Anti-inflammatory lipid nanoparticle compositions and uses

Abstract

The present invention provides a lipid nanoparticle comprising an entity that contains one or multiple sialic acid (SA) moieties, the entity is preferably a ganglioside, a ganglioside derivative, a ganglioside mimetic, or a combination thereof. The lipid nanoparticle of the present invention can effectively present SA moieties on the surface to bind Siglecs and complement factors to, inter alia, mitigate inflammation. The present invention also provides methods of using said lipid nanoparticle compositions described herein for pharmaceutical applications. For example, the lipid nanoparticles provided herein are useful for treating inflammatory and autoimmune diseases.

Description

[0001] ANTI-INFLAMMATORY LIPID NANOPARTICLE COMPOSITIONS AND USES

[0002] RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 617,218, filed on January 3, 2024. The entire teachings of the above application are incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Sialic acid (SA), also known as N-acetylneuraminic acid (or NANA), is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectin (Siglec). Sialic acid has mainly three derivatives: N-acetyl neuraminic acid (Neu5Ac), N-acetyl neuraminic acid hydroxyalkyl (Neu5Gc) and 3- deoxy-D-glycero-D-galacto-nonyl ketose (Kdn). There are other sialic acid derivatives that are further derived from these primary derivatives.

[0006] Most Siglecs have an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) that can mediate inhibitory signals upon binding to sialic acid and activate downstream inhibitory signaling through the recruitment of tyrosine phosphatases SHP-1 and SHP-2. Sialic acid can also regulate the alternative pathway of complement activation. Major serum protein complement factor H recognizes sialic acid as a “self’ marker, which helps to inhibit Clq / C3b fragment activation. Therefore, Sialic acid, when binding a Siglec receptor on immune cells or the complement factor H (CFH), serves as a self-associated molecular pattern (SMAP) to suppress over-reactive immune responses and maintain an overall homeostasis.

[0007] Therefore, it can be therapeutically beneficial to bind Siglecs with a chemical or biological entity that comprises sialic acid residue to modulate the immune activation and inhibition for the treatment of inflammatory diseases. To increase the avidity, valency and binding efficiency, a common strategy is to attach a sialic acid residue on the surface of a nanoparticle so that the nanoparticles can carry the sialic acid residue to reach targeted Siglecs for binding. Nanoparticles decorated with sialic acid residues also have the advantage of multivalency and can be more efficient than sialic acid molecules in binding Siglecs.

[0008] A commonly used method for attaching sialic acid to the surface of lipid nanoparticles is chemical conjugation. Typically, a sialic acid molecule is functionalized with a reactive group capable of forming a covalent bond with another reactive group on the nanoparticle surface. For example, Spence, S, et al. in Sci. Trans. Med., 2 Sep 2015, Vol 7, Issue 303 p. 303(140) described a Siglec-binding platform consisting of poly(lactic-co-gly colic acid) (PLGA) nanoparticles decorated with di(a2— >8) N-acetylneuraminic acid (a2,8 NANA-NP). They first prepared the PLGA nanoparticles by using an emulsification and solvent evaporation process. The PLGA nanoparticles were then activated with a carbodiimide (EDC) followed by conjugation with a2,8 NANA to form the a2,8 NANA-NP. In general, this type of method suffers from several shortcomings: 1) the multi-step process is complex and difficult to scale up; 2) low conjugation efficiency due to steric hinderance which leads to low ligand density on NP surface and thus low binding efficiency; 3) introduction of harmful chemical residues in the pharmaceutical formulation that are difficult to remove; and 4) such conjugation may cause unwanted immunogenicity.

[0009] Therefore, there is still an unmet need for novel approaches to presenting sialic acid ligands on nanoparticle surfaces to facilitate the binding of Siglecs and more effectively and safely treat inflammatory and autoimmune diseases.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention in part provides a lipid nanoparticle comprising an entity that contains one or multiple sialic acid (SA) moieties, the entity is preferably a ganglioside, a ganglioside derivative, a ganglioside mimetic, or a combination thereof. The lipid nanoparticle of the present invention can effectively present SA moieties on the surface to bind Siglecs and complement factors to, inter alia, mitigate inflammation. The present invention also provides methods of using said lipid nanoparticle compositions described herein for pharmaceutical applications. For example, the lipid nanoparticles provided herein are useful for treating inflammatory and autoimmune diseases.

[0012] Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked on a sugar chain. Preferred gangliosides include a ganglioside containing one sialic acid unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two sialic acid units such as GDla, GDlb, GD2, and GD3, or three sialic acid units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four salic acid units such as GQ1. Other non-limiting examples of gangliosides include ganglioside-total, C18:0(2-10NBD) GM1, NGcGM3, C18:0 GM3, C20:0 GM1, C17:0 GM1, and C18:0 GM1, commercially available at Avanti Polar Lipids, Birmingham, AL.

[0013] For gangliosides that comprise only a single sialic acid unit (such as in the case of GM1, GM2, and GM3), the sialic acid unit can be linked with its neighboring sugar ring via an a2,3, a2,6, a2,8, or a2,9 linkage. For gangliosides that comprise multiple sialic acid units, one sialic acid unit may be linked with its neighboring sialic acid unit via an a2,3, a2,6, a2,8, or a2,9 linkage.

[0014] Preferably, the lipid nanoparticles further comprise a pharmaceutical excipient selected from the group containing cholesterol and / or a phospholipid, or derivatives, mimetics, or combinations thereof.

[0015] In one embodiment, the lipid nanoparticle described herein comprises a ganglioside and a cholesterol or cholesterol derivative. Cholesterol can be natural in its native form or a natural or synthetic derivative.

[0016] In another embodiment, the anti-inflammatory lipid nanoparticle described herein comprises a ganglioside and one or more phospholipids.

[0017] In yet another embodiment, the anti-inflammatory lipid nanoparticle described herein comprises a ganglioside, a mixture of cholesterol or cholesterol derivatives, and one or more phospholipids.

[0018] The lipid nanoparticles of the present invention can be produced by co-precipitating said ganglioside with cholesterol or phospholipid. Preferably, they are produced using a microfluidic device.

[0019] The invention further relates to methods for the treatment of a disease or disorder, such as an inflammatory disease or autoimmune disease, in a subject in need thereof comprising administering to the subject the composition of the invention.

[0020] Anti-inflammatory lipid nanoparticles of the present invention are particularly useful in treating inflammatory diseases in the eye, including but not limited to age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, diabetic retinopathy, diabetic macular edema.

[0021] In another embodiment, the lipid nanoparticle of the current invention is used to treat dry AMD such as geographic atrophy.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1. Lipid Nanoparticles of the Present Invention Reduced the Expression of IL- 6, TNF-a, and IL-ip.

[0024] Figure 2. MTT Assay of Ml Microphages.

[0025] Figure 3. MTT Viability Assay of Ml Microphages. Figure 4. GD3-DSPC NPs Reduced the Expression of IL-6 in Cell Culture in a Dose- Dependent Manner. The bars, from left to right, are Control, 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml.

[0026] Figure 5 A. GD3-Chol NPs Reduced the Expression of IL-6 in Cell Culture in a Dose- Dependent Manner. The bars, from left to right, are Control, 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml.

[0027] Figure 5B. GD3-DSPC NPs Reduced the Expression of TNF-a in Cell Culture in a Dose-Dependent Manner. The bars, from left to right, are Control, 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml.

[0028] Figure 5C. GD3-DSPC NPs Reduced the Expression of IL-ip in Cell Culture in a Dose-Dependent Manner. The bars, from left to right, are Control, 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Definitions

[0031] As used herein, “pharmaceutically acceptable” includes those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for medical or veterinary use when in contact with the tissues of human beings and animals at the concentration, dosage or amount present in the product, without causing excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Preferably, a pharmaceutically acceptable material (e.g., polymer, excipient, surfactant, solvent, or microparticles / nanoparticles produced therefrom) is suitable or approved for human medical use.

[0032] As used herein, “nanoparticles” are preferably roughly round, sphere, or sphere-like in shape, and are generally within the size range of, e.g., between about 1-1,000 nm, between about 10-1,000 nm, or between about 50-1,000 nm, or between about 100-500 nm, as measured by laser diffraction, for example. The subject nanoparticles may also include particles that are less likely to clump in vivo.

[0033] Particle size and size distribution can be measured by a dynamic light scattering instrument, e.g., a Malvern Zetasizer. The particle size is typically reported as Z-average mean diameter. Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation. The term “nanoparticle” is not intended to convey any specific shape limitation. Such particles include, but are not limited to, those having a generally polyhedral or spherical geometry. Preferred particles are characterized by a spherical geometry typically produced by emulsion-based encapsulation processes. It is understood that the terms “microparticle” and “nanoparticle” are used interchangeably herein, unless accompanied by a specific description of size. For example, the term “microparticles” is intended to also embrace “nanoparticles” as if stated as “microparticles and / or nanoparticles” unless the context demands otherwise.

