Lipid nanoparticles and uses thereof

Abstract

The present disclosure describes compositions and methods including liposome or lipid nanoparticles (LNPs) including carbohydrate-based ligands that target the liver, lungs, and / or kidneys. More particularly, the present disclosure relates to compositions and methods including liposome or lipid nanoparticles (LNPs) including carbohydrate-based ligands, such as di-lactobionic acid (LBA) ligand, and a suitable cargo such as nucleic acids and small molecule to target the liver, lungs, and / or kidneys for treatment and / or diagnosis of diseases and conditions.

Description

LIPID NANOPARTICLES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims priority to U.S. Provisional Application No. 63 / 527,105, filed on July 17, 2023, the entire contents of which are incorporated herein in their entirety for all purposes.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides lipid- based delivery systems such as lipid nanoparticles (LNPs) or liposomes and methods for improved hepatic, pulmonary, and renal delivery of nucleic acids and small molecule drugs. More particularly, the present disclosure relates to lipid nanoparticles or liposomes including carbohydrate decorated compositions and methods including such LNPs to target hepatic, pulmonary, and renal delivery of cargoes such as small molecules or nucleic acids for treatment and / or diagnosis of diseases and conditions.BACKGROUND

[0003] Nanoparticle-based delivery systems have been an attractive approach for delivering nucleic acids and small molecule drugs to tumors due to the enhanced permeation rate (EPR) effect. However, to achieve organ-specific accumulation, targeting ligand moieties is required, and there is an urgent need for improved compositions and methods for liver, kidney, and lung targeting therapeutic modalities to achieve desired therapeutic outcomes.SUMMARY

[0004] In an aspect, disclosed is lipid-based delivery system such as a lipid nanoparticles (LNPs) or liposomes including carbohydrate-based ligands to target the liver, lungs, and / or kidneys as shown and described herein. In an aspect, the liposomes or LNPs have their surfaces decorated with carbohydrate-based ligands for targeted delivery of a one or more cargoes.

[0005] In an aspect, disclosed is a method for delivery of the therapeutic cargo, wherein the method includes a lipid nanoparticle (LNP) or liposome as shown and described herein.

[0006] In an aspect, disclosed is a method for preparation of a lipid nanoparticle (LNP) or liposome as shown and described herein.

[0007] In an aspect, disclosed is a composition for diagnosing, treating, or preventing a disease, a disorder, or symptom in a subject, the composition including at least one of the lipid nanoparticle (LNP) or liposome disclosed herein or a pharmaceutically acceptable salt thereof with at least one pharmaceutically acceptable vehicle and / or excipient.

[0008] In an aspect, disclosed is a method of diagnosing, treating, or preventing a disease, a disorder, or symptom associated with the disease or disorder in a subject in a need thereof, the method including: providing and administering a therapeutically effective amount of at least one of the a lipid nanoparticle (LNP) or liposome disclosed herein, a pharmaceutically acceptable salt thereof, or the composition disclosed herein to the subject.

[0009] These and other aspects of the present invention are described in more detail below.BRIEF DESCRIPTION OF THE DRAWINGS100101 The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0011] Figure 1 shows a synthesis scheme of DSPE-LBAAC using HATU / DIEA amide coupling reaction conditions.

[0012] Figure 2 shows a synthesis scheme of DSPE-LBAusing HATU / DIEA amide coupling reaction conditions.

[0013] Figure 3 shows a high performance liquid chromatography (HPLC) separation of crude DSPE-LBAAC conjugate and MALDLTOF mass spectrometry analysis of the purified fraction.

[0014] Figure 4 shows a preparation of liposomes using lipid film hydration technique.

[0015] Figure 5 shows a schematic representation of a chemical structure of ligand- lipid conjugates and their respective arrangement in liposomes.

[0016] Figure 6 illustrates a schematic of a microfluidic-based LNP synthesis system for the preparation of LNP formulations via microfluidic mixing of lipids, e.g., from Lipidschosen from formulation 1. D-Lin-MC3-DMA, 2. DMG-PEG 2000, 3. Cholesterol, and 4. DSPC.

[0017] Figure 7A, 7B, and 7C - Characterization and cellular uptake of mRNA LNPs. 7A) Characterization and comparison of ligand functionalized FLuc mRNA LNPs. 7B) Cellular uptake of ligand functionalized GFP mRNA LNPs in HK-2 cells using confocal microscopy. 7C) Comparative cellular uptake of ligand functionalized GFP mRNA LNPs in HK-2 cells using flow cytometry.

[0018] Figure 8A illustrates various non-PEG spacer conjugates and Figure 8B illustrates various PEG spacer conjugates.DETAILED DESCRIPTION

[0019] Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, or examples, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0020] Nanoparticle-based delivery systems have been an attractive approach for delivering nucleic acids and small molecule drugs to tumors due to the enhanced permeation rate (EPR) effect. However, to achieve organ-specific accumulation, targeting ligand moieties is required. Carbohydrate-based ligands such as galactose, fucose, mannose, and N- acetylgalactosamine have been employed for developing polymeric and lipid-based targeted nanoparticles. LBA delivery platform previously disclosed by inventors has shown enhanced liver retention until 24 hours, similar to GalNAc for the delivery of PNA (Kumar V et al. , Adv. Healthc. Mater. 2023). Whereas, being a major excretory organ, effective and efficient delivery of therapeutic modalities at the desired site in the kidney is a significant hurdle in various renal disorders. Inventors have shown that LBA acetylated constructs enhance kidney retention for up to 96 hours. Recently, inventors have also explored the lung targeting potential of LBA ligands by demonstrating enhanced pulmonary retention of LBA conjugated to 5-carboxy rhodamine (TAMRA) for up to 72 hours via intratracheal administration for the efficient delivery of a small molecule.

[0021] Different nanoformulations, including polymeric nanoparticles, liposomes, carbon nanotubes, and DNA nanocages, have been explored so far for organ-specific delivery. But the size-based non-specific biodistribution of nanoformulation is the key hurdlefor their successful clinical translation. Active organ / tissue targeting using functionalized nano-formulations will result in more precise site-specific delivery.

[0022] Disclosed herein are lipid-based delivery systems such as lipid nanoparticles (LNPs) or liposomes including carbohydrate decorated formulations for improved hepatic, pulmonary, and renal delivery of cargos such as nucleic acids and small molecule drugs. Any suitable cargo such as nucleic acids (for example, mRNA, siRNA, miRNA, antisense oligonucleotides, PNA, or DNA); and / or small molecule drugs (for example, drugs for pulmonary diseases, cancer, or any suitable drug targeting lung, kidney, and / or liver) can be delivered using these lipid nanoparticles.

[0023] Liposomes are lipid structures having one or more lipid bilayers and can range in diameters in the low nanometer range (~20 nm) to greater than 1000 nm. Liposomes can be unilamellar vesicles or multilamellar vesicles comprising two or more concentric bilayer structures. The lipid bilayer can surround an aqueous inner space.

[0024] Lipid nanoparticles (LNPs) are lipid structures having an average diameter size of about 10 to about 1000 nm. LNPs have an exterior lipid layer with a hydrophilic exterior surface. LNPs include an interior space that can be aqueous or non-aqueous. LNPs may have one or more membrane layers and may be lamellar or non-lamellar.

[0025] Described herein are lipid nanoparticles (LNPs) or liposomes including carbohydrate-based ligands for targeted delivery of one or more cargo such as nucleic acids and small molecule drugs to specific organs in a subject (for example, in mammals, in particular humans), and more in particular to the liver, lungs, and / or kidneys of a mammal such as a human. LNPs and liposomes are different delivery systems. The concept of including carbohydrate-based ligands in each of these lipid structures for targeted delivery of one or more cargos is fully included herein. Specifically, all descriptions of lipid nanoparticles (LNPs) including carbohydrate-based ligands and their uses as described herein are fully applicable to liposomes and fully incorporated herein. Likewise, all descriptions of liposomes including carbohydrate-based ligands and their uses as described herein are fully applicable to LNPs and fully incorporated herein.

[0026] In an embodiment, the liposomes or LNPs have their surfaces decorated with carbohydrate-based ligands for targeted delivery of one or more cargoes. In an aspect, the lipid nanoparticles (LNPs) or liposomes include one or more lipids which are covalently and / or non-covalently (for example, ionic interactions) bonded with the carbohydrate-based ligands to ensure organ specific delivery by receptor targeting. In an embodiment, the LNP or liposome includes a carbohydrate ligand covalently and / or non-covalently linked to a lipidvia linker backbone covalently bonded to both. The carbohydrate ligand can be selected to target the kidneys, lungs, and / or liver. In an aspect, the LNP or liposome selectively binds to specific receptors on cells to deliver the cargo such as a therapeutic agent to the cells bearing the receptors. In an aspect, the lipid nanoparticles (LNPs) or liposomes include one or more cargoes including one or more therapeutic agents such as nucleic acids, small molecules, or a combination thereof.

[0027] In an embodiment, a carbohydrate-based ligand such as di-lactobionic acid (LBA) ligand was utilized as an organ-targeting moiety to formulate organ -targeted liposomes by covalent conjugation with l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid. DSPE is a commonly known biocompatible, biodegradable and amphiphilic lipid that can be functionalized with various biomolecules for specific functions. The lipid may be of synthetic or natural origin. Any suitable lipid can be used, some non-limiting examples are, phospholipids e.g. a glycerophospholipid such as phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, or phosphoinositide, or phosphosphingolipids, ionizable lipids, PEGylated lipids, or a combination thereof. Other suitable lipids are described herein for the “L” group of Formula (I). In an embodiment, to prepare surface-modified liposomes (also called lipid nanoparticles, LNPs), first, LBA ligands were covalently conjugated to DSPE-PEG(2000) using amide coupling chemistry in the solution phase. Ligand-conjugated DSPE-PEG(2000) along with other lipids in different molar ratios were then used to prepare liposomes containing synthetic nucleic acids like peptide nucleic acids and small molecule drugs like doxorubicin.

