claims 1-16, wherein the glycan-containing lipid is a 2,6-linked sialic acid-containing lipid, wherein the 2,6-linked sialic acid-containing lipid is a natural or modified form.

26. The composition of any one of claims 1-25, wherein the lipid (L) is selected from one or more alkyl groups, wherein the alkyl groups can be saturated, unsaturated, branched, and / or unbranched, and can further comprise one or more heteroatoms including but not limited to N, O, Si, P, S, Cl, Br, and F.

27. The composition of any one of claims 1-26, wherein the glycan-containing moiety (S) is selected from natural forms of monosaccharides, disaccharides, oligosaccharides, andpolysaccharides or chemically modified forms of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

28. The composition of claim 27, wherein the chemically modified forms result from covalently added functional groups, such as phenyl group, carboxyl group, or benzene group, by chemical reactions.

29. The composition of claim 27, wherein the chemically modified forms result from chemoenzymatic reactions, graft-to, or graft-from chemical reactions.

30. The composition of any one of claims 1-29, wherein the glycan-containing moiety (S) is combined with a positively charged functional group to form a zwitterionic glycan- containing moiety.

31. The composition of claim 30, wherein the zwitterionic glycan-containing moiety is composed of single or repeat units of a negatively charged glycan-containing moiety and a positively charged functional group.

32. The composition of Claim 30, wherein the positively charged functional group can be quaternary amine, phosphonium, and sulfonium.

33. The composition of any one of claims 1-32, wherein the non-cationic lipid in component (ii) contains a zwitterionic moiety.

34. The composition of claim 33, wherein the zwitterionic moiety is selected from the group consisting of phosphobetaine, phosphatidylcholine, phosphoserine, carboxybetaine, sulfobetaine, trimethylamine N-oxide, and glutamic acid-lysine (EK)-containing peptide.

35. The composition of claim 33, wherein the non-cationic lipid is selected from the group consisting of poly(carboxybetaine) (PCB), a poly(sulfobetaine), a poly(phosphobetaine), poly(phosphatidylcholine), glutamic acid-lysine (EK) -containing polypeptide, a poly(trimethylamine N-oxide) polymer, a poly(zwitterionic phosphatidyl serine) and poly (ethylene glycol) (PEG).

36. The composition of claim 33, wherein the non-cationic lipid is selected from the group consisting of a dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monomethyl-phosphoethanolamine, 16-O-dimethyl-phosphoethanolamine, 18-1-trans-phosphoethanolamine, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1,2-dioleoyl-sn glycero-3-phophoethanolamine (transDOPE), and 1,2-disteroyl-sn-glycero- 3 -phosphocholine (DSPC).

37. The composition of claim 33, wherein said lipid moiety in component (i) is a diacylglyceride.

38. The composition of claim 33, wherein the zwitterionic polymer is selected from the group consisting of a poly(carboxybetaine) (PCB), a poly(sulfobetaine), a poly(phosphobetaine), poly(phosphatidylcholine), glutamic acid-lysine (EK) -containing polypeptide, a poly(trimethylamine N-oxide) polymer and a poly(zwitterionic phosphatidyl serine).

39. The composition of claim 33, wherein the zwitterionic polymer is a betaine polymer.

40. The composition of claim 39, wherein the betaine polymer is a poly(carboxybetaine), poly(sulfobetaine), or poly(phosphobetaine) polymer.

41. The composition of any one of claims 1-40, wherein components (i), (ii), and (iii) exclude a polyalkylene oxide segment.

42. The composition of any one of claims 1-40, wherein at least one of components (i), (ii), and (iii) includes a poly alkylene oxide segment.

43. The composition of any one of claims 1-32, wherein the non-cationic lipid in component (ii) is selected from a phospholipid.

44. The composition of claim 43, wherein the phospholipid is a phosphatidyl serine lipid.

45. The composition of any one of claims 1-44, wherein the cationic or ionizable lipid in component (iii) possesses a secondary, tertiary, or quaternary amino group.

46. The composition of any one of claims 1-44, wherein the cationic or ionizable lipid in component (hi) possesses a secondary, tertiary, or quaternary group along with a functional group which is negatively charged under physiological conditions.