[0034] The term “particle” encompasses both nanoparticle and microparticles. As used herein “a” or “an” means one or more unless otherwise specified.

[0035] As used herein, “about” generally means up to ±10% of the particular term being modified.

[0036] The terms “sialic acid residue” and “sialic acid moiety” as well as their plural referents, and the like, are used interchangeably herein.

[0037] As used herein, the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human. The terms “patient” and “subject” may be used herein interchangeably. “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing own or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) includes to clinical intervention to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, combinations of the invention are used to delay development of a disease or to slow the progression of a disease.

[0038] Ganglioside

[0039] Gangliosides are molecules composed of glycosphingolipid with one or more sialic acids linked on the sugar chain. They form lipid rafts in the outer leaflet of the cell plasma membrane, especially in neuronal cells in the central nervous system. Gangliosides have been found to be highly important molecules in immunology as they participate in cellular proliferation, differentiation, adhesion, signal transduction, cell-to-cell interactions, tumorigenesis, and metastasis. More than 60 gangliosides are known.

[0040] Ganglioside GTlb

[0041] Ganglioside GQ1

[0042] Gangliosides can be named based on the number of sialic acid (SA) units they have in the molecule. Thus, gangliosides having one SA unit are named “GM,” such as GM1, GM2, and GM3. Here, “G” stands for “ganglioside,” and “M” stands for “mono.” Similarly, “GD”, “GT” and “GQ” would refer to gangliosides having two (“di”), three (“tri”), and four (“quadruple”), respectively. As an illustration, the structures of gangliosides GM1, GD3, GT lb, and GQ1 are shown above.

[0043] As described above, gangliosides contain sialic acid (SA) residues in their molecular structures. The SA units in the gangliosides can bind sialic acid-binding immunoglobulin-like lectins (Siglec) receptors expressed on various types of cells. For example, many cell types, including T cells, macrophages, microglial, neutrophils, mast cells, eosinophils, and basophils, all express one or multiple types of Siglecs on their surfaces, and such expressions are upregulated when inflammations occur.

[0044] Most Siglecs have an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) that can mediate inhibitory signals upon binding to SA and activate downstream inhibitory signaling through the recruitment of tyrosine phosphatases SHP-1 and SHP-2. SA can also regulate the alternative pathway of complement activation. Major serum protein complement factor H recognizes sialic acid as a “self’ marker, which helps to inhibit Clq / C3b fragment activation. Therefore, Sialic acid, when binding a Siglec receptor on immune cells or the complement factor H (CFH), serves as a self-associated molecular pattern (SMAP) to suppress over-reactive immune responses and maintain overall homeostasis.

[0045] Thus, lipid nanoparticles incorporating a ganglioside molecule on the nanoparticle surface can provide high avidity and efficiency for binding Siglecs and CFH to mitigate inflammation.

[0046] The ganglioside selected in making the lipid nanoparticles of the present invention may be a ganglioside containing one SA unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two SA units such as GDla, GDlb, GD2 and GD3, or three SA units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four SA units such as GQ1.

[0047] Other non-limiting examples of gangliosides include ganglioside-total, Cl 8:0(2- NBD), GM1, NGcGM3, C18:0 GM3, C20:0 GM1, C17:0 GM1 and C18:0 GM1; commercially available at Avanti Polar Lipids, Birmingham, AL.

[0048] For gangliosides that comprise only a single SA unit (such as GM1, GM2 and GM3), the SA may be linked with its neighboring sugar ring via a2,3, a2,6, a2,8, or a2,9 linkage.

[0049] For gangliosides that comprise multiple sialic acid units, one sialic acid unit may be linked with its neighboring sialic acid unit via a2,3, a2,6, a2,8, or a2,9 linkage.

[0050] In a preferred embodiment, the ganglioside is selected from the group containing the gangliosides having one or two SA units in their molecular structures. In another embodiment, the ganglioside is selected from the group containing GM1, GM3, and GD3. The amount of the ganglioside can vary depending on the number of components. The gangliosides can be added to the formulation in an amount of at least about 0.1% molar percentage of the entire nanoparticle composition, preferably between 5-85%, or 20-75%, for example, in a two component system. The ganglioside preferably has 1, 2, 3, 4 or more SA units. Alternatively or additionally, the molar ratio of the ganglioside to total lipids can be between 0.1-50% or more, 0.5-20%, or 1-15% in a system having 3 or more components.

[0051] Cholesterol

[0052] Cholesterol can be natural cholesterol in its native form or synthetic derivatives. Natural cholesterol has a chemical structure shown below.

[0053] Cholesterol

[0054] Cholesterol derivatives include compounds comprising the four-cyclic-ring system of cholesterol, preferably a compound characterized by the formula shown below:

[0055] Wherein R1 is selected from hydrogen or a substituted or unsubstituted alkyl, such as a Cl to C4 alkyl; R2 is selected from a substituted or unsubstituted, saturated or unsaturated alkyl, such as a Cl to C12 alkyl or alkenyl; R3 is hydrogen, methyl or methylene, and each dashed line is independently a single or double bond. Cholesterol derivatives also include vitamin D and other open ring derivatives including cholecalciferol and ergocalciferol:

[0056] And compounds having the structure shown below: Wherein R1 is selected from hydrogen or a substituted or unsubstituted alkyl, such as a Cl to C4 alkyl; R2 is selected from a substituted or unsubstituted, saturated or unsaturated alkyl, such as a Cl to C12 alkyl or alkenyl; and the dashed line can be a single or double bond.

[0057] In a preferred embodiment, the lipid nanoparticle described herein further comprises cholesterol or a cholesterol derivative in addition to the ganglioside.

[0058] The cholesterol or cholesterol derivative can be added to the formulation in an amount of at least about 0.1% weight percentage of total solids in the nanoparticle composition. The molar ratio of the cholesterol or cholesterol derivative can preferably be between 1-50%, 10- 45%, or, more preferably between 15 and 40% or 20-40%, such as 35% of the total lipid composition. In a two component system the cholesterol accounts for the remainder of the composition with the ganglioside. The molar ratio of the cholesterol or cholesterol derivative can preferably be between 15-95%, 30- 70%, or, more preferably, between 30 and 40% of the total composition, such as in compositions containing 3 or more components.

[0059] Phospholipid

[0060] Phospholipids are a class of lipids that have in their molecular structures a hydrophilic "head" containing a phosphate group and one, two or more hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (such as a glycerol molecule). The phosphate group can be further substituted with various chemical moieties. Examples of such chemical moieties include serine, ethanolamine, choline, glycerol, inositol, and polyethylene glycol (PEG).

[0061] Phospholipids include but are not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanol amine (SOPE), l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (trans DOPE), phospholipid-PEG, the derivatives and combinations thereof.

[0062] In a preferred embodiment, the phospholipid is a phosphatidyl choline, such as distearoylphosphatidylcholine (DSPC).

[0063] In a preferred embodiment, the lipid nanoparticle described herein further comprises a phospholipid or its derivative in addition to the ganglioside. The phospholipid can be added in lieu of the cholesterol or in addition to it. In a two component system the phospholipid accounts for the remainder of the composition with the ganglioside.

[0064] The phospholipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the phospholipid can preferably be between 1-50%, 2-20%, 3-15%, 5-15% or about 10% of the total composition.

[0065] PEG-lipid

[0066] "PEG-lipid" refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids include PEG- modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG- DPPC, or a PEG-DSPE lipid.

[0067] PEG-lipid is also an essential component of traditional LNP systems. The PEG unit plays a key role in reducing the particle size and minimizing particle aggregation. For systemically administered LNPs, the PEG-lipid reduces clearance mediated by the kidneys and the mononuclear phagocyte system (MPS) and thus prolongs the blood circulation time so that the LNPs have enough time to reach their targeted site. In some cases, prolonged circulation can lead to improved disease targeting mediated by the enhanced permeability and retention (EPR) effect. However, PEG-lipid may be associated with immunogenicity. For example, it has been found that PEG may have caused anaphylaxis to mRNA COVID-19 vaccines in patients. The formation of anti-PEG antibodies can lead to accelerated blood clearance. PEG can also cause complement activation-related pseudo-allergies. The immunogenicity mediated by PEG can prevent repeated dosing, thus hindering drug development. In addition, the anti-PEG antibody may reduce the therapeutic effect of the API encapsulated in the LNP.