[0028] In an aspect, the surface of the liposomes is decorated with the carbohydrate- based ligands, such as the LBA ligand, can thus target various organs. In certain embodiments, the lungs can be targeted by intratracheal administration via receptor-mediated endocytosis. In certain embodiments, the liver can be targeted using these formulations via asialoglycoprotein receptors through subcutaneous / systemic administration. In certain embodiments, formulations decorated with acetylated LBA (LBAAC) ligands can be used for kidney-targeted delivery through subcutaneous / systemic administration. LBA ligands thus can achieve pulmonary and hepatic accumulation, whereas LBAAC ligands can achieve renal accumulation. These organ targeted liposomes namely, pulmonary targeted liposomes (PTL), liver-targeted liposomes (LTL), and kidney-targeted liposomes (KTL) can be used to develop drug delivery systems for various diseases such as fibrosis, cancer, and various genetic disorders.

[0029] The disclosed compositions and methods have numerous applications for the treatment and diagnosis of organ-associated diseases. In certain embodiments, the carbohydrate-based ligand decorated liposomes / LNPs, such as the LBA-decorated liposomes, can be encapsulated with nucleic acid and small-molecule drugs. These compositions can be used as a versatile delivery system for the treatment and diagnosis of various hepatic, pulmonary, and renal diseases such as renal fibrosis, liver fibrosis, pulmonary fibrosis, hepatocellular carcinoma, lung cancer, asthma, viral respiratory diseases, cystic fibrosis, and other liver, kidney and lung genetic disorders. Herein, by utilization of the carbohydrate- based ligand, such as the LBA ligand, on the surface of liposomes, multiple cargoes such as nucleic acids and small molecule drugs can be encapsulated and delivered to the target organs. These organ targeted liposomes can be used as a delivery system when developing drugs and gene-based therapeutic modalities to specifically target and treat liver, kidney, and lung disorders.

[0030] Disclosed carbohydrate ligands can functionalize diverse formulations for active targeting of the liver, kidney, and lungs. The small size, high specificity, stability, and ease of formulation of disclosed ligands and liposomes (LNPs) are key advantages for successful clinical translation. Moreover, the carbohydrate based ligand, lactobionic acid, is safe and has been reported as an excipient in various pharmaceutical formulations. Like other ligand-targeting moieties such as GalNAc, LBA has shown robust safety without any toxicides.

[0031] Inventors have shown efficient delivery of peptide nucleic acids directly conjugated to LBA to target organs such as the liver, kidney, and lungs. Herein, disclosed are the carbohydrate based ligand decorated liposomes, such as LBA-decorated liposomes, to deliver both nucleic acids such as peptide nucleic acids / siRNA / mRNA / allele specific oligonucleotides (ASOs), small molecule drugs, and / or combination thereof. Compared to covalent conjugates, disclosed liposomes / LNPs / formulations offer added benefits of multiple cargo loading and selective uptake to tumors by the enhanced permeation rate (EPR) effect. Also, including PEGylated lipids in liposomes improves the circulation half-life, further increasing organ accumulation. Furthermore, this is the first report of optimized / equivalent carbohydrate-based ligands for active targeting of kidneys and lungs.

[0032] Disclosed herein are new formulations and methods including a lipid nanoparticles (LNPs) or liposomes including carbohydrate-based ligands, such as di- lactobionic acid (LBA) ligand, to target hepatic, pulmonary, and renal delivery of cargoes such as small molecules and / or nucleic acids. In an embodiment, the liposomes or LNPs havetheir surfaces decorated with carbohydrate-based ligands for targeted delivery of a one or more cargoes. Advantageously, this formulation results in improved toxicity and specific organ targeting along with optimum delivery of the therapeutic cargo to the target site. This is the first report of optimized / equivalent carbohydrate-based ligands for active targeting of kidneys and lungs. The disclosed carbohydrate-based ligands can be monovalent, divalent, tri valent, tetravalent, or polyvalent. Thus, the disclosed lipid nanoparticles (LNPs) or liposomes including carbohydrate-based ligands, such as di-lactobionic acid (LBA) ligand, result in cost effective and efficient hepatic, pulmonary, and / or renal targeting for safe delivery of therapeutic agents. The formulations and techniques disclosed may be applied for the delivery of nucleic acids and / or small molecules with greater hepatic, pulmonary, and renal retention to target various kidney, lung, and liver diseases.

[0033] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed materials and methods provide improvements in hepatic, pulmonary, and / or renal targeting along with optimum delivery of the therapeutic cargo to the target site. For example, the carbohydrate-based ligands, such as di-lactobionic acid (LBA) ligand, of the disclosure as described herein are capable of greater hepatic, pulmonary, and / or renal retention to target various hepatic, pulmonary, and renal diseases. In addition, the liposomes or LNPs including carbohydrate-based ligands of the disclosure as described herein are less toxic and show less unwanted side effects to due active organ-targeting compared to the noncarbohydrate based conventional lipid nanoparticles used for the same purpose.

[0034] The benefits of the liposomes or LNPs including carbohydrate-based ligands of the disclosure as described herein address a number of current and important drug delivery hurdles present in the art, such as toxicity, unwanted side effects, and the ability to easily functionalize the ligand of a drug delivery vehicle for therapeutic or targeted delivery applications, biodegradability, and the capacity for combination therapy.

[0035] In addition, achieving efficient targeting is highly desired for a therapeutic response, which could help to reduce the dose required to achieve effect. Carbohydrate ligands such as lactobionic acid show longer retention in the lungs and require simple conjugation steps for its synthesis, making scale up easier. The liposomes or LNPs including carbohydrate-based ligands as described herein, can carry and deliver various cargoes including therapeutic agents such as nucleic acids and small molecules providing a versatile approach for the treatment and diagnosis of hepatic, pulmonary, and renal diseases such as renal fibrosis, liver fibrosis, pulmonary fibrosis, hepatocellular carcinoma, asthma, viralrespiratory diseases, lung cancer, cystic fibrosis and other liver, kidney and lung genetic disorders.

[0036] In an aspect, disclosed is a composition / system including a liposome / LNP including a carbohydrate-based ligand / s and a genetic sequence and / or a small molecule as a cargo (a therapeutic agent). The system / composition can be used for treatment of a subject in need thereof ex vivo or in vivo. The system / composition can be used to target hepatic, pulmonary, and renal delivery of cargoes such as small molecules or nucleic acids for treatment and / or diagnosis of diseases and conditions.

[0037] This invention has numerous applications for treating and diagnosis of hepatic, pulmonary, and renal diseases. The carbohydrate-based ligands disclosed herein, such as LB A, can be conjugated with both nucleic acid and small molecules providing a versatile approach for the treatment and diagnosis of hepatic, pulmonary, and renal diseases such as renal fibrosis, liver fibrosis, pulmonary fibrosis, hepatocellular carcinoma, asthma, viral respiratory diseases, lung cancer, cystic fibrosis and other liver, kidney and lung genetic disorders. Also, carbohydrate-based ligands, such as the LBA ligand, can be used for the functionalization of lipids, biopolymers, and synthetic polymers for enhanced hepatic, pulmonary, and renal delivery of various therapeutic and diagnostic agents. This technology can be used for developing drugs and gene-based therapeutic modalities to specifically target and treat hepatic, pulmonary, and renal disorders.

[0038] Carbohydrate based ligands, such as LBA, are a safe alternative to be used for hepatic, pulmonary, and renal delivery without toxicity. In addition, carbohydrate based ligands can be further modified using carbohydrate chemistry for more specific or broader hepatic, pulmonary, and renal distribution. The designed ligand is highly versatile to conjugate with diverse functional groups and nucleic acid analogs. Scale-up for can be done easily with simple chemistry steps.

[0039] The liposomes and / or ligands disclosed herein can include a cargo that is a genetic sequence (GS) having a 3’ end and a 5’end, which can be a PNA or an oligonucleotide such as an mRNA sequence, an siRNA sequence, or a DNA sequence. Each genetic sequence can be natural or optionally modified, for example in an order of nucleotides or via modifications as a gamma-serine modified gamma peptide nucleic acid, an alanine gamma peptide nucleic acid, a clamp G- modified peptide nucleic acid, a locked nucleic acid (LNA), a phosphorothioates (PS), a phosphorodiamidate morpholino (PMO), a 2’-O-methyl (2’-0-Me), , 2’-O-methoxyethyl (2’-0-M0E), 2’-flouro (2’F), a 5’- methylcytosine, or a combination thereof. In an aspect, the genetic sequence is a PNA. ThePNA can be modified as described below. The liposomes disclosed herein can include a small molecule, some nonlimiting examples of the small molecules are ivacaftor, elexacaftor, tezacaftor, lumacaftor, pirfenidone, N-acetylcysteine, Nintedanib, cisplatin, carboplatin, docetaxel, paclitaxel, pemetrexed, azathioprine, mycophenolate mofetil, tocilizumab, cyclophosphamide, saracatinib, or a combination thereof. Additional suitable small molecules include a therapeutic agent, such as any one of the therapeutic agents described herein.

