Novel lipid nanoparticle compositions and uses thereof

Abstract

The present invention provides a lipid nanoparticle (LNP) formulation, the lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)-containing lipid. The LNP preparation can effectively bind Siglecs on the surface of a cell, transfect certain cells in vitro, and target certain cells in vivo. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided by the invention can be used for delivering nucleic acid therapeutic agents into cells of a subject.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 356,557, filed on June 29, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Nucleic acids, such as DNA, mRNA, siRNA and antisense oligonucleotides (ASO), have become a new class of promising new therapeutic agents for the prevention and treatment of various diseases. However, there are still many unmet needs in converting nucleic acids into drugs. For example, the delivery of nucleic acid cargoes into target cells relies heavily on viral vectors, which leads to many safety and efficacy issues. In addition, the safe, effective and stable in vivo delivery of nucleic acid therapy remains a challenge. It is crucial to add nucleic acids to formulations that protect them from degradation and allow cellular uptake and intracellular cargo release.

[0004] Lipid nanoparticles (LNPs) are promising vehicles for delivering nucleic acid therapeutics because LNPs can effectively protect nucleic acid cargo and deliver it to cells. For example, Onpattro, an LNP-based siRNA drug, has been approved by the US FDA for the treatment of polyneuropathy caused by hereditary transthyretin amyloidosis. LNP-mRNA vaccines are currently being used clinically to combat COVID-19. These successes pave the way for the clinical development of many other nucleic acid-based therapies enabled by LNP delivery.

[0005] LNP preparation is usually composed of nucleic acid goods and four kinds of main lipid components, and the four kinds of lipid components are cation or ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol (PEG) lipid.Cation or ionizable lipid interact with anionic nucleic acid in particle formation process, and promote cell entry when administration.Phospholipid has stabilized the structure of particle, and has regulated the biodistribution of LNP to a certain extent.Cholesterol has regulated the integrity and rigidity of cell membrane, thereby further stabilizing particle and improving the efficiency of in vivo delivery.

[0006] PEG-lipids are also an important component of LNP systems. The PEG unit plays a key role in reducing particle size and minimizing particle aggregation. For systemically administered LNPs, PEG-lipids can reduce kidney and mononuclear phagocyte system (MPS)-mediated clearance, thereby prolonging blood circulation time, allowing LNPs sufficient time to reach their target sites. In some cases, prolonged circulation can improve disease targeting through the enhanced permeation and retention (EPR) effect.

[0007] Although traditional lipid nanoparticles have achieved great success in delivering siRNA and mRNA, there are still some defects. For example, traditional LNPs are less efficient in transfecting lymphocytes (such as T cells) and natural killer (NK) cells and macrophages. T cells, NK cells and macrophages are target cells for many treatments, such as CAR-T, CAR-NK and CAR-macrophage therapy. Traditional LNPs also have the problem of lack of in vivo targeting, especially targeting immune cells and tumor cells. It is also important to target immune cells and complement systems as sources of inflammation to treat inflammatory diseases. However, this may not be achieved using traditional LNPs.

[0008] Furthermore, systemically administered LNPs tend to accumulate primarily in the liver. Therefore, most LNP-based therapeutics target the liver and liver-related diseases. Therefore, “extrahepatic” delivery for the treatment of non-liver-related diseases is an unmet need in the biopharmaceutical field.

[0009] Recently, it has been reported that PEG-lipids in traditional LNP formulations may lead to the formation of anti-PEG antibodies, which may cause allergic reactions, accelerated blood clearance (ABC), and unwanted immunogenicity. Therefore, it is desirable to replace PEG-lipids in LNP formulations with non-immunogenic entities.

[0010] These disadvantages limit the application of LNPs in certain fields, such as adoptive cell therapy, in vivo cell engineering, and gene therapy.

[0011] Sialic acid, also known as N-acetylneuraminic acid, is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectins (Siglec). There are three main derivatives of sialic acid, including N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid hydroxyalkyl (Neu5Gc), and 3-deoxy-D-glycero-D-galactosyl-nonylketose (Kdn). There are also other sialic acid derivatives derived from these main derivatives. An important sialic acid derivative is ganglioside, which is found in the brain and other human tissues.