[0068] There have been attempts to replace PEG-lipid in the LNP formulations. However, most existing PEG-replacing approaches use synthetic polymers similar to PEG and may thus also cause immunogenicity. Certain natural or biocompatible substances proposed to replace the PEG-lipid, such as hyaluronic acid and polysialic acid, must be chemically linked to lipids or LNPs. However, these conjugates might induce an immune response, and the conjugation process can produce harmful byproducts. Additionally, some of these "PEG Substitutes" may be too large and hydrophilic, which could compromise the integrity and stability of the LNPs. It is to be noted that the addition of PEG-lipid is optional in the present invention as the ganglioside-containing LNP of the present invention can be made without the use of PEG-lipids. Thus, in embodiments, the LNPs are free of or substantially free of PEG-lipids. “Substantially free” in this context is intended to mean a non-immunogenic amount and can preferably be less than 1%, such as less than 0.5% total weight. The ganglioside in the lipid nanoparticles of the current invention can act to stabilize the nanoparticles without the presence of PEG. Gangliosides are naturally occurring and widely found in animals and humans. Therefore, gangliosides are safe and less likely to be immunogenic. Additionally, nanoparticles with an SA moiety may facilitate the RES (reticuloendothelial system) escape and render the nanoparticles prolonged circulation in the bloodstream.

[0069] Cationic and Ionizable Lipid

[0070] In a particular embodiment, the lipid nanoparticle further comprises one or more cationic or ionizable lipids. A cationic lipid is a lipid having a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0071] In some embodiments, the cationic lipid can be selected from, for example Dioleoyl- 3- trimethylammonium propane (DOTAP), l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l- piperazineethanamine (KL10), Nl-[2-(didodecylamino)ethyl]-Nl,N4,N4-tridodecyl-l,4- piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]- di oxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4- (dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)- [1,3] -di oxolane (DLin-KC2-DMA), 1,2- dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12- dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)- cholest-5-en-3- yloxy]octyl})oxy)-N,N-dimethyl-3-[(9- Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8- [(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z- ,12Z)-octadeca-9,12- dien-1- yloxy]propan-l- amine (Octyl-CLinDMA (2S)).

[0072] An ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH but will be protonated to become positively charged at lower pHs. Examples of commercially available ionizable lipids include DLin- KC2-DMA, DLin-MC3- DMA, DLin-DMA, LP-01, DODMA, DODAP, ALC- 0315, SM-102, SS-OP, SS-EC, etc. The ionizable lipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the cationic or ionizable lipid can preferably be between 1-50%, 10-45%, or, more preferably between 15 and 40% of the total lipid composition.

[0073] The ionizable lipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the cationic or ionizable lipid can preferably be between 1-75%, 10-60%, or, more preferably, between 20 and 50% of the total lipid composition.

[0074] Molar Ratios

[0075] It has been discovered that selecting molar ratios of the lipid components can improve properties of the LNPs, including tissue or cell targeting, anti-inflammatory properties and the like. Formulations with the stated molar ratios have been made:

[0076] Table. Characteristics of Exemplary of Lipid Nanoparticles

[0077] Table. Various lipid nanoparticles of the present invention exhibited medium to strong binding toward Siglec-5, 7, 9, 11, and CFH

[0078] The formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM1, or GM3) and a phospholipid (e.g., a phosphatidyl choline or DSPC). The molar ratio of such LNPs are preferably between 1 : 1 to 1 : 100, more preferably about 1 :2, molar ratio of ganglioside to phospholipid. The formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM3, or GM1) and cholesterol or a derivative thereof (preferably cholesterol). The molar ratio of such LNPs are preferably between 1 : 1 to 1 : 100, more preferably about 1 :3, molar ratio of ganglioside to cholesterol or derivative. The formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3, GM3, or GM1), a phospholipid (e.g., a phosphatidyl choline or DSPC) and cholesterol or a derivative thereof. The molar ratio of such LNPs are preferably between 1 : 1 to 1 :3, more preferably about 1 :2, molar ratio of ganglioside to phospholipid; and preferably between 1 :2 to 1 :8, more preferably about 1 :3 to 1 :7, more preferably about 1 :6, molar ratio of ganglioside to cholesterol. The formulations include lipid nanoparticles that consist of a ganglioside (e.g., GD3,

[0079] GM3, or GM1) and a phospholipid (e.g., a phosphatidyl choline or DSPC), cholesterol or a derivative thereof, and an ionic lipid, such as MC3, and, optionally a PEG-lipid. The molar ratios of these multicomponent lipid systems can be characterized as a molar percentage of the total lipids in the formulation. For example, the ganglioside can be between 3% and 15% (5% to 10% is preferred) of the composition where the balance of the composition is phospholipid, ionic lipid, cholesterol and, optionally, PEG-lipid. In these formulations, the molar amount of the ionic lipid is typically greater than the other components (individually), the molar amount of cholesterol (or a derivative thereof) is less than the ionic lipid but more than the phospholipid. When a PEG-lipid is added, it is typically a minor amount of the formulation, e.g., less than 5%, preferably less than 2%, of the total lipids. Active Agent

[0080] The anti-inflammatory lipid nanoparticle described herein can optionally further comprise an active agent, such as a small molecule, a peptide, a protein, or a nucleic acid. The active agent can be encapsulated within said lipid nanoparticle. The amount of the active agent can be about 0.01 to about 50% (w / w) of the nanoparticle total solids, or about 0.05 to about 25%, about 0.1 to about 10%, about 0.2 to about 5%, about 0.5 to about 3%, about 1 to about 5%, or about 2 to about 5% (w / w) of the nanoparticle total solids. The weight ratio of lipids to nucleic acid can be about 1 to 20, preferably 3 to 15, more preferably 4 to 10.

[0081] The concentration of nucleic acids to LNPs can be characterized by a N:P ratio. For example, N refers to the number of nitrogen atoms in the ionizable or cationic lipid (typically 1 nitrogen per molecule). P refers to the number of phosphates in the nucleic acid molecule. The nucleic acid molecule can be added to the lipid nanoparticle in a ratio preferably between 1 :2 to 10: 1, such as between 1 : 1 to 10: 1, more preferably between 3: 1 to 8: 1, such as 6: 1.

[0082] In certain aspects, the active agent is advantageously an anionic drug (also referred to herein as an active pharmaceutical ingredient, or API). However, active agents that are non- therapeutic, such as diagnostics, can also be included as part of the particles according to the methods. Preferred active ingredients are oligonucleotides, nucleic acid molecules and mimics thereof, such as DNA, RNA, PNA, siRNA, microRNA, circular RNA, antisense, oligonucleotide, aptamer, and a combination thereof. The term “API” and “cargo” are used interchangeably herein.

[0083] In embodiments, the nanoparticles are free of active agents and, optionally, consist essentially of the lipid compositions described herein. In particular, the nanoparticles are free of an active agent that is independently anti-inflammatory.

[0084] Production of the Particles

[0085] The lipid nanoparticle described herein can be manufactured by a coprecipitation process. In general, the ganglioside can be dissolved along with other lipids (cholesterol or phospholipid) in a preferably water-miscible organic solvent, such as an alcohol. The organic solution containing the ganglioside and other lipids is combined slowly (e.g., dropwise) or with mixing (e.g., via a microfluidic device) with an aqueous buffer solution to incur nanoprecipitation. For example, a small amount of organic solution can be added to the aqueous phase with mixing. The particles may also be manufactured using a pre-assembled device such as a T- mixer, or an automated, microfluidic device such as NanoAssemblr Ignite of Cytivia and iNano L Series of Micro&Nano Biologies.

[0086] Exemplary solvents miscible with water include methanol, ethanol, isopropyl alcohol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0087] Such obtained nanoparticle suspension (either by manual co-precipitation or with a microfluidic system) can be further purified with dialysis, centrifugation, or tangential flow.

[0088] Cryoprotectants, such as sucrose, lactose, glucose, and mannitol, can be added to keep the LNP stable at low temperatures. Preferably, said cryoprotectant is added to the LNP suspension at 1-30% (weight / volume) based on the total volume of the LNP suspension, more preferably at 5-15%.

[0089] In another embodiment, the lipid nanoparticle described herein can be manufactured by an emulsification process. In general, the ganglioside can be dissolved along with other lipids (such as DSPC and cholesterol) in a preferably water-immiscible organic solvent, such as dichloromethane, chloroform, and ethyl acetate. The organic solution containing the ganglioside and other lipids is combined with water or an aqueous buffer followed by emulsification. The emulsification process can be accomplished by stirring (magnetic or mechanical), probe sonication, homogenization, microfluidization, high-pressure homogenization, or in-line homogenization. After emulsification, solvent is then removed and / or particles collected, for example, by evaporation, solvent exchange, centrifugation or filtration, dialysis, tangential flow filtration, followed by dehydration, e.g., concentration or lyophilization.