[0040] In an embodiment, the liposomes bear 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 carbohydrate residues or ligands. In an embodiment, the carbohydrate-based ligands (also called as carbohydrate ligands) can be a monovalent, a divalent, a trivalent, a tetravalent, or a polyvalent carbohydrate ligands. In an embodiment, the carbohydrate-based ligands can be a monovalent, a divalent, a trivalent, or a tetravalent carbohydrate ligands. In an embodiment, the carbohydrate-based ligands can be a monovalent or a divalent carbohydrate ligands. In an embodiment, the carbohydrate-based ligand is a divalent carbohydrate ligands. The number and type of carbohydrate ligands are selected to target the liver, kidneys, and lungs. For example, the carbohydrate ligand can be selected to target the Asialglycoprotein receptor (ASPGR) expressed on cells. ASGPR is a C-type lectin, primary expressed on the sinusoidal surface of hepatocytes. In an embodiment, the liposomes bear 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 galactose ligands to target the ASPGR on lung cells. In an aspect the liposomes bear 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4 galactose amine (GalNAc) ligands to target the target the mannose and other carbohydrate receptors on lung cells and alveolar macrophages. In an embodiment, the liposomes bear 1 to 8, or 1 to 5, or 2 to 5, or 2 to 4, or 2 to 3 acetylated lactobionic acid ligands to target the megalin and tubulin receptors present on kidney cells.

[0041] The carbohydrate ligand(s) of the disclosed LNPs or liposomes can be fully or partially acylated on a hydroxy or amino group thereof with a C2 to C15 acyl group. For example, the carbohydrate ligand can be fully or partially acetylated on a hydroxy or amino group. The acetylation of a carbohydrate ligand can be performed using an acetylating reagent such as acetic anhydride, acetyl chloride, mixed anhydrides, acids with coupling agents such as DCC or like reagents and a base such as triethyl amine, pyridine, DIEA, DMAP or the like, or an organic, inorganic, or polymeric base as used in the art. In an embodiment, the carbohydrate ligand is a GalNAc residue that is fully or partially acylated, preferably acetylated, preferably fully acetylated. Alternatively, the carbohydrate ligand is a lactobionic acid residue that is fully or partially acylated, preferably acetylated, preferably fully acetylated.

[0042] In an aspect, disclosed is a liposome or LNP including carbohydrate-based ligands to target the liver, lungs, or kidneys. In an aspect, the liposome or LNP includes a ligand- lipid conjugate having a Formula (I):CL-linker-L (I) wherein L is a lipid optionally functionalized with a functional moiety, “linker” is a linking group or a bond, and CL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide, specifically wherein CL comprises a carbohydrate residue / ligand derived from a monosaccharide, a disaccharide, or a polysaccharide, optionally wherein the carbohydrate ligand is fully or partially acylated on a hydroxy or amino group thereof with a C2 to C15 acyl group, specifically wherein the carbohydrate ligand is fully or partially acetylated on a hydroxy or amino group thereof. In an embodiment, CL comprises a lactobionic acid. The lipid group can be any one of the lipid groups described herein, optionally substituted with a functional moiety, also described further herein. The “linker” group can comprise a variety of backbones, but in general contain at least two functional groups, for example at least two amino groups, one or more for reaction with the carbohydrate ligand(s) and one or more for reaction with the lipid(s), for example through the formation of amide bonds. The amino groups can be selectively protected as known in the art. The backbone can include moieties to modify properties such as solubility. The “linker” can comprise 1 to 200, specifically 1 to 100, and more specifically 1 to 20 carbon atoms, and optionally one or more reactive groups such as hydroxy, carboxy, thio, or amino. In an embodiment, the “linker” comprises a residue of a polyethylene glycol, a polypropylene glycol, or a polyethylene-propylene glycol. The PEG group can contain 1 to 100 ethylene glycol residues (-OCH2CH2-), specifically 5 to 90, more specifically 10 to 80, yet more specifically 20 to 70, still yet more specifically 30 to 60, and still more specifically 40 to 50 ethylene glycol residues that can terminate in a hydroxy, amino, ether, or like functional moiety, which is bonded to a carbohydrate ligand or a lipid. In another aspect, the “linker” can comprise a polypropylene glycol (PPG) moiety. In yet another aspect, the “linker” can comprise a combination of PEG and PPG moieties.

[0043] In an aspect, the liposome or LNP includes a ligand-lipid conjugate having a Formula (II):wherein L is a lipid, R1and R2are each independently H or a substituted or unsubstituted Ci to Ce alkyl, X1is O, S, NR3, C=O, or C(R3)2 where R3is H or a substituted or unsubstituted Ci to Ce alkyl, X2is O, S, NR3, or C(R3)2 where R3is H or a substituted or unsubstituted Ci to Ce alkyl, G1is a direct bond or a group linking the lipid to the linker or carbohydrate ligand, G2is H or a functional moiety, CL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide, preferably wherein CL comprises a carbohydrate residue / ligand derived from a monosaccharide, a disaccharide, or a polysaccharide, optionally wherein the carbohydrate ligand is fully or partially acylated on a hydroxy or amino group thereof with a C2 to C15 acyl group, preferably wherein the carbohydrate ligand is fully or partially acetylated on a hydroxy or amino group thereof, n1is 1 to 20, and n2is 0 to 20.

[0044] In an embodiment, the CL carbohydrate ligand comprises a lactobionic acid.

[0045] The “L” lipid moiety can be of synthetic or natural origin. Any suitable lipid can be used, some non-limiting examples include phospholipids e.g. a glycerophospholipid such as phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, or phosphoinositide; phosphosphingolipids; ionizable lipids; PEGylated lipids; cholesterol; or a combination thereof. Other suitable lipids include, for example, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, pegylated derivatives thereof, or a combination thereof. Particular suitable lipids include 1 ,2- diarachidonoyl-sn-glycero-3-phosphocholine; l,2-diarachidonoyLsn-glycero-3- phosphoethanolamine; 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine; 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; l,2-dihexanoyl-sn-glycero-3- phosphocholine (DHPC); l,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE); l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); l,2-dilinoleoyl-sn-glycero-3- phosphocholine; 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine; 1 ,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC); 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); dimyristoylphosphatidylglycerol (DMPG); l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC); l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2- dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1 ,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS); l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE); l,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE); 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC); 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1 -palmitoyl-2-oleoyl phosphatidylglycerol; l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE); egg phosphatidylcholine (EPC); glycerol monooleate (GMO); partially hydrogenated soy phosphatidylchloline (PHSPC); phosphatidylethanolamine (PE); phosphatidylglycerol (PG); phosphatidylinositol (PI); phosphatidylserine (PS); soybeanphosphatidylcholine (SPC);PEGylated derivatives thereof; or a combination thereof. PEGylated derivatives thereof may contain 1 to 100 ethylene glycol residues (-OCH2CH2-), specifically 5 to 90, more specifically 10 to 80, yet more specifically 20 to 70, still yet more specifically 30 to 60, and still more specifically 40 to 50 ethylene glycol residues. The PEG portion of PEGylated lipids may correspond to the G1moiety of Formula (II).

[0046] In an embodiment, the liposome can include cargoes such as small molecules or nucleic acids. In an embodiment, the cargo can be a small molecule, for example, ivacaftor, elexacaftor, tezacaftor, lumacaftor, pirfenidone, N- acetylcysteine, Nintedanib, cisplatin, carboplatin, docetaxel, paclitaxel, pemetrexed, or those small molecules described herein, or a combination thereof.

[0047] In an embodiment, the liposome can include cargoes such as a genetic sequence. In an embodiment, the genetic sequence can be a peptide nucleic acid or an oligonucleotide such as an mRNA sequence, an siRNA sequence, ASOs, or a DNA sequence, optionally wherein each genetic sequence is natural or modified, for example, comprises a gamma-serine modified gamma peptide nucleic acid, an alanine gamma peptide nucleic acid, a clamp G-modified peptide nucleic acid, a locked nucleic acid (LNA), a phosphorothioate (PS), a phosphorodiamidate morpholino (PMO), a 2’-O-methyl (2’-0-Me), 2’-O- methoxyethyl (2’-0-M0E), 2’-flouro (2’F), a 5 ’-methylcytosine, or a combination thereof, the genetic sequence having a 3’ end and a 5’ end.

[0048] The carbohydrate ligands “CL” can be covalently attached to the lipid “L” by a backbone linker as shown in Formula (II), i.e. groups X1through G1. A variety of backbones can be used, but in general contain at least two functional groups, for example at least two amino groups, one or more for reaction with the carbohydrate ligand(s) and one or more for reaction with the lipid / s, for example through the formation of amide bonds. The amino groups can be selectively protected as known in the art. The backbone can includemoieties to modify properties such as solubility. For example, lysine and arginine residues can be present in a backbone. In an embodiment, the carbohydrate ligands can be a monovalent, a divalent, a trivalent, a tetravalent, or a polyvalent carbohydrate ligands.

[0049] In an aspect, as shown in Formula (II), a group G1or G2can be optionally present. G1can be a linker from the backbone to the lipid, for example a linker having 1 to 200, specifically 1 to 100, and more specifically 1 to 20 carbon atoms, and optionally one or more reactive groups such as hydroxy, carboxy, thio, or amino. In another aspect, each G1or G2independently can be a functional moiety. The functional moiety G1, G2can provide a structural feature to the liposomes that can impart a desired function such as stearic separation from a binding ligand, enhancing hydrophilicity or hydrophobicity, facilitating absorption, of the liposomes, facilitating distribution of the liposome in the body, or other functions advantageous in medicinal chemistry and drug design. The functional moiety can be linked between the backbone and the lipid or at a terminal end of the lipid, or both. In an embodiment, a functional moiety G1, G2is, for example, a residue of a polyethylene glycol, a polypropylene glycol, or a polyethylene-propylene glycol. In an aspect, G1or G2, or both can be a polyethylene glycol (PEG) group. The PEG group can contain 1 to 100 ethylene glycol residues (-OCH2CH2-), specifically 5 to 90, more specifically 10 to 80, yet more specifically 20 to 70, still yet more specifically 30 to 60, and still more specifically 40 to 50 ethylene glycol residues that can terminate in a free hydroxy, amino, ether, or like functional moiety, which is optionally bonded to a ligand, a backbone or structure of a liposome. In another aspect, G1or G2, or both can be a polypropylene glycol (PPG) group. In yet another aspect, G1or G2, or both can be a combination of PEG and PPG groups.