47. The composition of claim 46, where the cationic or ionizable lipid comprises the following structure:wherein:R I / R2= H or an alkyl group and wherein the alkyl group can be saturated, unsaturated, branched, and / or unbranched, and can further comprise 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, wherein the covalent linker group can comprise one or more of -CH2-, -CH2CH(OH)-, -CH2CHC1CH2-, -CH2OCH2-, - CH2SCH2-, -CH2SSCH2-, -CH2COOCH2-, C, H, and can further comprise one or more heteroatoms including but not limited to N, O, F, Si, P, S, Cl, Br, and F; andA-(X)n is a functional group that is negatively charged under certain pH conditions and can comprise:(i) A(X)n is a carboxylic acid group (A = -COOH and n=0) or(ii) A(X)n is a phosphate, where A =and n = 1. X = H or an alkyl group that is saturated or unsaturated, branched or not branched, all-carbon or containing heteroatoms such as but not limited to N, O, F, Si, P, S, Cl, Br, and F or(iii) A(X)n is a sulfonic acid group, where A =and n = 0(iv) A(X)n is a sulfonamide group, where A =and n = 1 , X = H or an alkyl group that is saturated or unsaturated, branched or not branched, all carbon and hydrogen or containing heteroatoms such as but not limited to N, O, F, Si, P, S, C1, Br, and F.

48. The composition of claim 46, wherein the cationic or ionizable lipid is selected from the group consisting of:wherein n=l to 10.

49. The composition of any one of claims 1-48, wherein the lipid nanoparticle composition further comprises cholesterol or a derivative thereof.

50. The composition of any one of claims 1-49, wherein two or three of components (i)- (iii) are chemically combined.

51. The composition of any one of claims 1 -49, wherein the gly can-containing lipid and cationic lipid are chemically combined into one lipid.

52. The composition of any one of claims 1-49, wherein the glycan-containing lipid and non-cationic lipid are chemically combined into one lipid.

53. The composition of any one of claims 1-49, wherein the glycan-containing lipid, cationic lipid, and non-cationic lipid are chemically combined into one lipid.

54. The composition of claim 49, wherein the glycan-containing lipid and cholesterol or a derivative are chemically combined into one lipid.

55. The composition of claim 49, wherein the glycan-containing lipid, cationic lipid and cholesterol or a derivative are chemically combined into one lipid.

56. The composition of claim 49, wherein the glycan-containing lipid, non-cationic lipid and cholesterol or a derivative are chemically combined into one lipid.

57. The composition of claim 49, wherein the glycan-containing lipid, non-cationic lipid, cationic lipid and cholesterol or a derivative are chemically combined into one lipid.

58. The composition of any one of claims 1-57, wherein the lipid is selected from “simple lipids,” which include fats, oils, and waxes; “compound lipids,” which include phospholipids and glycolipids; or “derived lipids,” such as steroids, such as, for example, a phospholipid, sphingolipid, or a sterol; or a synthetic lipid.

59. The composition of any one of claims 1-58, wherein the lipid in component (i) is a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, or a cerebroside.

60. The composition of any one of claims 1-58, wherein the lipid in component (i) is a phosphatidylethanolamine (PE), a phosphatidylglycerol (PG), phosphatidic acid (PA), or a phosphatidylinositol (PI).

61. The composition of any one of claims 1-60, wherein the LNP composition is chemically modified with a targeting ligand.

62. The composition of claim 61, wherein the targeting ligand is in the glycan- containing lipid.

63. The composition of claim 61, wherein the targeting ligand is an antibody (or Fab or Fc).

64. The composition of any one of claims 1-63, wherein the glycan-containing moiety (S) is chemically conjugated to or fused to an extracellular vesicle (EV).

65. The composition of any one of claims 1-63, wherein the gly can-containing moiety(S) is chemically conjugated to an adeno-associated virus (AAV).

66. A method of delivering a therapeutic substance to a subject, the method comprising administering to the subject a lipid nanoparticle composition according to any one of claims 1-65.

67. The method of claim 66, comprising administering to said subject a lipid nanoparticle composition comprising:(i) at least one glycan- containing lipid;(ii) at least one non-cationic lipid selected from charged and uncharged lipids; and(iii) at least one cationic or ionizable lipid containing a secondary, tertiary, or quaternary amino group; and(iv) at least one therapeutic substance.

68. The method of any one of claims 66-67, wherein the lipid nanoparticle composition is delivered to cells of the subject.