[0090] Preferably, after the solvent is removed, the nanoparticle suspension is further washed and concentrated to the desired concentration. The washing and concentrating step can be accomplished by centrifugation, filtration, dialysis, tangential flow filtration, or a combination thereof.

[0091] Preferably, concentrated nanoparticle suspension is stored below 0°C, such as at - 20°C or -80°C.

[0092] Preferably, a cryoprotectant is added to the nanoparticle suspension to prevent them from aggregating during storage. Examples of cryoprotectants include sugars such as lactose, glucose, sucrose, and mannitol. Particle Sizes

[0093] The size of the subject nanoparticles is from about 1 nm to about 10 pm, preferably from about 10 nm to about 2 pm, and more preferably from about 20 nm to about 1 pm, and most preferably from about 40 nm to about 500 nm. For example, the nanoparticles may have an average size between about 50 and 900 nm, such as about 50, 75, 100, 300, 500, 700, or 900 nm.

[0094] As used herein, particle size can be determined by any conventional particle size measuring techniques well known to those skilled in the art. Such techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation.

[0095] Binding interactions between nanoparticles and Siglecs and between nanoparticles and Complement Factor H

[0096] The novel lipid nanoparticles described herein are designed to selectively target Siglecs present on the membranes of immune cells, as well as Complement Factor H (CFH). The sialic acid-containing entities such as gangliosides used in the nanoparticle composition are chosen for their ability to bind specific Siglec receptors. Agonistic binding of these Siglecs and CFH is expected to have downstream effects on the immune response and reduce both complement-mediated and cellular-mediated inflammation. The presentation of sialic acids in a nanoparticle format is propitious, as it provides avidity and clustering for trans interactions. Furthermore, there is potential for downstream integration of the gangliosides into the cellular lipid membrane, which could effectuate cis signaling.

[0097] To this end, quantification of the binding affinities between the nanoparticles and various Siglecs and CFH is integral to elucidating the mechanistic basis of the technology and screening candidate nanoparticles. Bio-layer interferometry (BLI) and surface plasmon resonance (SPR) are two widely used label-free assays used to assess binding events. Both techniques are used to evaluate binding of these lipid nanoparticles to relevant Siglecs and CFH in a comparative manner. In these binding assays, the ligand is either CFH or one of the following recombinant human Siglecs: Siglec-5, -7, -9, or -11. The analytes are the nanoparticle test articles of the present invention.

[0098] BLI is an optical analytical technique for real-time measurements. This technology works by detecting shifts in the interference pattern of white light that are proportional to the changes in the quantity of molecules bound to a biosensor tip. In a BLI assay, the selected ligand is immobilized to a biosensor probe tip. The probe is first immersed in a solution containing the analyte to obtain the association rate, then dipped in buffer solution to measure the dissociation rate. When there is binding of the analyte to the ligand, there is a corresponding increase in optical thickness at the probe tip. The thickness of the biological layer is thereby determined with the resultant wavelength shift. The BLI assay provides data such as sensorgrams depicting visual binding results, as well as dissociation rates (Kdiss) which are a concentration-independent metric which can be used for comparative analysis even when particle concentrations are unknown. The Sartorius Octet® BLI platform is often used to perform BLI binding assays.

[0099] Surface plasmon resonance (SPR) is also a useful method for evaluating molecular binding interactions. The ligand is first immobilized onto the surface of a sensor chip. When the analyte is flowed over the chip surface, binding events are registered as changes in the refractive index at the surface of the gold chip, measured as resonance units (RU). Since the RU signal is proportional to the mass and number of bound analytes, fitting algorithms can be applied to find the equilibrium dissociation constant KD as well as kinetic rate constants. The BIACORE™ system is popular for executing SPR assays.

[0100] Pharmaceutical Composition

[0101] One aspect of the present invention provides pharmaceutical compositions comprised of the subject lipid nanoparticles and, optionally, a pharmaceutically acceptable carrier or excipient. It will also be appreciated that certain of the subject particles of the present invention can exist in free form for treatment or, where appropriate, as a pharmaceutically acceptable derivative thereof.

[0102] A pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington ’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0103] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; com oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;

[0104] Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, adjuvants including but not limited to Alum, MF59, AS04, and CPG can also be present in the composition, according to the judgment of the formulator.

[0105] The pharmaceutical compositions comprising the lipid nanoparticles described herein can optionally further comprise one or more additional therapeutic agents. Alternatively, the subject particles of the current invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for conjoint administration or inclusion in a pharmaceutical composition with a compound of this invention may be an approved anti-inflammatory agent, an immunotherapeutic agent, or a chemotherapeutic agent, or it may be any one of a number of agents undergoing approval in the Food and Drug Administration.

[0106] In certain embodiments, the subject is a human patient. In certain embodiments, the subject is a non-human mammal, such as a non-human primate, a livestock animal (horse, mule, cattle, bull, cow, sheep, goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.), or a pet (cat, dog).

[0107] In one embodiment, the method includes administering the subject composition or pharmaceutical composition comprising the subject lipid nanoparticles by any suitable means or routes, such as orally, nasally, intravitreally, intravenously, intraperitoneally, intrathecally, intracranially, intramuscularly, ocularly, transdermally, or subcutaneously. In a particular embodiment, the particles are administered intravitreally. In still another embodiment, the particles are administered locally such as in an eye drop.

[0108] Modes of ocular delivery include a local administration, preferably selected from an intraocular administration, a subretinal administration, and an administration to the cornea, more preferably an intravitreal administration, even more preferably in the vicinity of the retina of the respective eye of said patient. Exemplary Uses

[0109] The lipid nanoparticles and compositions thereof have numerous applications including in therapeutic methods.

[0110] Preferably, the nanoparticles or the composition comprising the nanoparticles can be used in a method of treating a disease or condition in a subject in need thereof, or a method of reducing the duration or severity of the disease or condition in the subject in need thereof, wherein the disease or condition is treatable with the particles (and optionally with a specific (API), comprising administering a composition or a pharmaceutical composition comprising the particles to the subject, thereby treating the disease or condition. Where the particles comprise (for example, encapsulate) an API, the particles can be used in a method of administering or delivering the API to a subject in need thereof and / or for a method of treating a subject suffering from a disease or condition that can be treated with the API. For example, when the API is an anti-cancer agent, the particles can be administered to a subject from a cancerous condition.

[0111] The lipid nanoparticles described herein can be used to treat an inflammatory condition. Examples of such diseases and conditions include, but are not limited to, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, celiac disease, hyper-IgM immunodeficiency, arteriosclerosis, atherosclerosis, coronary artery disease, sepsis, myocarditis, encephalitis, transplant rejection, hepatitis, thyroiditis (e.g. Hashimoto's thyroiditis, Graves disease), osteoporosis, polymyositis, dermatomyositis, Type I diabetes, Type II diabetes, gout, dermatitis, alopecia areata, systemic lupus erythematosus, Sjogren's syndrome, lichen sclerosis, scleroderma, ulcerative colitis, diabetic retinopathy, pelvic inflammatory disease, periodontal disease, arthritis, juvenile chronic arthritis (e.g. chronic iridocyclitis), psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic inflammatory disease, chronic inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis, liver fibrosis, chronic inflammatory lung disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, peritonitis, cardiovascular disease, reperfusion injury, ischemia injury, stroke, burns, and other acute and chronic inflammatory diseases of the Central Nervous System (CNS; e.g. multiple sclerosis), gastrointestinal system, the skin and associated structures, the immune system, the hepato-biliary system, or any site in the body where pathology can occur with an inflammatory component. Inflammatory diseases also include diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis); diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumonoultramicroscopicsilicovolcanoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoa and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasculitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, autoimmune myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet’s syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Reiter’s syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.

[0112] Diseases that can be treated also include allergic disorders or conditions, including allergic disease, allergy, eczema, asthma, allergic rhinitis or skin hypersensitivity. The disease to be treated can also be a viral infection, including, for example, a hepatitis virus infection, a West Nile virus infection, a flavivirus, an influenza infection, a rhinovirus infection, a papillomavirus infection, a paramyxovirus infection, or a parainfluenza virus infection. Preferably, the viral infection infects the central nervous system of said subject. Preferably, the viral infection causes viral encephalitis or viral meningitis. In yet other aspects, the disease to be treated is a bacterial infection. Exemplary bacterial infections are staphylococcus infections, streptococcus infections, mycobacterial infections, bacillus infections, Salmonella infections, Vibrio infections, spirochete infections, and Neisseria infections. Preferred are bacteria that infect the central nervous system of the subject. Most preferred are bacteria that cause encephalitis or meningitis.