[0050] In another aspect, the functional moiety can include a therapeutic agent. For example, kielin, tolvaptan, nintedanib, paclitaxel, bleomycin, cyclosporin, cisplatin, romidepsin, doxorubicin, docetaxel, danunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, prednisolone, cytarabine, or pirfenidone.

[0051] Additionally examples of the functional moiety as a therapeutic agent include beta-2 agonists (e.g., albuterol (Salbutamol), salmeterol, formoterol), anticholinergics (ipratropium bromide, tiotropium, methylxanthines, theophylline) anti-inflammatory agents (e.g., corticosteroids, beclomethasone, budesonide, fluticasone), leukotriene modifiers (e.g., montelukast), mast cell stabilizers (e.g., cromolyn sodium, nedocromil), antibiotics and antimicrobials (e.g., tobramycin, aztreonam, colistin (Polymyxin E), amphotericin B, voriconazole); antiviral agents (e.g., zanamivir, oseltamivir, ribavirin); mucolytics (e.g., N-acetylcysteine, pulmozyme); immunomodulators; monoclonal antibodies (e.g., dupilumab (for asthma) omalizumab (for allergic asthma), cyclosporine (investigational for lung transplant patients)); gene therapy and nucleic acid-based therapies (e.g., adenoviral vectors, lentiviral vectors); siRNA and antisense oligonucleotides; pulmonary surfactants (e.g., beractant, poractant alfa); analgesics (e.g., fentanyl); anticancer agents (chemotherapeutic agents such as cisplatin, paclitaxel in nano-formulation form for inhalational delivery); antioxidants and anti-fibrotic agents (e.g., pirfenidone, nintedanib); or a combination thereof.

[0052] Other examples of G1or G2include deoxyribonuclease I (rhDNase) (domase alfa), catalase, superoxide dismutase (SOD), glucocerebrosidase, alpha- 1 -antitrypsin, lipase, hyaluronidase, or alpha-galactosidase.

[0053] Examples of other functional moieties that can be used as a therapeutic agent include vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, actinomycin D, daunorubicin, doxorubicin, penicillin V, penicillin G, ampicillin, amoxicillin, cephalosporin, tetracycline, doxycycline, minocycline, demeclocycline, erythromycin, aminoglycoside antibiotics, polypeptide antibiotics, nystatin, griseofulvin, and idarubicin), anthracy clines, mitoxantrone, bleomycins, plicamycin, mithramycin and mitomycin, enzymes (L-asparaginase, which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine), antiplatelet agents such as G(GP) Ilb / IIIa inhibitors and vitronectin receptor antagonists, anti-proliferative / antimitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkyl sulfonates- busulfan, nitrosoureas (e.g., carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC), anti- proliferative / antimitotic antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., fluorouracil, floxuridine, cytarabine), purine analogs and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine {cladribine}), platinum coordination complexes (e.g., cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide, hormones (e.g., estrogen), anti-coagulants (e.g., heparin, synthetic heparin salts and other inhibitors of thrombin), fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab, antimigratory, antisecretory (e.g., breveldin), anti-inflammatory: such as adrenocortical steroids (e.g., cortisol, cortisone, fludrocortisone, prednisone, prednisolone, 6a-methylprednisolone, triamcinolone, betamethasone, and dexamethasone), non-steroidalagents (e.g., salicylic acid derivatives such as aspirin, para- aminophenol derivatives such as acetaminophen, indole and indene acetic acids (e.g., indomethacin, sulindac, etodalac), heteroaryl acetic acids (e.g., tolmetin, diclofenac, ketorolac), arylpropionic acids (e.g., ibuprofen and derivatives), anthranilic acids (e.g., mefenamic acid, meclofenamic acid), enolic acids (e.g., piroxicam, tenoxicam, phenylbutazone, oxyphenthatrazone), nabumetone, gold compounds (e.g., auranofin, aurothioglucose, gold sodium thiomalate), immunosuppressives (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (e.g., rapamycin, azathioprine, mycophenolate mofetil), angiogenic agents such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiotensin receptor blockers, nitric oxide donors, anti-sense oligionucleotides and combinations thereof, cell cycle inhibitors, mTOR inhibitors, and growth factor receptor signal transduction kinase inhibitors, retinoids, cyclin / CDK inhibitors, HMG co-enzyme reductase inhibitors (statins) or protease inhibitors.

[0054] In an embodiment, the small molecule (SM) can facilitate inhalational therapy of the lungs and comprises antioxidants and mucolytes (e.g., N-acetylcysteine), tyrosine kinase inhibitors (e.g., nintedanib), cystic fibrosis modulators (elexacaftor, tezacaftor, lumacaftor and ivacaftor), antifolate antineoplastic agents (e.g., pemetrexed), microtubule stabilizers (e.g., paclitaxel), chemotherapy drugs (e.g., doxorubicin, cisplatin and carboplatin), antimetabolites (e.g., methotrexate), immunosuppressants (e.g., tacrolimus, mycophenolate mofetil), NSAIDs (e.g., piroxicam), corticosteroids (e.g., prednisone), or a combination thereof.

[0055] Other therapeutic agents can be found in the Merck Index published by the Royal Society of Chemistry published in print and online at https: / / www.rsc.org / merck- index. For example, G1can be a linker between the backbone and the lipid, and include a therapeutic agent covalently bound thereto. Alternatively, or in addition, the group G2can be a therapeutic agent covalently bound to the lipid either directly or by a linker. Although not shown in Formula (II), it is also possible for a functional moiety such as a therapeutic agent to be linked to the backbone using a linkage similar to that linking the carbohydrate residue.

[0056] Lactobionic acid (LBA) is a disaccharide formed from gluconic acid and galactose. In some embodiments, lactobionic acid is derivatized as part of a liposome or LNP. Lipid-lactobionic acid conjugate of Formula (Ila):wherein LBA is a lactobionic acid residue, X1is NR3, 0, or C(R3h where R3is H or a substituted or unsubstituted Ci to Ce alkyl, each X3is independently O, S, NR3, or C(R3)z where R3is H or a substituted or unsubstituted Ci to Ce alkyl, n3is 0 to 20, and n4is 1 to 8. Each of R1, R2, n1, n2, X2, G1, G2, and L are as defined above for Formula (II). Preferably, G1is a group linking the lipid to the linker, R1and R2are each H, X1, X2, and X3are each NH, and n1=6, n2=2, and n3=4. In an embodiment, R1and R2are each H and n3=4.

[0057] For example, the lipid-lactobionic acid conjugate can be of Formula (Ila- 1):wherein L, G1, and G2are as defined above, preferably wherein G1is a functional moiety linking the lipid to the linker and G2is a functional moiety. Optionally in any of the Formulas (Ila) and (IIa-1), the hydroxyl groups can be fully or partially acylated with an acyl group having from 2 to 15 carbon atoms or 2 to 8 carbon atoms, preferably acetylated, more preferably fully acetylated as described above.

[0058] In another aspect, the ligand-lipid conjugate can be of Formula (lib):wherein X1is C=0 or C(R3)2 where R3is H or a substituted or unsubstituted Ci to G, alkyl, X4is O, S, NR3, or C(R3)2 where R3is H or a substituted or unsubstituted Ci to Ce alkyl, CL is a carbohydrate residue linked to CH by a 1 to 30 atom linker chain comprising a substituted or unsubstituted Ci to C12 alkyl or a Ce to C12 aryl comprising an amide, ester, or ether group, and n4is 2 or 3. Each of R1, R2, n1, n2, X2, G1, G2, and L are as defined above for Formula (II). The carbohydrate residue in Formula (lib) can be derived from N- acetylgalactosamine and can be a fully or partially acylated carbohydrate residue wherein the acyl groups have 2 to 15 carbon atoms or 2 to 8 carbon atoms, for example a fully or partially acetylated carbohydrate residue, such as fully acetylated.

[0059] In an embodiment, the ligand-lipid conjugate can be of Formula 3 (DSPE- LBAAC conjugate):

[0060] In an embodiment, the ligand-lipid conjugate can be of Formula 5 (DSPE- LBA conjugate):

[0061] Additional conjugates include the non-PEG spacer conjugates in FIG. 8A and PEG spacer conjugates in FIG. 8B. The non-PEG spacer conjugates of FIG. 8 A include Glycerol phospholipids 1 ,2-dioleoyl-sn-glycero-3 -phosphocholine conjugate and 1,2- dioleoyl-sn-Glycero-3-Phospho(Ethylene Glycol) conjugate; Glycerol lipids 1,2-dioleoyl-sn- glycero-3-choline conjugate and l,2-dioleoyl-sn-Glycero-3-Ethylene Glycol conjugate; Sphingolipids Ceramide conjugate and Sphingosine conjugate; and Sterol lipids Cholesterol conjugate and Ergosterol conjugate. The PEG spacer conjugates of FIG. 8B include Glycerol phospholipids l,2-dioleoyl-sn-glycero-3-phosphocholine conjugate and 1,2-dioleoyl-sn- Glycero-3-Phospho(Ethylene Glycol) conjugate; Glycerol lipids l,2-dioleoyl-sn-glycero-3- choline conjugate and l,2-dioleoyl-sn-Glycero-3 -Ethylene Glycol conjugate; Sphingolipids Ceramide conjugate and Sphingosine conjugate; and Sterol lipids Cholesterol conjugate andErgosterol conjugate, each shown having 43 ethylene glycol units. All corresponding conjugates having 1 to 100 ethylene glycol units are included herein. Additionally, the nonacetylated analogs of the conjugates of FIG. 8A and FIG. 8B are further included herein.

[0062] In an embodiment, the liposome or LNP comprises a ligand-lipid conjugate as described herein, and further comprises an additional lipid that does not have a carbohydrate ligand covalently attached.