[0012] Siglec, which is mainly expressed by various immune cells, has a main intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) that can mediate inhibitory signals based on sialic acid binding and activate downstream inhibitory signals through the recruitment of tyrosine phosphatases SHP-1 and SHP-2. There are several Siglecs that have an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) that can mediate stimulatory signals and activate downstream stimulatory signals when bound to sialic acid. Sialic acid can also regulate the alternative pathway of complement activation. The major serum protein complement factor H recognizes sialic acid as a "self" marker, which helps to inhibit the activation of Clq / C3b fragments. In addition, sialic acid also binds to carbohydrate-binding lectins that are overexpressed in several types of cancer.

[0013] Abnormal interactions between sialic acid and Siglecs are associated with a variety of conditions, including infection, autoimmunity, inflammation, and cancer. Therefore, it is therapeutically beneficial to bind Siglecs on specific types of cells to chemical or biological entities that include sialic acid residues to modulate immunosuppression or immune activation to treat conditions including infection, autoimmunity, inflammation, and cancer. Binding to cell surface Siglecs also allows endocytosis, and therefore it can be used for cell targeting and intracellular delivery of therapeutic agents.

[0014] However, it is difficult to deliver such sialic acid molecular entities to target cells in vivo. A common strategy is to attach sialic acid or polysialic acid molecules to the surface of nanoparticles so that the nanoparticles can carry the sialic acid entities to target cells.

[0015] In addition, since sialic acid binds to Siglecs on certain types of immune cells, chemical entities including sialic acid moieties can be attached to particles as ligands to guide the particles to immune cells and bind to Siglecs on cells. This binding can promote the entry of particles into cells through receptor-mediated endocytosis. In this way, nanoparticles loaded with therapeutic agents and coated with sialic acid or sialic acid-containing entities on the surface can target immune cells and deliver therapeutic agents into cells.

[0016] The conventional method of attaching sialic acid to the surface of lipid nanoparticles is through chemical conjugation. For example, the sialic acid molecule can be functionalized with a reactive group that can form a covalent bond with another reactive group on the surface of the nanoparticle. However, this method has the problem of low conjugation efficiency and side reactions leading to the production of unwanted byproducts in the drug formulation.

[0017] Therefore, a new method is needed to attach sialic acid-containing ligands to the surface of nanoparticles, especially LNPs, to facilitate cell transfection and targeting via the binding of Siglecs by SA-containing ligands on LNPs. Summary of the invention

[0018] The present invention provides, in part, a lipid nanoparticle (LNP) formulation comprising at least one lipid containing sialic acid (SA), wherein the LNP formulation can effectively bind to cell surface Siglecs, transfect certain cells in vitro and target certain cells in vivo. The present invention also provides a method for using the LNP composition described herein for pharmaceutical applications. For example, the LNP provided herein can be used to deliver a nucleic acid therapeutic agent to a subject's cell.

[0019] The SA-containing lipid may be synthetic or naturally derived and may include a plurality of sialic acid residues selected from Neu5Ac, Neu5Gc and Kdn or a combination thereof. The SA-containing lipid may have a molecular weight of 200 to 500,000Da, 500 to 100,000Da and 1,000 to 50,000Da.

[0020] In some embodiments, the SA-containing lipid is formed by coupling a chemical entity comprising at least one SA unit with a fatty acid, wherein the fatty acid can be a saturated or unsaturated fatty acid, and can have 2-100 carbons, preferably 10-30 carbons, more preferably 13-21 carbons. In some embodiments, the SA-containing lipid can be linear and only include one lipid unit.

[0021] In some embodiments, the SA-containing lipid may be branched and include multiple lipid units.

[0022] In some embodiments, the SA-containing lipid is a naturally occurring lipid.

[0023] In some embodiments, the SA-containing lipid is a ganglioside, a ganglioside derivative, a mimetic, or a combination thereof.

[0024] Gangliosides are molecules composed of glycosphingolipids and one or more sialic acids attached to a sugar chain. The gangliosides may be synthetic or natural.

[0025] The ganglioside used in the LNP composition of the present invention can be a ganglioside containing one SA unit, such as GM1, GM2, GM3, desialo-GM1, GAI, desialo-GM2, GA2, or a ganglioside with two SA units, such as GDIa, GDlb, GD2 and GD3, or a ganglioside with three SA units, such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or a ganglioside with four SA units, such as GQ1.

[0026] Other non-limiting examples of gangliosides include ganglioside-total, C18:0(2-NBD)GM1, NGcGM3, C18:0GM3, C20:0GM1, C17:0GM1, and C18:0GM1; commercially available from Avanti Polar Lipids, Inc., Birmingham, Alabama.