[0113] Age-Related Macular Degeneration (AMD)

[0114] AMD is one type of inflammatory disease that affects a patient’s central vision. AMD can result in severe loss of central vision. It happens when aging causes damage to the macula, the part of the eye that controls sharp, straight-ahead vision. AMD is a common condition and a leading cause of vision loss for older adults. Although AMD doesn’t cause complete blindness losing the central vision can dramatically affect a patient’s life quality.

[0115] There are dry AMD and wet AMD. Most people with AMD have dry AMD. Dry AMD develops when the macula gets thinner with aging. Dry AMD happens in 3 stages: early, intermediate, and late. It usually progresses slowly over several years. There’s no treatment for late dry AMD.

[0116] Wet AMD is less common than dry AMD. It is a type of late AMD that usually causes faster vision loss. Any stage of dry AMD can turn into wet AMD. Wet AMD happens when abnormal blood vessels grow in the back of the eye and damage the macula.

[0117] EXEMPLIFICATION GENERAL METHODS

[0118] Materials and Abbreviations Formulation of Anti-Inflammatory Lipid-Based Nanoparticles

[0119] The anti-inflammatory lipid-based nanoparticles are prepared using one of two general processes. A simple self-assembly technique is employed. Lipid moieties solubilized in an organic solvent (e.g., ethanol) are added in a steady, drop-wise manner to a magnetically stirred aqueous solution, such as PBS or distilled water.

[0120] In a preferred embodiment, the lipid particles of the present invention are manufactured using a pre-assembled device such as a T-mixer or an automated microfluidic device such as NanoAssemblr™ (Ignite, Blaze, etc.) of Cytiva, Automated Nanoparticle System of Particle Works, and the iNano L series of Micro&Nano Biologies. Specifically, lipids (such as ganglioside and cholesterol) dissolved in a water-miscible solvent (such as ethanol) are rapidly mixed with an aqueous solution, precipitating the hydrophobic lipids. Equipment used to facilitate this mixing includes, but is not limited to, a T-mixer conjoined with syringe pumps (in-line macromixing) and automated microfluidic systems.

[0121] The organic solvent is removed from the nanoparticle suspension by solvent evaporation and dialysis against aqueous solutions. Following the dialysis, the nanoparticles can be further purified and concentrated to the desired concentration. The solvent removal and nanoparticle purification can be accomplished by dialysis, centrifugation, tangential flow filtration (TFF), and a combination thereof.

[0122] The anti-inflammatory lipid nanoparticle composition described herein may optionally further comprise a cationic or ionizable lipid and a nucleic acid cargo.

[0123] Example 1.1: Preparation of lipid nanoparticles comprising GD3 and DSPC by precipitation

[0124] A DSPC stock solution at a concentration of 20 mg / mL was prepared by dissolving an appropriate amount of solid DSPC in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. 105 pL of the DSPC stock solution and 200 pL of the GD3 solution were combined for a 1 :2 GD3:DSPC molar ratio and further diluted to 5 mL in ethanol. A 1-mM solution of citrate buffer was prepared by diluting 0.5 M citrate buffer in PBS, and 20 mL of this aqueous solution was magnetically stirred at 400 rpm, 40° C in a 50 mL glass beaker. A 10 mL syringe with a 21 G needle was positioned above the stirring aqueous solution using a clamp. The stopper was removed from the syringe, and the DSPC / GD3 solution in ethanol was loaded into the syringe for a steady, drop-wise addition. After 1 hour of stirring, this was diluted by adding 40 mL PBS. The nanoparticle suspension was transferred to a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 20K molecular weight cut-off (MWCO) and dialyzed against PBS for 6 hours, with the buffer solution replaced with fresh PBS after 3 hours. Nanoparticles were concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 2.5% w / v sucrose solution. The nanoparticles were characterized by a Malvern Panalytical Zetasizer Lab and found to have a Z-average size of 84.14 nm with a poly dispersity of 0.21.

[0125] Example 1.2: Preparation of lipid nanoparticles comprising GD3 andDSPC by precipitation (PPT)

[0126] Stock solutions of DSPC at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed to ensure complete solubilization. The lipid mix was made by adding 473.1 pL of the GD3 solution and 118.5 pL of the DSPC solution to 458.0 pL ethanol. This corresponds to a molar GD3 to DSPC ratio of 1 :2 and a total starting lipid quantity of 4.736 mg.

[0127] 7 mL UltraPure distilled water, warmed to 37°C, was added to a 10-mL glass beaker and magnetically stirred with a stir bar at 350 rpm on a stir plate. A 1-mL syringe with the stopper removed was fastened to a 25-G needle and positioned above the stirring solution of water using a clamp. The lipid mix was added to the syringe using a pl 000 pipette, which enabled a steady, dropwise addition into the stirring aqueous phase. When the dropwise addition of the lipid mix was complete, 0.5 mL of pure ethanol was added to rinse the syringe, and the stirring was continued for an additional 15 minutes.

[0128] The resulting nanoparticle suspension was transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 24 hours (changing the dialysis buffer halfway), concentrated in a 30K MWCO Amicon Ultra-15 centrifugal filter unit, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 198.3 nm and a PDI of 0.17.

[0129] Example 1.3: Preparation of lipid nanoparticles comprising GD3 and DSPC with a microfluidic device

[0130] Stock solutions of DSPC at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed to ensure complete solubilization. The lipid mix was made by adding 473.1 pL of the GD3 solution and 118.5 pL of the DSPC solution to 458.0 pL ethanol. This corresponds to a molar GD3 to DSPC ratio of 1 :2 and a total starting lipid quantity of 4.736 mg.

[0131] The lipid mix was loaded into a 1-mL Luer-lock syringe, and warm UltraPure distilled water into a 5-mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 15 mL / min; FRR = 4 (water): 1 (lipid). Total volume = 5.0 mL (1 mL lipid + 4 mL water); start waste = 0.2 mL. The nanoparticles were collected from the iNanoL+ mixer, transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 24 hours (changing the dialysis buffer halfway), concentrated in a 30K MWCO Amicon Ultra-15 centrifugal filter unit, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 27.1 nm and a PDI of 0.41.

[0132] Example 1.4: Preparation of lipid nanoparticles comprising GD3 and DSPC with a microfluidic device

[0133] Stock solutions of DSPC at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed at 40°C to ensure complete solubilization. The lipid mix was made by adding 189.2 pL of the GD3 solution and 55.3 pL of the DSPC solution to 458.0 pL. This corresponds to a molar GD3 to DSPC ratio of 30:70 and a total starting lipid quantity of 2.052 mg.

[0134] The lipid mix was loaded into a 1-mL Luer-lock syringe, and PBS buffer into a 3-mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 15 mL / min; FRR = 4 (PBS): 1 (lipid). Total volume = 1.25 mL (0.25 mL lipid + 1.0 mL PBS); start waste = 0.2 mL. The nanoparticles were collected from the iNanoL+ mixer, transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS at room temperature for 6 hours, concentrated in a 100K MWCO Amicon Ultra- 15 centrifugal filter unit at 2000 RCM for 5 minutes to a volume of 550 mL, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 37.8 nm and a PDI of 0.26.

[0135] Example 2.1: Preparation of lipid nanoparticles comprising GD3 and Choi by PPT

[0136] A cholesterol stock solution at a concentration of 20 mg / mL was prepared by dissolving appropriate amount of solid cholesterol in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. 38.5 pL of the Choi stock solution and 100 pL of the GD3 solution were combined for a 1 :3 GD3 :Chol molar ratio and further diluted to a volume of 5 mL in ethanol.

[0137] In a 50 mL glass beaker, 20 mL of PBS was magnetically stirred at 300 rpm. A 10 mL syringe with a 21 G needle was positioned above the stirring aqueous solution using a clamp. The stopper was removed from the syringe, and the Chol / GD3 solution in ethanol was loaded into the syringe for a steady, drop-wise addition. After 30 minutes of stirring, this was diluted by adding 40 mL PBS. The nanoparticle suspension was transferred into a pre-hydrated Slide-A- Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against PBS for 6 h, replacing with fresh buffer solution midway through. Nanoparticles were concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 51.42 nm with a poly dispersity of 0.202.

[0138] Example 2.2: Preparation of lipid nanoparticles comprising GD3 and Choi by PPT

[0139] Stock solutions of cholesterol (Choi) at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed to ensure complete solubilization. The lipid mix was made by adding 473.0 pL of the GD3 solution and 87.0 pL of the Choi solution to 490.0 pL ethanol. This corresponds to a molar GD3 to Choi ratio of 1 :3 and a total starting lipid quantity of 4.105 mg.