[0063] A method of preparing the liposomes and LNPs includes, for example, the film hydration method, reverse phase evaporation, homogenization (high-shear homogenization, high-speed homogenization), ultrasonication, extrusion, and microfluidization. The film hydration method generally involves dissolving lipids in an organic solvent followed by removal of the solvent to form a thin film. The thin film is hydrated, with or without agitation, to form a liposomal dispersion. The liposomal dispersion can be extruded through filters having defined pore sizes to produce liposomes of targeted diameters. The reverse phase evaporation method generally involves forming a water-in-oil emulsion with an aqueous phase and a lipid-containing organic phase followed by removal of the organic phase under reduced pressure.

[0064] A method of incorporating a genetic sequence and / or small molecules into the liposome is described. The method includes drying of lipids to constitute / form a lipid film; and hydrating the lipid film using aqueous solvents containing genetic sequence for encapsulation. The method of encapsulation of genetic sequence (GS) and / or small molecules can be passive loading as described for the lipid film hydration or by means of pH based loading wherein pH gradient is maintained to enable influx of the GS and / or small molecules into the pre-formed liposomes.

[0065] In an embodiment, a lactobionic acid residue can be coupled to a backbone comprising a lysine residue by its alpha and epsilon amino groups. The lysine carboxyl group is in turn coupled to an amino group on an alkyl diamine, and the other amino group is coupled to a succinyl COOH group linked to a peptide nucleic acid. In other embodiments, the alkyl diamine can be substituted by an alkane diol to form a backbone with ester linkages. Alternatively, the succinic acid at the 5 ’ end can be replaced by a substituted or unsubstituted C to C20 dicarboxylic acid.

[0066] Particularly when GalNAc is used, the GalNAcs can be linked to the backbone by groups bearing an alkyl ether, amide, ester residues to provide the carbohydrate ligand. Some of these ligands are available commercially or can be synthesized using chemical synthesis methods familiar to one of ordinary skill in the art. General methods for chemicalsynthesis may be found in, among other sources, “Comprehensive Organic Transformations: A Guide to Functional Group Preparations,” Richard C. Larock, Wiley-VCH: 1999 and in “March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, Jerry March & Michael Smith, John Wiley & Sons Inc.: 2001. Of course, other carbohydrate residues can be similarly linked to the backbone by a group, e.g., a chain, bearing alkyl ether, amide, or ester residues to form the ligand.

[0067] In an aspect, disclosed are methods for the use of the liposomes / LNPs including carbohydrate-based ligands, and / or the systems or compositions including the liposomes to target the liver, lungs, and / or kidneys. In an embodiment, the lungs are targeted using a pulmonary delivery including the disclosed liposomes / LNPs and / or the systems or compositions. In an embodiment, the liver is targeted using a hepatic delivery including the disclosed liposomes / LNPs and / or the systems or compositions. In an embodiment, the kidneys are targeted using a renal delivery including the disclosed liposomes / LNPs and / or the systems or compositions. For example, the liposomes / LNPs and / or the systems or compositions can be used to treat cancers in the lung, kidney, and / or liver. In an embodiment, the liposomes can be used to treat non-small cell lung cancer. In an embodiment, the liposomes can be used to treat small-cell carcinoma. In an embodiment, the liposomes can be used to treat large cell carcinoma. In an embodiment, the liposomes can be used to treat lung adenocarcinoma. In an embodiment, the liposomes can be used to treat lung mesothelioma. In an embodiment, the liposomes can be used to treat squamous cell carcinoma. In an embodiment, the liposomes can be used to treat renal fibrosis. In an embodiment, the liposomes can be used to treat pulmonary fibrosis. In an embodiment, the liposomes can be used to treat liver fibrosis. The formulations can be administered directly to a subject for in vivo gene therapy.

[0068] In an embodiment, the compositions / systems including a liposome including a genetic sequence cargo (a therapeutic agent) can be used for treatment of a subject in need thereof ex vivo or in vivo. The methods typically include contacting a cell ex vivo or in vivo with an effective amount of the composition, to deliver a therapeutic agent, for example to modify the expression of an RNA. In an embodiment, the method includes contacting a population of target cells with an effective amount of the composition, to modify the expression of RNA to achieve a therapeutic result.

[0069] In an embodiment, the compositions / systems including a liposome including a small molecule cargo (a therapeutic agent) can be used for treatment of a subject in need thereof ex vivo or in vivo. The methods typically include contacting a cell ex vivo or in vivowith an effective amount of the composition, to deliver a therapeutic agent, for example to modify the expression of an RNA. In an embodiment, the method includes contacting a population of target cells with an effective amount of the composition, to modify the expression of RNA to achieve a therapeutic result.

[0070] In an embodiment, the compositions / systems including a liposome including a small molecule and genetic sequence cargoes (therapeutic agents) can be used for treatment of a subject in need thereof ex vivo or in vivo. The methods typically include contacting a cell ex vivo or in vivo with an effective amount of the composition, to deliver a therapeutic agent, for example to modify the expression of an RNA. In an embodiment, the method includes contacting a population of target cells with an effective amount of the composition, to modify the expression of RNA to achieve a therapeutic result.

[0071] In another aspect, a method for targeting mRNA, microRNA, DNA and gene editing in a health disorder in a subject comprises: providing to a cell of the subject in vivo or ex vivo a composition including the liposome and a cargo as disclosed herein, wherein the mRNA, microRNA, DNA cargo targeted to the cell modulates expression of a gene.100721 The liposome is generally provided as a formulation including an effective amount of a conjugate and a polymer, lipid, protein, or other pharmaceutical excipient for the organ- specific delivery (liver, lungs, and / or kidneys). Pharmaceutically acceptable carrier (also referred to as an excipient in the art), where the formulation is selected to suit the mode of administration. Pharmaceutically acceptable carriers are determined in part by the particular liposome being administered, as well as by the particular method used to administer the liposome. For example, the formulations may be for administration topically, locally, or systemically in a suitable pharmaceutical carrier. Accordingly, there is a wide variety of suitable formulations for the liposomes. Remington's Pharmaceutical Sciences, 15th Edition by E. W. Martin (Mark Publishing Company, 1975), discloses typical carriers and methods of preparation. For example, the formulations can include pharmaceutically acceptable carriers such as salts, carriers, buffering agents, emulsifiers, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, or stabilizers. The liposomes can also be encapsulated in suitable biocompatible microcapsules, microparticles, nanoparticles, or microspheres formed of biodegradable or non-biodegradable polymers or proteins for targeting liver, kidney, and / or lung cells. The particles can be capable of controlled release of the active agent. The particles can be microparticle(s) and / or nanoparticle(s). The particles can include one or more polymers. One or more of the polymers can be a synthetic polymer.T1The particle or particles can be formed by, for example, single emulsion technique or double emulsion technique or nanoprecipitation. Such systems are well known to those skilled in the art and may be optimized for use with the appropriate nucleic acid.

[0073] The liposomes, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosols for delivery to the respiratory system are created through various methods that generate fine particles or droplets capable of being inhaled into the lungs. These methods involve specialized devices and formulations designed to produce aerosols with the appropriate particle size and characteristics for effective respiratory delivery. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and air. For administration by inhalation, the compounds are delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant.

[0074] In an embodiment, the composition may be aerosolized for inhalational therapy using compressed air or oxygen to convert the composition into a mist. In an embodiment, a high-velocity airstream passes through a narrow opening, creating a low- pressure area that draws up the liquid composition (the liposome or LNP) and breaks it into fine droplets. In another manner, ultrasonic nebulization may be used to provide a therapeutic dose to a subject via the respiratory system. High-frequency ultrasonic waves may be used to create vibrations in the liquid composition, generating a mist of fine droplets. In yet another embodiment, a mesh nebulizer with a vibrating mesh having microscopic holes may be used to produce a fine mist from the liquid medication. These are compact, efficient, and produce consistent particle sizes.

[0075] An effective amount or therapeutically effective amount of the liposome and / or a composition including the liposome can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or to otherwise provide a desired pharmacologic and / or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder. The precise dosage will vary according to a variety of factors such as formulation and subject-dependent variables (e.g., age, immune system health, clinical symptoms etc.).

[0076] The liposomes, in particular a formulation including the liposome, can be administered to or otherwise contacted with target cells once, twice, or three time daily; one, two, three, four, five, six, seven times a week, one, two, three, four, five, six, seven or eighttimes a month. For example, in some embodiments, the composition is administered every two or three days, or on average about 2 to about 4 times week.

[0077] In an aspect, disclosed are methods for the production of the liposomes. In an aspect, disclosed is a pharmaceutical composition including the liposome that includes a carbohydrate-based ligand, a cargo, and a pharmaceutical excipient. The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be oral, parental (intradermal (IM), subcutaneous (SQ), intramuscular (IM), and intravenous (IV)), topical (e.g., by a transdermal patch), intranasal, intratracheal, epidermal and transdermal.

[0078] The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, emulsions or microemulsions, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers. The pharmaceutical composition of the present disclosure may also include a pharmaceutical carrier or excipient. A pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more cargoes such as nucleic acids to a subject.

[0079] In an aspect, the pharmaceutical composition comprises the liposome that includes a carbohydrate-based ligand, a cargo, and a pharmaceutical excipient for parental (intradermal (IM), subcutaneous (SQ), intramuscular (IM), and intravenous (IV)) administration. Compositions for parenteral administration include, for example, pharmaceutically acceptable and sterile emulsions, microemulsions, solutions, or suspensions. Liquid compositions may comprise, for example, water or other parenterally acceptable solvents, antioxidants, buffers, solubilizing agents, emulsifiers, or a combination thereof. Suitable vehicles and solvents include sterile water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils can be as a solvent or suspending medium, including mono- or diglycerides, or fatty acids such as oleic acid, e.g., for the preparation of non-aqueous sterile injectables. In an aspect, the present invention also provides kits for treating, preventing or diagnosing liver, kidney, and lung related disorder or condition in a subject in need thereof.