[0027] For gangliosides comprising only a single SA unit (e.g., in the case of GM1, GM2, and GM3), the SA may be linked to its adjacent sugar ring via an α2,3, α2,6, α2,8, or α2,9 bond. For gangliosides comprising only one SA unit, an α2,3 bond is preferred in the present invention.

[0028] The present invention also provides LNP preparations and methods of use thereof including at least one SA lipid (e.g., ganglioside) described herein. In addition to nucleic acid cargo (e.g., DNA, mRNA, or siRNA), the LNP preparations may also include other types of lipids, including but not limited to cationic lipids or ionizable lipids, cholesterol, PEG-lipids, and phospholipids.

[0029] Cationic lipids are pharmaceutically acceptable generally, and can be natural or synthetic lipids. An example of the cationic lipid is an ammonium lipid, or a lipid characterized by a positively charged nitrogen moiety. For example, the cationic lipid can be substituted by a tertiary ammonium group, such as a trialkylammonium, preferably trimethylammonium. The cationic lipid can be further substituted by one or more substituted or unsubstituted long-chain alkyls or alkenyls, such as C4-C20 alkyls or alkenyls. The example of conventional lipids includes multivalent cationic lipids, DOTMA, ethyl PC, DDAB, pH sensitive lipids, diol-3-trimethylammonium propane (DOTAP), DC-cholesterol and GL67.

[0030] Ionizable lipids are a class of lipid molecules that are neutral and nonionic at physiological pH, but can be protonated and positively charged at lower pH. Ionizable lipids can also form complexes with SA-containing entities, while promoting endosome escape and reducing toxicity. Examples of commercially available ionizable lipids include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, ALC-0315, SM-102, DODMA, and DODAP.

[0031] In order to increase the stability, functionality and other performance characteristics of the composite nanoparticles, other chemical entities commonly used in lipid nanoparticle (LNP) preparations, such as structural lipids, PEGylated lipids, cholesterol, phospholipids, etc., can be added to the nanoparticle preparations of the present invention.

[0032] The invention provides a lipid nanoparticle composition comprising at least one ganglioside capable of binding to Siglecs on the cell surface for treating a disease or condition, such as cancer, autoimmune disease and inflammatory disease. The invention also includes administering the lipid nanoparticle composition to a subject.

[0033] The present invention also provides methods of delivering an active pharmaceutical ingredient (eg, a nucleic acid) to cells of a subject in need thereof. The present invention includes administering the composition to a subject.

[0034] The present invention further relates to a method for treating a disease or condition, such as cancer or an autoimmune disease or an inflammatory disease, in a subject in need thereof, the method comprising administering to the subject a composition of the present invention.

[0035] The present invention also relates to methods of preparing lipid nanoparticles capable of delivering nucleic acid cargo into certain cells in vitro and targeting certain cells in vivo via systemic administration.

[0036] In addition, the present invention also provides lipid nanoparticle compositions that do not include PEG-lipids to reduce or eliminate the possibility of forming anti-PEG, allergic reactions, accelerated blood clearance, and undesirable immunogenicity.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A is a sketch of the LNP nanoparticle of the present invention, and Figure 1B Schematic diagram of LNP nanoparticles and their interaction with target cells.

[0039] Figure 2A and 2B is a graph of GAPDH knockdown in cells with SiRNA loaded Sia-carrying lipid LNPs.

[0040] Specific embodiments of the present invention

[0041] A description of preferred embodiments of the present invention follows.

[0042] definition

[0043] As used herein, "pharmaceutically acceptable" includes those compounds, materials, compositions and / or dosage forms that are suitable for medical or veterinary use within the scope of reasonable medical judgment, and when they are in contact with the tissues of humans and animals at the concentrations, dosage forms or doses present in the product, they will not produce excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio. Preferably, pharmaceutically acceptable materials (e.g., polymers, excipients, surfactants, solvents or microparticles / nanoparticles prepared therefrom) are suitable for or approved for human medical use.

[0044] As used herein, "nanoparticles" are preferably roughly round, spherical, or spheroidal in shape and typically, for example, within a size range of about 1-1,000 nm, about 10-1,000 nm, or about 50-1,000 nm, or about 100-500 nm as measured by, for example, dynamic light scattering. The nanoparticles may also include particles that are less likely to aggregate in vivo.