[0140] 7 mL UltraPure distilled water, warmed to 37°C, was added to a 10-mL glass beaker and magnetically stirred with a stir bar at 350 rpm on a stir plate. A 1-mL syringe with the stopper removed was fastened to a 25-G needle and positioned above the stirring solution of water using a clamp. The lipid mix was added to the syringe using a pl 000 pipette, which enabled a steady, dropwise addition into the stirring aqueous phase. When the dropwise addition of the lipid mix was complete, 0.5 mL of pure ethanol was added to rinse the syringe, and the stirring was continued for an additional 15 minutes.

[0141] The resulting nanoparticle suspension was transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 24 hours (changing the dialysis buffer halfway), concentrated in a 30K MWCO Amicon Ultra-15 centrifugal filter unit, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 84.6 nm and a PDI of 0.14.

[0142] Example 2.3: Preparation of lipid nanoparticles comprising GD3 and Choi with a microfluidic device

[0143] Stock solutions of cholesterol (Choi) at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed to ensure complete solubilization. The lipid mix was made by adding 473.0 pL of the GD3 solution and 87.0 pL of the Choi solution to 490.0 pL ethanol. This corresponds to a molar GD3 to Choi ratio of 1 :3 and a total starting lipid quantity of 4.105 mg.

[0144] The lipid mix was loaded into a 1-mL Luer-lock syringe, and warm UltraPure distilled water into a 5-mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 15 mL / min; FRR = 4 (water): 1 (lipid). Total volume = 5.0 mL (1 mL lipid + 4 mL water); start waste = 0.2 mL. The nanoparticles were collected from the iNanoL+ mixer, transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 24 hours (changing the dialysis buffer halfway), concentrated in a 30K MWCO Amicon Ultra-15 centrifugal filter unit, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 27.7 nm and a PDI of 0.12.

[0145] Example 2.4: Preparation of lipid nanoparticles comprising GD3 and Choi with a microfluidic device

[0146] Stock solutions of cholesterol (Choi) at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GD3 at 5 mg / mL was prepared by dissolving a proper amount of GD3 in methanol. The lipid solutions were vortexed and warmed at 40°C to ensure complete solubilization. The lipid mix was made by mixing 189.2 pL of the GD3 solution with 27.2 pL of the Choi solution. This corresponds to a molar GD3 to Choi ratio of 30:70 and a total starting lipid quantity of 3.92 mg.

[0147] The lipid mix was loaded into a 1-mL Luer-lock syringe, and PBS buffer into a 3-mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 15 mL / min; FRR = 4 (PBS): 1 (lipid). Total volume = 1.05 mL (0.21 mL lipid + 0.84 mL PBS); start waste = 0.2 mL. The nanoparticles were collected from the iNanoL+ mixer, transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS at room temperature for 6 hours, concentrated in a 100K MWCO Amicon Ultra- 15 centrifugal filter unit at 2000 RCM for 5 minutes to a volume of 550 pL, followed by filtration through a 0.22 mm syringe filter. The nanoparticle sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 26.6 nm and a PDI of 0.22.

[0148] Example 3: Preparation of lipid nanoparticles comprising GM 3 and Choi

[0149] A cholesterol stock solution at a concentration of 20 mg / mL was prepared by dissolving appropriate amount of solid cholesterol in ethanol. A GM3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GM3 in ethanol. 45.7 pL of the Choi stock solution and 100 pL of the GM3 solution were combined for a 1 :3 GM3 :Chol molar ratio and further diluted to a volume of 5 mL in ethanol.

[0150] In a 50 mL glass beaker, 20 mL of PBS was magnetically stirred at 300 rpm. A 10 mL syringe with a 21 G needle was positioned above the stirring aqueous solution using a clamp. The stopper was removed from the syringe, and the Chol / GM3 solution in ethanol was loaded into the syringe for a steady, drop-wise addition. After 30 minutes of stirring, this was diluted by adding 40 mL PBS. The nanoparticle suspension was transferred into a pre-hydrated Slide-A- Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against PBS for 6 h, replacing with fresh buffer solution midway through. Nanoparticles were concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 99.36 nm with a poly dispersity of 0.239. Example 3.1: Preparation of lipid nanoparticles comprising GM1 and Choi with a microfluidic device

[0151] Stock solutions of cholesterol (Choi) at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipid in ethanol. A stock solution of ganglioside GM1 at 5 mg / mL was prepared by dissolving a proper amount of GM1 in methanol. The lipid solutions were vortexed and warmed to ensure complete solubilization. The lipid mix was made by adding 469.2 pL of the GM1 solution and 87.0 pL of the Choi solution to 494.0 pL ethanol. This corresponds to a molar GM1 to Choi ratio of 1 :3 and a total starting lipid quantity of 4.086 mg.

[0152] The lipid mix was loaded into a 1-mL Luer-lock syringe, and 4 mL of PBS into a 5- mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 15 mL / min; FRR = 4 (water): 1 (lipid). Total volume = 5.0 mL (1 mL lipid + 4 mL PBS); start waste = 0.2 mL. The nanoparticles were collected from the iNanoL+ mixer, transferred into a 15-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 4 hours (changing the dialysis buffer halfway), concentrated in a 100K MWCO Amicon Ultra-15 centrifugal filter unit. The nanoparticle sample was collected and stored at 4° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 29.8 nm and a PDI of 0.15.

[0153] Example 4: Preparation of lipid nanoparticles comprising GD3, DSPC, and Choi

[0154] Stock solutions of DSPC and cholesterol at concentrations of 10 mg / mL and 20 mg / mL, respectively, were prepared by dissolving appropriate amounts of the solid lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving 5 mg of solid GD3 in 0.5 mL methanol. For a 1 :2:6 GD3:DSPC:Chol molar ratio, 40 pL of the GD3 stock solution, 42 pL of the DSPC stock solution, and 30.85 pL of the Choi stock solution were combined and diluted to a total volume of 513 pL with ethanol.

[0155] In a 10 mL glass beaker, 2 mL of a 1-mM citrate buffer solution in PBS (measured at a pH of 5) was magnetically stirred at 300 rpm, 40°C. The GD3 / DSPC / Chol solution was added dropwise to the aqueous solution using a p200 pipette. After stirring for 20 minutes, the nanoparticle suspension was transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS overnight. Nanoparticles were concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized by the Zetasizer Lab by Malvern Panalytical. The Z-average size was 75.34 nm with a poly dispersity of 0.09.

[0156] Example 5: Preparation of lipid nanoparticles comprising 5% GD3, Choi, DSPC, and an ionizable lipid

[0157] 5.1 - in a molar ratio ofMC3:Chol:DSPC:GD3=46.5:38.5:10: 5

[0158] 20 mg / mL stock solutions of MC3, Choi, and DSPC were prepared by dissolving an appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving a proper amount of solid GD3 in methanol. The lipid packet was made by combining 198.1 pL MC3 solution, 98.8 pL Choi solution, 52.4 pL DSPC solution, and 100 pL GD3 solution; the volume was brought up to 2.5 mL with ethanol, and the lipid mixture was vortexed. Polyadenylic acid (Poly A) was solubilized at 5 mg / mL in distilled water. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet, 142.9 pL of the dissolved Poly A was diluted to 7.5 mL in 10 mM citrate buffer.

[0159] The lipid and cargo packets were loaded into 10 mL Luer-lock syringes which were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted to 30 mL with PBS, then transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against PBS for 6 hours. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z-average size was 149.3 nm with a poly dispersity of 0.10.

[0160] 5.2 - in a molar ratio ofMC3:Chol:DSPC:GD3=49:37:9:5

[0161] 20 mg / mL stock solutions of MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made with 1.2x excess by combining 35.6 pL MC3 solution, 16.2 pL Choi solution, 8.05 pL DSPC solution, and 17.04 pL GD3 solution; the volume was brought up to

[0162] 1.2 mL with ethanol, and the lipid mixture was vortexed. Anti-GAPDH siRNA was dissolved in distilled water to a 0.7 mg / mL stock concentration. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in 1.2x excess), 125 pL of the siRNA solution was diluted to 3.6 mL in 10 mM citrate buffer.

[0163] The lipid and cargo packets were loaded into 10 mL Luer-lock syringes which were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 20 mL / min; FRR = 3: 1; cargo flow rate = 15 mL / min, lipid flow rate = 5 mL / min. 4 mL of suspended nanoparticles were collected from the T-mixer in a 15 mL conical tube. The LNP suspension was diluted by adding 10 mL PBS, then transferred into a pre-hydrated Slide- A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS for 5 h, replacing with fresh buffer solution after 2 h. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 2.5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z-average size was 199.5 nm with a poly dispersity of 0.178.

[0164] Example 6: Preparation of lipid nanoparticles comprising MC 3, Choi, DSPC, and 10%GD3 6.1 in a molar ratio ofMC3:Chol:DSPC:GD3=46.5:36: 7.5:10 20 mg / mL stock solutions of MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 99.1 pL MC3 solution, 46.2 pL Choi solution, 19.7 pL DSPC solution, and 100 pL GD3 solution; the volume was brought up to 2.5 mL with ethanol and the lipid mixture was vortexed.