[0080] In an aspect, disclosed is a method for delivery of the therapeutic cargo, wherein the method includes a liposome as shown and described herein. In an embodiment, the method of delivery is a pulmonary delivery. In an embodiment, the method of delivery is an intratracheal delivery. In an embodiment, the method of delivery is a renal delivery. In an embodiment, the method of delivery is a hepatic delivery.

[0081] Disclosed herein is a method of delivering a liposome to the lungs of a subject in need thereof, comprising inhalationally administering to the subject an effective amount of the liposome detailed above.

[0082] In an aspect, disclosed is a method for reducing expression of a targeted RNA involved in a health disorder in a subject, the method includes: providing to a cell of the subject in vivo or ex vivo a liposome including carbohydrate-based ligand (such as lactobionic acid) and a suitable cargo as described herein, wherein the binding of the cargo to the targeted RNA reduces expression of the targeted RNA, in particular where the targeted RNA is a microRNA.

[0083] In an aspect, disclosed is a method for targeting DNA and gene editing in a health disorder in a subject, the method including providing to a cell of the subject in vivo or ex vivo a liposome including carbohydrate-based ligand and a suitable cargo (a DNA in this case) as described herein, wherein the DNA of the cargo targeted to the cell modulates expression of a gene.

[0084] The present disclosure is illustrated and further described in more detail with reference to the following non-limiting examples.EXAMPLES

[0085] This disclosure is illustrated by the following Examples, which are not intended to limit the claims.Synthesis of DSPE-LBAAC and DSPE-LBA

[0086] To develop LBA-decorated liposomes, l,2-Distearoyl-sn-glycero-3- phosphoethanolamine-PEG (2000) (DSPE-PEG) lipid was utilized, which is an amphiphilic lipid commonly used for developing lipid conjugates. The carboxy terminated DSPE-PEG (1) was conjugated to di-lactobionic acid acetylated ligand (LBAAC) (2) by amide coupling using diisopylethanolamine (DIEA) and Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) reagents in dimethylformamide (DMF) (Figure 1). The conjugated product DSPE-LBAAC (3) was purified using HPLC and MALDI-TOF (Figure 3). Similarly, DSPE-LBA was synthesized using DSPE-PEG (1) using deacetylated di-lactobionic acid (LBA) ligand (4) to form the conjugated product DSPE-LBA conjugate (5) (Figure 2).Formulation of DSPE-LBA liposomes

[0087] Following the synthesis of our ligand-conjugated DSPE-PEG lipid, liposomes were formulated using thin-film lipid hydration technique (Figure 4 & Figure 5). A combination of four different lipids were utilized, l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l ,2-Distearoyl-sn-glycero-3-phospho-L-serine (DSPS), DSPE-PEG (2000) (DSPE), and DSPE-PEG (2000)-LBA (DSPE-ligand) at an optimum lipid mole ratio. The general process for the preparation of liposomes using lipid film hydration technique, also referred to as the “film hydration method’- (Figure 4) involves solubilizing the lipid composition in an organic solvent; solvent evaporation to cause lipid film formation; hydration of the lipid film for the encapsulation of cargos; and extrusion through filters to form unilamellar liposomes with defined pore size. Figure 5 shows a schematic representation of chemical structures of ligand-lipid conjugates and their respective arrangement in liposomes. The scheme shows LBA or the acetylated analog LBAAC coupled to a lipid via amide coupling reaction. The formed LBA-Lipid conjugate and LBAAc-Lipid conjugate products are prepared into LBA-decorated and acetylated-LBA-decorated liposomes, respectively, encapsulating cargos such as small molecule drugs and peptide nucleic acids / ASOs. The LBA-Lipid conjugate liposomes encapsulating cargo (e.g., nucleic acids, small molecule drugs, etc.) are suitable for targeting pulmonary (pulmonary targeted liposomes (PTL)) and / or liver (liver-targeted liposomes (LTL)), which can be administered by intratracheal or subcutaneous administration. The LBAAc-Lipid conjugate liposomes encapsulating cargo are suitable for targeting kidney (kidney-targeted liposomes (KTL)), which can be administered by subcutaneous administration.

[0088] Figure 6 illustrates a schematic of a microfluidic -based LNP synthesis system for the preparation of LNP formulations via microfluidic mixing of lipids, e.g., from Lipids chosen from formulation 1. D-Lin-MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)- l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester; CAS# 1224606-06-7); 2. DMG-PEG 2000 (l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol); 3. Cholesterol; and 4. DSPC. The LNP formulations were prepared by ethanol-dilution method using the microfluidic -based instrument FLEX-M by Precigenome LLC (California, USA). The ethanolic lipid solution and aqueous mRNA buffer solutions were introduced into the microfluidic device, where positively-charged lipids and negatively-charged mRNAs formedcomplexes via electrostatic interactions. The mRNA-cationic lipid complexes were then assembled with other lipids to form LNPs. Lipids dissolved in ethanol were self- assembled by diluting ethanol with a buffer solution, ligand conjugated lipid with the lipid excipients prior to formulating the LNPs.

[0089] Figure 7A illustrates the characterization and comparison of ligand functionalized FLuc mRNA LNPs (“FLuc Ligand-LNPs”) to FLuc LNPs using DLS. Results highlight that incorporation of ligand-lipid conjugates in LNPs does not impact the particle size and encapsulation FLuc mRNA cargo inside the LNPs. Figure 7B illustrates the cellular uptake of ligand functionalized GFP mRNA LNPs in HK-2 cells using confocal microscopy exhibiting that GFP mRNA can be efficiently encapsulated in LNPs which could translate to GFP protein. Figure 7C illustrates the comparative cellular uptake of ligand functionalized GFP mRNA LNPs in HK-2 cells using flow cytometry highlighting increased transfection from mRNA ligand-LNPs as opposed to mRNA LNPs.

[0090] The following terms are used to describe the invention of the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0092] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0093] Compounds and materials are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The following terms are used to describe the invention of the present disclosure. In instanceswhere a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0094] As used herein, the term “hydrocarbyl” and “hydrocarbon” refer to a substituent comprising carbon and hydrogen, optionally with 1 to 3 heteroatoms, for example, oxygen, nitrogen, halogen, silicon, sulfur, or a combination thereof; “alkyl” refers to a straight or branched chain, saturated monovalent hydrocarbon group; “alkylene” refers to a straight or branched chain, saturated, divalent hydrocarbon group; “alkylidene” refers to a straight or branched chain, saturated divalent hydrocarbon group, with both valences on a single common carbon atom; “alkenyl” refers to a straight or branched chain monovalent hydrocarbon group having at least two carbons joined by a carbon-carbon double bond; “cycloalkyl” refers to a non-aromatic monovalent monocyclic or multicyclic hydrocarbon group having at least three carbon atoms, “cycloalkenyl” refers to a non-aromatic cyclic divalent hydrocarbon group having at least three carbon atoms, with at least one degree of unsaturation; “aryl” refers to an aromatic monovalent group containing only carbon in the aromatic ring or rings; “arylene” refers to an aromatic divalent group containing only carbon in the aromatic ring or rings; “alkylaryl” refers to an aryl group that has been substituted with an alkyl group as defined above, with 4-methylphenyl being an exemplary alkylaryl group; “arylalkyl” refers to an alkyl group that has been substituted with an aryl group as defined above, with benzyl being an exemplary arylalkyl group; “acyl” refers to an alkyl group as defined above with the indicated number of carbon atoms attached through a carbonyl carbon bridge (-C(=O)-); “alkoxy” refers to an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge (-O-); and “aryloxy” refers to an aryl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge (-O-).

[0095] Unless otherwise indicated, each of the foregoing groups can be unsubstituted or substituted, provided that the substitution does not significantly adversely affect synthesis, stability, or use of the compound. The term “substituted” as used herein means that at least one hydrogen on the designated atom or group is replaced with another group, provided that the designated atom’s normal valence is not exceeded. When the substituent is oxo (i.e., =0), then two hydrogens on the atom are replaced. Combinations of substituents or variables are permissible provided that the substitutions do not significantly adversely affect synthesis or use of the compound. Exemplary groups that can be present on a “substituted” position include, but are not limited to, cyano; hydroxyl; nitro; azido; alkanoyl (such as a C2-6alkanoyl group such as acyl); carboxamido; Ci-6 or C1-3 alkyl, cycloalkyl, alkenyl, and alkynyl (including groups having at least one unsaturated linkages and from 2 to 8, or 2 to 6 carbon atoms); C1-6 or C1-3 alkoxys; C6-10 aryloxy such as phenoxy; C1-6 alkylthio; C1-6 or C1-3 alkylsulfinyl; C1-6 or C1-3 alkylsulfonyl; aminodi(Ci-6 or Ci-3)alkyl; C6-12 aryl having at least one aromatic rings (e.g., phenyl, biphenyl, naphthyl, or the like, each ring either substituted or unsubstituted aromatic); C7-19 arylalkyl having 1 to 3 separate or fused rings and from 6 to 18 ring carbon atoms; or arylalkoxy having 1 to 3 separate or fused rings and from 6 to 18 ring carbon atoms, with henzyloxy being an exemplary arylalkoxy. The indicated number of carbon atoms of a group do not include any substituents.

[0096] As used herein, a peptide nucleic acid (PNA) is an artificially synthesized polymer with a backbone comprising repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-) to a nitrogen on the backbone as shown in Figure 1. By convention, PNA is represented with the N-terminus upward (or side to the left) and the C-terminus downward (or side to the right) as in peptides. A PNA is not a peptide or a nucleic acid in the formal sense, but rather a hybrid of the two.