69. The method of any one of claims 66-68, wherein the therapeutic substance is a nucleic acid molecule, and administration thereof results in gene therapy of the subject.

70. The method of any one of claims 66-68, wherein the therapeutic substance is a nucleic acid molecule, and administration thereof results in vaccination of the subject.

71. A method for targeted delivery of a therapeutic agent to a secondary lymphoid organ (SLO) in a subject, the method comprising administering an effective amount of a lipid nanoparticle composition according to any one of claims 1-65 to the subject, wherein the therapeutic agent in the lipid nanoparticle is targeted to a secondary lymphoid organ (SLO) of the subject.

72. The method of claim 71, wherein the therapeutic agent comprises a nucleic acid molecule.

73. The method of claim 71, wherein the SLO is selected from spleen and lymph nodes.

74. A method for targeted delivery of a therapeutic agent to the brain of a subject, the method comprising administering an effective amount of a lipid nanoparticle composition according to any one of claims 1-65 to the subject, wherein the therapeutic agent in the lipid nanoparticle is targeted to the brain of the subject.

75. A method for targeted delivery of a therapeutic agent to the bone marrow of a subject, the method comprising administering an effective amount of a lipid nanoparticle composition according to any one of claims 1-65 to the subject, wherein the therapeutic agent in the lipid nanoparticle is targeted to the bone marrow of the subject.

76. The method according to any one of claims 71-75, wherein the targeted delivery of the therapeutic agent provides cancer immunotherapy, autoimmune disease immunotherapy, vaccination and / or gene editing.

77. A method for targeted delivery of a therapeutic agent to specific cells (e.g., immune cells, precursor cells, or stem cells) in a subject, the method comprising administering an effective amount of a lipid nanoparticle composition according to any one of claims 1-65 to the subject, wherein the therapeutic agent in the lipid nanoparticle is targeted to specific cells of the subject.

78. The method of claim 77, wherein the therapeutic agent comprises a nucleic acid molecule.

79. The method of any one of claims 77-78, wherein the targeted delivery of the therapeutic agent provides cancer immunotherapy, autoimmune disease immunotherapy, vaccination and / or gene editing.

80. The method of any one of claims 77-79, wherein the specific cells are selected from lymphoid cells, myeloid cells, and stem cells.

81. The method of any one of claims 77-79, wherein the specific cells are immune cells selected from natural killer cells, natural killer B / T cells, dendritic cells, macrophages, monocytes, B cells, T cells, neutrophils, and microglia cells.

82. The method of any one of claims 77-79, wherein the specific cells are selected from liver sinusoidal endothelial cells, astrocytes, neurons, precursor cells, and pluripotent stem cells.

83. The method of any one of claims 77-79, wherein the LNP is used to deliver nucleic acids encoding chimeric antigen receptor or an exogenous T cell receptor into immune cells.

84. The method of any one of claims 66-83, wherein the LNP is injected directly in vivo and delivered into the host cells in vivo.

85. The method of any one of claims 66-83, wherein the LNP is transfected into the host cells ex vivo and the resulting cells are then infused in vivo.

86. The method of any one of claims 66-83, wherein the LNP is administered to a mammal intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

87. The method of any one of claims 66-83, wherein the LNP is used for multiple in vitro and in vivo applications, including, but not limited to, protein replacement therapy, cancer immunotherapy, cell therapy, cancer vaccines, infectious disease vaccines, gene therapy, autoimmune disease treatment and cancer diagnosis.

88. The method of any one of claims 66-87, wherein the LNP is used for in vitro and in vivo production of extracellular vesicles.

89. The method of any one of claims 66-87, wherein the LNP is used for clustered regularly interspaced short palindromic repeats-Cas endonuclease (CRISPR-Cas) gene editing, prime editing, based editing, and RNA editing in vitro and in vivo.

90. The method of any one of claims 66-87, wherein the LNP is used for vaccines against coronavirus (e.g., SARS-CoV-2).

91. The method of any one of claims 66-87, wherein the LNP is delivered along with checkpoint inhibitor (e.g., anti- Programmed death-ligand 1 (anti-PD-Ll) antibody, anti- cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4)) to treat cancer.

92. The method of any one of claims 66-91, wherein the LNP is delivered along with cytokines (e.g., Interleukin- 12, Interleukin-35, and Interferon alpha) to treat a disease.

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