[0165] The lipid packet was loaded into a 10 mL Luer-lok syringe, and a second syringe was filled with 7.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in- house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; aqueous flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted to 30 mL with distilled water, then transferred into a pre- hydrated Slide- A-Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against distilled water for 6 hours. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20°C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 173.7 nm with a poly dispersity of 0.068.

[0166] 6.2 - in a molar ratio ofMC3:Chol:DSPC:GD3=46:36:8:10

[0167] 20 mg / mL stock solutions of MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 321 pL MC3 solution, 142.5 pL Choi solution, 62.5 pL DSPC solution, 317 pL GD3 solution, and 2.5 mL ethanol. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water.

[0168] The lipid packet was loaded into a 10 mL Luer-lock syringe, and a second syringe was filled with 9 mL citrate buffer. These were then fastened to a T-junction mixer fabricated inhouse and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; aqueous flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted by adding 40 mL PBS, then transferred into a pre- hydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS water for 6 hours, replacing with fresh buffer after 3 h. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 2.5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 154.4 nm with a poly dispersity of 0.089.

[0169] 6.3 - in a molar ratio ofMC3:Chol:DSPC:GD3=47.5:35: 7.5:10

[0170] 20 mg / mL stock solutions of MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 64.2 pL MC3 solution, 28.5 pL Choi solution, 12.5 pL DSPC solution, 63.4 pL GD3 solution, and 1032 pL ethanol.

[0171] The lipid packet was loaded into a 5 mL Luer-lok syringe, and a second 5 mL syringe was filled with 3.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 20 mL / min; FRR = 3: 1; aqueous flow rate = 15 mL / min, lipid flow rate = 5 mL / min. Lipid nanoparticles were collected from the T-mixer in a 15 mL conical tube. The LNP suspension was diluted by adding 10 mL distilled water, then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 66.23 nm with a poly dispersity of 0.23.

[0172] Example 7: Preparation of a traditional LNP comprising MC 3, Choi, DSPC, and PEG-lipid in a molar ratio of 50:38.5:10:1.5

[0173] 20 mg / mL stock solutions of MC3, Choi, and DSPC and a 10 mg / mL stock solution of PEG-lipid were prepared by dissolving the appropriate amount of weighed lipids in ethanol. The lipid packet was made by combining 64.2 pL MC3 solution, 29.7 pL Choi solution, 15.8 pL DSPC solution, and 15.0 pL PEG-lipid solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed.

[0174] The lipid packet was loaded into a 5 mL Luer-lock syringe, and a second 5 mL syringe was filled with 3.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 20 mL / min; FRR = 3: 1; aqueous flow rate = 15 mL / min, lipid flow rate = 5 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted by adding 36 mL distilled water, then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 2.5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 106.1 nm with a poly dispersity of 0.30.

[0175] Example 8: Preparation of lipid nanoparticles comprising MC 3, Choi, DSPC, PEG-lipid, and 5%GD3 in a molar ratio of 45:38.5:10:1.5:5

[0176] 20 mg / mL stock solutions of MC3, Choi, and DSPC and a 10 mg / mL stock solution of PEG-lipid were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 191.8 pL MC3 solution, 98.8 pL Choi solution, 52.4 pL DSPC solution, and 100 pL GD3 solution; the volume was brought up to 2.5 mL with ethanol and the lipid mixture was vortexed.

[0177] The lipid packet was loaded into a 10 mL Luer-lock syringe, and a second syringe was filled with 7.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in- house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; aqueous flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted to 30 mL with distilled water, then transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against distilled water for 6 h. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z-average size was 73.38 nm with a poly dispersity of 0.257.

[0178] Example 9: Preparation of lipid nanoparticles comprising MC 3, Choi, DSPC, PEG-lipid, and 10% GD3

[0179] 9.1 - in a molar ratio ofMC3:Chol:DSPC:PEG-lipid:GD3=45:36: 7.5:1.5:10

[0180] 20 mg / mL stock solutions of MC3, Choi, and DSPC and a 10 mg / mL stock solution of PEG-lipid were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 95.9 pL MC3 solution, 46.2 pL Choi solution, 19.7 pL DSPC solution, and 100 pL GD3 solution; the volume was brought up to 2.5 mL with ethanol and the lipid mixture was vortexed.

[0181] The lipid packet was loaded into a 10 mL Luer-lok syringe, and a second syringe was filled with 7.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in- house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; aqueous flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted to 30 mL with distilled water, then transferred into a pre- hydrated Slide-A-Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against distilled water for 6 h. The lipid nanoparticles were then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z-average size was 66.29 nm with a poly dispersity of 0.104. 9.2 - in a molar ratio ofMC3:Chol:DSPC:PEG-lipid:GD3=47.5:34: 7:1.5:10

[0182] 20 mg / mL stock solutions of MC3, Choi, and DSPC and a 10 mg / mL stock solution of PEG lipid were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made by combining 64.2 pL MC3 solution, 27.7 pL Choi solution, 11.6 pL DSPC solution, 15.8 pL PEG-lipid, 63.4 mL GD3 solution, and 1018 pL ethanol.

[0183] The lipid packet was loaded into a 5 mL Luer-lok syringe, and a second 5 mL syringe was filled with 3.5 mL distilled water. These were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 20 mL / min; FRR = 3: 1; aqueous flow rate = 15 mL / min, lipid flow rate = 5 mL / min. Lipid nanoparticles were collected from the T-mixer in a 15 mL conical tube. The LNP suspension was diluted by adding 10 mL distilled water, then concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. The nanoparticle size was characterized with the Zetasizer Lab by Malvern Panalytical. The Z- average size was 102.2 nm with a poly dispersity of 0.35.

[0184] Table 1. Characteristics of Exemplary of Lipid Nanoparticles

[0185] BIOLOGICALLY RELEVANT EXPERIMENTS AND METHODS

[0186] Binding Affinity of Exemplary Nanoparticles to Siglec Receptors and Complement Factor H CFH) - BLI assay performed using the Octet RED96 platform

[0187] An Octet binding study was carried out to examine the binding affinity of lipid nanoparticles with Siglecs and CFH. Seven unique lipid nanoparticles (analytes) were each tested against recombinant human Siglec-5, -7, -9, and -11 Fc Chimera proteins and recombinant human Complement Factor H with His tag (ligands). The lipid nanoparticle formulations that were included in this experiment were Examples 1, 3, 5, 6, 7, and 9.

[0188] To immobilize the Siglec proteins, Anti -Human IgG Fc Capture (AHC) biosensors were used. For immobilization of CFH, anti-penta-HIS antibody (HIS IK) biosensors were used. After capturing the Siglec protein or CFH on the biosensor, the probe was dipped in a nanoparticle suspension and the association rate was recorded. The probe was next dipped in buffer solution to measure the dissociation rate. Binding sensorgrams depict the spectral shift as a change in wavelength versus time. Each nanoparticle test article was tested first at a 1 : 10 dilution and then at a 2nddilution selected based on the response [nm] shift seen at the 1 : 10 dilution. These data were used to quantify the dissociation rate (kdiss) for each molecular binding interaction examined in the study. The table below summarizes the Octet binding assay results for each nanoparticle analyte / protein ligand combination. Many of the exemplary lipid nanoparticles described herein displayed the desired strong binding to Siglecs and CFH, whereas the vehicle control LNP (Example 7) was measured to have no ligand binding. A very low kdiss indicates strong binding. Compared to published data using different nanoparticle platforms, several of the exemplary lipid nanoparticles described herein exhibited superior binding affinities, up to 5 orders of magnitude stronger. In fact, some of the nanoparticle formulations tested herein bound Siglecs and CFH with such great affinity that they reached the Octet instrument’s limit of detection, with dissociate rates below 1.0 x 10'7. There is reason to believe that binding affinity could be clinically relevant and perhaps enable dosing regimens that are easier to adhere to. For example, this could alleviate the burden of frequent intravitreal injections as a dry-AMD treatment. The relationship between kdiss rates and the half-life of the binding interaction is shown below. If, as is suspected, decreased cellular and complement- mediated inflammation is effectuated by ample and durable binding of Siglecs and CFH, then these lipid nanoparticle formulations could promote persistent antiinflammatory effects, lasting on the order of months.