[0097] In some aspects, the PNA monomers forming a PNA oligomer are modified at the gamma position in the polyamide backbone (yPNAs) as illustrated below (wherein “B” is a nucleobase and “R” is a substitution at the gamma position).Chiral yPN

[0098] Substitution at the gamma position creates chirality and provides helical preorganization to the PNA oligomer, yielding substantially increased binding affinity to the target RNA. Other advantageous properties can be conferred depending on the chemical nature of the specific substitution at the gamma position (the “R” group in the chiral yPNAabove). The synthesis of yPNAs is described in U.S. Patent No. 10,221,216, incorporated herein by reference for the disclosure of yPN A and methods of synthesis of yPNA.

[0099] Examples of y substitution with other side chains include that of alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, and the derivatives thereof. The “derivatives thereof’ herein are defined as those chemical moieties that are covalently attached to these amino acid side chains, for instance, to that of serine, cysteine, threonine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, and arginine.

[0100] In an aspect, the PNA oligomer forming a PNA / RNA / PNA triplex is a yPNA with a tail clamp, or a ytcPNA.

[0101] Chemical modifications of the basic PNA structure are known and can be used. For example, fluorine-modified, cyclopentyl-modified, mini-peg-modified, guanidinium-modified, pyrrolidinyl-modified, and 2-aminopyridien-modified PNAs are known in the art and can be chosen for preparation of the PNA oligomer to improve cell permeability or increase RNA binding affinity. Mini-PEG-containing y-PNAs and their methods of synthesis are described in US. Patent No. 10,793,605,

[0102] The PNA oligomers can also include other positively charged moieties to increase the solubility of the PNA, for increased cell permeability, and / or to increase the affinity of the PNA for the target RNA. Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. Lysine and arginine residues can be added to a tcPNA linker or can be added to the carboxy or the N-terminus of a PNA oligomer strand.

[0103] Exemplary modifications to PNA include, but are not limited to, incorporation of charged amino acid residues, such as lysine at the termini or in the interior part of the oligomer; inclusion of polar groups in the backbone, carboxymethylene bridge, and in the nucleobases; chiral PNAs bearing substituents on the original N-(2-aminoethyl)glycine backbone; replacement of the original aminoethylglycyl backbone skeleton with a negatively- charged scaffold; conjugation of high molecular weight polyethylene glycol (PEG) to one of the termini; fusion of PNA to RNA to generate a chimeric oligomer, redesign of the backbone architecture, conjugation of PNA to DNA or RNA. These modifications improve solubility but often result in reduced binding affinity and / or sequence specificity.

[0104] Gamma- PNA modifications include serine modified, lysine modified, glutamic acid modified, or alanine modified. Particularly when the PNAs are serine gammamodified, the yPNAs target RNA more efficiently compared to the conventional full length PNAs based on their binding affinity.

[0105] Phosphorothioate analogues of DNA, RNA and OMe-RNA have sulfur in place of oxygen as one of the non-bridging ligands bonded to phosphorus.

[0106] A morpholino, also known as a morpholino oligomer and as a phosphorodiamidate morpholino oligomer (PMO), is a type of oligomer used in molecular biology to modify gene expression. Its molecular structure contains DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholines block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholines are used as research tools for reverse genetics by knocking down gene function.

[0107] Locked nucleic acid is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformation restriction increases binding affinity for complementarity sequences and provides a chemical approach for the control of gene expression and optimization of microarrays.

[0108] 2'-O-methylation is a nucleoside modification of RNA, where a methyl group is added to the 2' hydroxyl of the ribose moiety of a nucleoside, producing a methoxy group. 2'-O-methylated nucleosides are mostly found in ribosomal RNA and small nuclear RNA and occur in the functionally essential regions of the ribosome and spliceosome.

[0109] Like the 2’-OMe nucleoside modification of RNA, the 2’-O-methoxyethyl- RNA (2’-M0E) backbone provides enhanced duplex stability, significant nuclease resistance.

[0110] 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation.

[0111] 5 ’-Methylcytosine is a methylated form of the DNA base cytosine (C) that regulates gene transcription and takes several other biological roles. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered

[0112] The G-clamp heterocycle modification, a cytosine analog that clamps on to guanine by forming an additional hydrogen bond, was rationally designed to enhance oligonucleotide / RNA hybrid affinity. PNAs containing internally-linked guanidinium moieties (GPNAs) are readily taken-up by mammalian cells, and bind to DNA and RNA with high affinity and sequence specificity.

[0113] A protecting group is a functional group that transforms a reactive functional group in an organic molecule so that it does not undergo a reaction meant for another functional group in the structure. Protecting groups are widely used in various forms in organic synthesis. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 4d. Ed., Wiley & Sons, 2007. Adjustments to the protecting groups and formation and cleavage methods described herein may be adjusted as necessary in light of the various substituents.

[0114] A residue is used to describe any of the parts that integrate to make up a larger molecule such as a conjugate. For example, a lysine residue refers to a lysine amino acid structure integral to a conjugate covalently bonded to an alkyl diamine via the lysine carboxyl group (by an amide function) and covalently bonded by its alpha and epsilon amino groups to lactobionic acid molecules (by amide bonds) as shown in the drawings herein. A residue may also be referred to as a moiety.|0115| RNA as used herein includes different types of RNA that serve different functions including messenger RNA, transfer RNA, ribosomal RNA, and microRNA. Micro RNA (miRNA) is involved in gene expression. miRNA is a non-coding region of mRNA that is believed to be important in the either promotion or inhibition of gene expression. These may involve small sequences of about 25 nucleotides.

[0116] A sequence of bases is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides forming alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequences are usually presented from the 5’ end to the 3' end. For DNA, the sense strand is used. Because nucleic acids are normally linear (unbranched) polymers, specifying the sequence is equivalent to defining the covalent structure of the entire molecule.

[0117] Gene expression is the process by which a genes coded information is converted into the structures present and operating in the cell. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs). miRNA is a non-coding region of mRNA that is believed to be important in the either promotion or inhibition of gene expression.

[0118] Below are the names or brief descriptions of some gene names and micro RNAs and genes known in the art. The nonlimiting examples of the microRNAs includemiR-33, miR-145, miR-143, miR-21, miR-182, or a combination thereof. The nonlimiting examples of the genes include c-myc, PTEN, PI3K, KRAS, TP53, EGFR, MET, LKB1, PRAF, PIK3CA, ALK, RET, ROS 1, CFTR, PDGFR, TGF-fh or a combination thereof. Other information can be found on the website https: / / www.genecards.org / which is incorporated herein by reference.

[0119] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. By way of example, "an element" means one element or more than one element.

[0120] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0121] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0122] The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0123] The terms “about” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within + 10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention anddoes not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0124] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0125] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0126] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionallyincluding more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0127] The phrase "one or more," as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0128] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as being modified in all instances by the term "about," meaning within 10% of the indicated number (e.g., "about 10%" means 9%- 11% and "about 2%" means 1.8%-2.2%).

[0129] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Generally, unless otherwise expressly stated herein, "weight" or "amount" as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising the ingredient, wherein the raw material may be described herein to comprise less than and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.

[0130] All ranges and amounts given herein are intended to include subranges and amounts using any disclosed point as an end point. Thus, a range of "1% to 10%, such as 2% to 8%, such as 3% to 5%," is intended to encompass ranges of "1% to 8%," "1% to 5%," "2% to 10%, " and so on. All numbers, amounts, ranges, etc., are intended to be modified by the term "about," whether or not so expressly stated. Similarly, a range given of "about 1% to 10%" is intended to have the term "about" modifying both the 1% and the 10% endpoints. Further, it is understood that when an amount of a component is given, it is intended to signify the amount of the active material unless otherwise specifically stated.

[0131] As used herein, the term “administering” means the actual physical introduction of a composition into or onto (as appropriate) a subject, a host, or cell. Any and all methods of introducing the composition into the subject, host or cell are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to thoseskilled in the art, and also are exemplified herein. “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0132] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0133] As used herein, the term “pharmaceutically acceptable” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human or a non-human subject. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0134] As used herein, the terms “treat,” “treating,” and “treatment” include inhibiting the pathological condition, disorder, or disease, e.g., arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or relieving the pathological condition, disorder, or disease, e.g., causing regression of the pathological condition, disorder, or disease or its clinical symptoms. Treatment means any way the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, preferably a human. Treatment also means providing an active compound to a patient in an amount sufficient to measurably reduce any cancer symptom, slow cancer progression or cause cancer regression. These terms also encompass therapy and cure. In certain embodiments treatment of the cancer may be commenced before the patient presents symptoms of the disease.

[0135] As used herein, the term "effective amount" or “therapeutically effective amount” refers to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy e.g., prophylactic or therapeutic agent). An effective amount can require more than one dose. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0136] As used herein, the term “dose” or “dosage” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0137] Effective amounts may vary depending upon the biological effect desired in the individual, condition to be treated, and / or the specific characteristics of the composition according to the present invention and the individual. In this respect, any suitable dose of the composition can be administered to the patient (e.g., human), according to the type of disease to be treated. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman’s: The Pharmacological Bases of Therapeutics , 8th ed., Pergamon Press, 1990; and Remington’s Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0138] The term “subject” or “patient” is used herein to refer to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird e.g., a duck or a goose), and a shark. In an embodiment, the subject or patient is a human subject or a human patient, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In one embodiment, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45- 50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 years of age. Values and ranges intermediate to the above recited ranges are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above-recited values as upper and / or lower limits are intended to be included. As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0139] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.

[0140] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.101411 All compounds are understood to include all possible isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers and encompass heavy isotopes and radioactive isotopes. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon includenC,nC, and14C. Accordingly, the compounds disclosed herein may include heavy or radioactive isotopes in the structure of the compounds or as substituents attached thereto. Examples of useful heavy or radioactive isotopes include18F,15N,18O,76Br,125I and131I.

[0142] “Pharmaceutical compositions” means compositions comprising at least one active agent, such as a compound or salt of Formula (I), etc., and at least one other substance, such as a carrier. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0143] “Carrier” means a diluent, excipient, or vehicle with which an active compound is administered. A “pharmaceutically acceptable carrier” means a substance, e.g., excipient, diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.

[0144] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s T-test, where p < 0.05.

[0145] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A liposome or lipid nanoparticle, comprising: a ligand-lipid conjugate having a Formula (I):CL-linker-L (I) wherein L is a lipid optionally functionalized with a functional moiety, “linker” is a linking group or a bond, and CL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

2. The liposome or lipid nanoparticle of claim 1 , whereinCL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide, preferably wherein CL comprises a carbohydrate residue / ligand derived from a monosaccharide, a disaccharide, or a polysaccharide, optionally wherein the carbohydrate ligand is fully or partially acylated on a hydroxy or amino group thereof with a C2 to C15 acyl group, preferably wherein the carbohydrate ligand is fully or partially acetylated on a hydroxy or amino group thereof.

3. The liposome or lipid nanoparticle of claim 1, wherein the ligand-lipid conjugate is according to Formula (II):whereinL is a lipid;R1and R2are each independently H or a substituted or unsubstituted Ci to G> alkyl;X1is O, S, NR3, C=O, or C(R3)z where R3is H or a substituted or unsubstituted Ci to C6alkyl;X2is O, S, NR3, or C(R3h where R3is H or a substituted or unsubstituted Ci to Ce alkyl;G1is a direct bond or a linking group;G2is H or a functional moiety;CL is a carbohydrate ligand comprising 2 to 16 carbohydrate residues derived from a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide, preferably wherein CLcomprises a carbohydrate residue / ligand derived from a monosaccharide, a disaccharide, or a polysaccharide, optionally wherein the carbohydrate ligand is fully or partially acylated on a hydroxy or amino group thereof with a C2 to C15 acyl group, preferably wherein the carbohydrate ligand is fully or partially acetylated on a hydroxy or amino group thereof; n1is 1 to 20; and n2is 0 to 20.

4. The liposome or lipid nanoparticle of claim 3, wherein the ligand-lipid conjugate iswhereinLBA is lactobionic acid; each X3is independently O, S, NR3, or C(R3)2 where R3is H or a substituted or unsubstituted Ci to Ce alkyl; n3is 0 to 20; n4is 1 to 8.

5. The liposome or lipid nanoparticle of claim 3, wherein the ligand-lipid conjugate is according to Formula (IIa-1 ):

6. The liposome or lipid nanoparticle of claim 3, wherein the ligand-lipid conjugate is according to Formula (lib):whereinX4is O, S, NR3, or C(R3)2 where R3is H or a substituted or unsubstituted Ci to G, alkyl;CL is a carbohydrate residue linked to CH by a 1 to 30 atom linker chain comprising a substituted or unsubstituted Ci to C12 alkyl or a Ce to C12 aryl comprising an amide, ester, or ether group; and n4is 2 or 3.

7. The liposome or lipid nanoparticle of claim 3 or 6, wherein CL is derived from N- acetylgalactosamine (GalNAc), galactose, lactobionic acid or an acetylated ester thereof, preferably wherein the carbohydrate residue is a fully or partially acetylated product of N- acetylgalactos amine (GalNAc), galactose, or lactobionic acid.

8. The liposome or lipid nanoparticle of any one of claims 3-7, wherein G1is a direct bond and G2is H.

9. The liposome or lipid nanoparticle of any one of claims 1-7, wherein L is a glycerophospholipid; a phosphosphingolipid; an ionizable lipid; a PEGylated lipids; cholesterol; a phosphatidylcholine; a phosphatidylethanolamine; a phosphatidylserine; a phosphatidylglycerol; or a combination thereof.

10. The liposome or lipid nanoparticle of any one of claims 1-7, wherein L is 1,2- diarachidonoyl-sn-glycero-3-phosphocholine; 1 ,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine; 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine; 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; l,2-dihexanoyl-sn-glycero-3- phosphocholine (DHPC); l,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE); 1 ,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1 ,2-dilinoleoyl-sn-glycero-3- phosphocholine; 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine; 1 ,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC); 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); dimyristoylphosphatidylglycerol (DMPG); l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC); l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2- dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1 ,2-dioleoyl-sn-glycero-3-(phospho-L- serine) (DOPS); l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE); l,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE); 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC); 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1 -palmitoyl-2-oleoyl phosphatidylglycerol; l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE); egg phosphatidylcholine (EPC); glycerol monooleate (GMO); partially hydrogenated soy phosphatidylchloline (PHSPC); phosphatidylethanolamine (PE); phosphatidylglycerol (PG); phosphatidylinositol (PI); phosphatidylserine (PS); soybeanphosphatidylcholine (SPC); PEGylated derivatives thereof; or a combination thereof.

11. The liposome or lipid nanoparticle of any one of claims 3-7, wherein G1is a residue of a polyethylene glycol, a polypropylene glycol, or a polyethylene-propylene glycol.

12. The liposome or lipid nanoparticle of any one of claims 3-7, wherein G1is a residue of a polyethylene glycol having 1 to 100 ethylene glycol residues, specifically 5 to90, more specifically 10 to 80, yet more specifically 20 to 70, still yet more specifically 30 to 60, and still more specifically 40 to 50 ethylene glycol residues.

13. The liposome or lipid nanoparticle of any one of claims 1-12, further comprising a cargo, wherein the cargo is a nucleic acid, a small molecule, or a combination thereof.

14. The liposome or lipid nanoparticle of claim 13, wherein the cargo is a nucleic acid that is an mRNA sequence, an siRNA sequence, ASOs, or a DNA sequence; a small molecule that is a beta-2 agonist, an anticholinergic, an anti-inflammatory agent, a leukotriene modifier, a mast cell stabilizer, an antibiotic, an antimicrobial, an antiviral agent, a mucolytic, an immunomodulator, a monoclonal antibody, an analgesic, an anticancer agent, an anti-fibrotic agent, a vinca alkaloid, an epidipodophyllotoxin, an anthracy cline, a bleomycin, a pyrimidine analog, a platinum coordination complex aminoglutethimide, a hormone, an anti-coagulant, a fibrinolytic agent, antimigratory, an antiinflammatory, a non-steroidal agent, an immunosuppressive, an angiogenic agent, an angiotensin receptor blocker, a nitric oxide donor, a statin, a protease inhibitor, a tyrosine kinase inhibitor, a cystic fibrosis modulator; a small molecule that is ivacaftor, elexacaftor, tezacaftor, lumacaftor, pirfenidone, N- acetylcysteine, nintedanib, cisplatin, carboplatin, docetaxel, paclitaxel, pemetrexed, kielin, tolvaptan, bleomycin, cyclosporin, romidepsin, doxorubicin, danunorubicin, vincristine, methotrexate, cyclophosphamide, venetoclax, hydroxyurea, mercaptopurine, cytarabine, albuterol, salmeterol, formoterol, pratropium bromide, tiotropium, methylxanthine, theophylline, a corticosteroid, beclomethasone, budesonide, fluticasone, montelukast, cromolyn sodium, nedocromil, tobramycin, aztreonam, colistin (Polymyxin E), amphotericin B, voriconazole, zanamivir, oseltamivir, ribavirin, pulmozyme, dupilumab, omalizumab, cyclosporine, beractant, poractant alfa, fentanyl, vinblastine, vinorelbine, etoposide, teniposide, dactinomycin, actinomycin D, daunorubicin, penicillin V, penicillin G, ampicillin, amoxicillin, cephalosporin, tetracycline, doxycycline, minocycline, demeclocycline, erythromycin, nystatin, griseofulvin, idarubicin, mitoxantrone, plicamycin, mithramycin, mitomycin, fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine, pentostatin, 2- chlorodeoxyadenosine, procarbazine, hydroxyurea, mitotane, aspirin, acetaminophen, indomethacin, sulindac, etodalac dipyridamole, ticlopidine, clopidogrel, abciximab, breveldin, cortisol, cortisone, fludrocortisone, prednisone, prednisolone, 6a- methylprednisolone, triamcinolone, betamethasone, dexamethasone, tolmetin, diclofenac,ketorolac, mefenamic acid, meclofenamic acid, piroxicam, tenoxicam, phenylbutazone, oxyphenthatrazone, nabumetone, auranofin, aurothioglucose, gold sodium thiomalate, tacrolimus (FK-506), sirolimus, rapamycin, azathioprine, mycophenolate mofetil; or a combination thereof.

15. The liposome or lipid nanoparticle of any one of claims 1-14, further comprising an additional lipid.

16. The liposome or lipid nanoparticle of any one of claims 1-14, wherein the ligand-a partially acetylated, or a non- acetylated analog thereof.

17. A composition comprising, the liposome or lipid nanoparticle of any one of claims 1-16, a cargo, and a pharmaceutically acceptable excipient.

18. A method for delivery of a therapeutic cargo to liver, lungs, kidneys, or a combination thereof, comprising administering the liposome or lipid nanoparticle of any one of claims 1-16 or the composition of claim 17 to a subject in need thereof.

19. The method of claim 18, comprising parenteral, intratracheal, or pulmonary administration.

20. A liposome or lipid nanoparticle, comprising: lactobionic acid linked to a lipid through a linker group, and wherein the liposome or lipid nanoparticle targets the liver, lungs, and / or kidneys.21 . The liposome or lipid nanoparticle of claim 20, further comprising a cargo, wherein the cargo is a nucleic acid, a small molecule, or a combination thereof.

22. A method for delivery of a therapeutic cargo to liver, lungs, kidneys, or a combination thereof, comprising administering the liposome or lipid nanoparticle of claim 20 or 21 to a subject in need thereof.

Images