[0189] Table 2. Various lipid nanoparticles of the present invention exhibited medium to strong binding toward Siglec-5, 7, 9, 11, and CFH

[0190] Table 3. Relationship between Kdiss and half-life of the complex formed via binding

[0191] Effect of nanoparticles on the cytokine profde of macrophages To examine the ability of exemplary nanoparticles to modulate an inflammatory response, macrophages were cultured with the nanoparticles and their cytokine profiles were characterized. THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng / mL lipopolysaccharide (LPS) and 20 ng / mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng / mL IL-4 for the M2 phenotype. After 48 hours, differentiation to the desired macrophage subtypes was confirmed with flow cytometry as well as morphological analysis with a microscope. Cells were washed and replaced with fresh media containing the differentiation cytokines and lipid nanoparticle treatments. The Ml cells were incubated for 24 h with 125 pg / 200 pL of nanoparticles. At this point the experiment was terminated. Supernatants were collected and relevant protein levels were measured using the Luminex platform. Specifically, levels of the cytokines IL-6, TNF-a, and IL-ip were quantified and compared to levels in untreated MO, Ml, and M2 macrophages. Nanoparticles tested in this assay included the following compositions: traditional LNP without ganglioside as vehicle control (NP7), GD3 / Chol (NP2), MC3 / Chol / DSPC / 5%GD3 (NP5), and MC3 / Chol / DSPC / 10%GD3 (NP6). As shown in Figure 1, all three nanoparticles comprising GD3, NP2, NP5 and NP6, demonstrated the ability to reduce the level of IL-6, TNF-a, and IL-ip, whereas NP7 is the vehicle control and does not comprise any ganglioside failed to show any inhibitive effect.

[0192] Hazard assessment of exemplary nanoparticles

[0193] To evaluate potential toxicity of these nanoparticles, an MTT assay for cell viability and proliferation was carried out in vitro. The MTT assay is widely used in vitro to characterize cytotoxicity and cellular metabolism. In short, MTT dye (3-(4,5-dimethylthiazol- 2-yl)-2,5- diphenyltetrazolium bromide) is added to the cell culture, and in the presence of metabolically active cells, NAD(P)H-dependent cellular oxidoreductase enzymes reduce the MTT to the water-insoluble formazan, which can be measured for at 570 nm. A relevant cell type involved in chronic inflammatory diseases is the Ml phenotype of macrophages. The MTT assay was performed with macrophages differentiated from the THP-1 monocyte cell line.

[0194] Differentiation of THP-1 monocytes to Ml macrophages was achieved with a protocol using PMA, LPS, and IFN-y. THP-1 cells at 93% viability were counted and resuspended to 2 x 105cells / mL in complete growth medium (RPML1640 base medium with 0.05 mM 2- mercaptoethanol, 10% fetal bovine serum (FBS), and IX penicillin / streptomycin) supplemented with 25 nM PMA. The cells were seeded in a 96-well tissue culture-treated plate at 4 x 104cells per well and incubated (37 °C, 5% CO2) for 24 h. After 48 h, cells were differentiated to the Ml phenotype with 20 ng / mL LPS and 20 ng / mL IFN-y. Microscopic observation supported the successful differentiation, as the cells had a characteristic Ml morphology. These activated macrophages were treated with escalating doses of exemplary lipid nanoparticles. Nanoparticles at concentrations of 5 pg, 25 pg, and 125 pg lipids per 200 pL were incubated with cells for 24 h. This experimental protocol was repeated with additional batches of nanoparticles at 125 pg / 200 pL.

[0195] A 5 mg / mL solution of the MTT reagent was prepared and filtered through a 0.22 pm syringe filter. Media was removed so that cells were in 100 pL / well. 10 pL of the MTT solution was added to each well, and the plate was incubated for 4 hours. Black formazan crystals were visible in positive control and experimental wells, not negative control conditions. 100 pL of isopropanol with 0.04 N HC1 was added per well and pipette-mixed until formazan crystals dissolved. A Varioskan™ LUX multimode microplate reader was used to measure absorbance at 570 nm and a reference wavelength of 630 nm. The MTT assay and viability results were shown in Figure 2 and Figure 3.

[0196] Testing the anti-inflammatory effect of GD3-DSPC nanoparticles

[0197] THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng / mL lipopolysaccharide (LPS) or 20 ng / mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng / mL IL-4 for the M2 phenotype. After 48 hours, differentiated Ml macrophages were washed and replaced with fresh media containing the differentiation cytokines and lipid nanoparticle treatments. The Ml macrophages were incubated for 24 h with 0. Img / mL, 0.5 mg / mL, and 1 mg / mL of nanoparticles. The supernatants were collected, and relevant inflammatory cytokines levels were measured using ELISA. Specifically, levels of the cytokines IL-6, TNF-a, and IL-ip were quantified and compared to levels in untreated Ml macrophages.

[0198] The nanoparticles tested in this essay were GD3 / DSPC prepared by PPT (NP 1.2). As shown in Figure 4, all nanoparticles tested demonstrated the ability to reduce the concentrations of IL-6.

[0199] Comparing the anti-inflammatory effect of nanoparticles prepared using PPT method and microfluidic device

[0200] THP-1 monocytes were differentiated to Ml and M2 macrophages by culturing with 25 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours, and then adding either 20 ng / mL lipopolysaccharide (LPS) or 20 ng / mL interferon-gamma (IFN- y) for the Ml phenotype or 20 ng / mL IL-4 for the M2 phenotype. After 48 hours, differentiated Ml macrophages were washed and replaced with fresh media containing the differentiation cytokines and lipid nanoparticle treatments. The Ml macrophages were incubated for 24 h with 0. Img / mL, 0.5 mg / mL, and 1 mg / mL of nanoparticles. The supernatants were collected, and relevant inflammatory cytokines levels were measured using ELISA. Specifically, levels of the cytokines IL-6, TNF-a, and IL-ip were quantified and compared to levels in untreated Ml macrophages.

[0201] The nanoparticles tested in this assay included the following two compositions: GD3 / Chol prepared by PPT (NP 2.2) and GD3 / Chol prepared by iNano L+ (NP2.3). As indicated by Figure 5A (IL-6), 5B (TNF-a), and 5C (IL-ip), both nanoparticles tested demonstrated the ability to reduce the concentrations of IL-6, TNF-a, and IL-ip, and the two preparation methods did not significantly differ in their anti-inflammatory effect.

[0202] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. All articles, publications and patents referenced herein are incorporated by reference in their entirety.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A pharmaceutically acceptable lipid nanoparticle composition comprising a ganglioside, and one or more of other lipid components selected from the group containing cholesterol, phospholipid, PEG-lipid, and cationic or ionizable lipid, said lipid nanoparticle has an average particle size of 5-1,000 nm, preferably 20-500 nm, or 30-200 nm.

2. The lipid nanoparticle of claim 1, wherein said other lipid component is cholesterol.

3. The lipid nanoparticle of claim 1, wherein said other lipid component is a phospholipid.

4. The lipid nanoparticle of claim 1, wherein said other lipid component is a mixture of cholesterol and a phospholipid.

5. The lipid nanoparticle of claim 4 further comprises a PEG-lipid.

6. The lipid nanoparticle of any one of claims 1-5 further comprises a cationic or ionizable lipid.

7. The composition of any preceding claim, wherein the molar percentage of ganglioside out of the total lipid composition is between 1-50%, 1 to 15% or 10-40%.

8. The composition of claim 6, wherein the molar percentage of cholesterol out of total lipid in the composition is 1-50%, 20-40%, or 35% of the total lipid composition.

9. The composition of claim 3, wherein the phospholipid is DSPC.

10. The composition of claim 3, wherein the molar percentage of phospholipid out of total lipid in the composition is 1-50%, 2-20%, 3-15%, 5-15% or about 10% of the total lipid composition.

11. The composition of claim 6, wherein the molar percentage of ionizable lipid out of total lipid in the composition is 1 -50%, 10-45%, or, more preferably between 15 and 40%.

12. The composition of claim 1, further comprising an anionic active ingredient.

13. The composition of claim 12, wherein the anionic active ingredient is a nucleic acid.

14. The composition of claim 13, wherein the nucleic acid is a DNA, MRA, a siRNA, a microRNA, or an aptamer, or a gene-editing agent.

15. A method for ocular administration of a nucleic acid to a subject in need thereof comprising administering to said subject the composition of any one of claims 1-14.

16. A method for the treatment of an ocular disease or disorder in a subject in need thereof comprising administering to said subject the composition of any one of claims 1-14.

17. The method of claim 16, wherein the disease age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, diabetic retinopathy, and diabetic macular edema.

18. A method of decreasing cytokine production in a subject in need thereof comprising administering to said subject the composition of any one of claims 1-14.

19. A method of treating inflammation in a subject in need thereof comprising administering to said subject the composition of any one of claims 1-14.

20. A method of treating an autoimmune disease in a subject in need thereof comprising administering to said subject the composition of any one of claims 1-14.

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