[0045] Particle size and particle size distribution can be measured by dynamic light scattering instrument, for example, using Malvern nanometer particle sizer (MalvernZsizer). The particle size is usually reported in the form of z-average diameter. The alternative technology includes, for example, sedimentation field flow classification, photon correlation spectroscopy, light scattering, light diffraction and disc centrifugation. The term "nanoparticle" is not intended to express any specific shape limitation. These particles include, but are not limited to, particles with a common polyhedral or spherical geometry. The preferred particles are characterized by a spherical geometry usually produced by an emulsion-based encapsulation method. It should be understood that the terms "microparticles" and "nanoparticles" are used interchangeably herein unless a specific description of size is attached. For example, unless the context requires otherwise, the term "microparticles" is also intended to include "nanoparticles", just like "microparticles and / or nanoparticles".

[0046] Although the size of each nanoparticle is generally sufficient to trigger phagocytosis of antigen presenting cells (APCs), other mononuclear macrophage system (MPS) cells or lymphocytes, the size of each nanoparticle does not have to be uniform. Preferably, the diameter of the nanoparticle is sufficient to trigger phagocytosis of antigen presenting cells (APCs), macrophages, NK cells, T cells and / or other MPS cells.

[0047] The term "particles" includes nanoparticles and microparticles.

[0048] As used herein, "a" or "an" means one or more than one unless expressly limited otherwise.

[0049] As used herein, "about" generally means up to ±10% of the particular term being modified.

[0050] The terms "sialic acid residue" and "sialic acid moiety" and their plural forms and the like are used interchangeably herein.

[0051] The term "subject" as used herein refers to an animal, preferably a mammal, including a human or non-human animal. The terms "patient" and "subject" are used interchangeably.

[0052] "Treatment" or "therapy" of a subject refers to any type of intervention or interventional process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications, disease-related conditions or biochemical indicators. As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") includes clinical intervention to change the natural course of the disease in the individual being treated, and can be used for prevention or in the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or alleviating the disease state, alleviating or improving prognosis. In some embodiments, the combination of the present invention is used to delay the development of the disease or slow down the progression of the disease.

[0053] Gangliosides

[0054] Gangliosides are molecules composed of glycosphingolipids and one or more sialic acids attached to sugar chains. They form lipid rafts in the outer layer of the plasma membrane, especially in the neurons of the central nervous system. Gangliosides have been found to be very important molecules in immunology because they are involved in cell proliferation, differentiation, adhesion, signal transduction, cell-cell interactions, tumorigenesis and metastasis. There are more than 60 known gangliosides.

[0055]

[0056] Ganglioside GM1

[0057]

[0058] Ganglioside GD3 disodium salt

[0059] Gangliosides can be named according to the number of SA units in their molecules. Thus, gangliosides with 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" refer to gangliosides with two ("di"), three ("tri"), and four ("quadruple") SA units, respectively. For illustration purposes, the structures of gangliosides GM1 and GD3 are shown above.

[0060] The SA unit in ganglioside molecules can bind to Siglecs expressed on various types of cells, including immune cells and tumor cells. Therefore, lipid nanoparticles with ganglioside molecules bound to the nanoparticle surface can promote the binding of Siglecs to treat diseases and deliver nucleic acid cargo into cells through receptor-mediated endocytosis.

[0061] In a specific embodiment of the present invention, gangliosides having SA units and α2,8 bonds can bind to Siglec-7 expressed on natural killer (NK) cells, and thus can be incorporated into specific lipid nanoparticle formulations to facilitate delivery of nucleic acid cargo into NK cells. Non-limiting examples of such gangliosides include GM2b, GD3, GT1a, GT3, and GQ1b.

[0062] In another embodiment, lipid nanoparticles incorporated with other gangliosides can be used to target other immune cells, such as monocytes, macrophages, dendritic cells, B cells, neutrophils, T cells, etc.

[0063] In addition, since sialic acid can also bind to a variety of receptors on tumor cells, LNPs containing gangliosides can be targeted to tumor sites through the high-affinity binding of sialic acid to lectins.

[0064] Gangliosides may be added to the formulation in an amount of at least about 0.1% (molar percentage of total lipids in the composition) (including any PEG-lipids that may be added as explained in more detail below), preferably between 0.1-50%, 0.5-20% or 1-10%. The gangliosides preferably have 1, 2, 3, 4 or more sialic acid units.

[0065] Cationic and ionizable lipids

[0066] Therefore, lipids may have a positive charge or a partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino) lipids. Lipids may also be zwitterionic, i.e. neutral molecules with both positive and negative charges.

[0067] In some embodiments, the lipid can be selected from, for example, dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(docosylamino)-N1, N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(docosylamino)ethyl]-N1, N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tridecyl-15 ,18,21,24-tetraaza-octadecane (KL25), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatrienoic acid-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dioleoyl-4-(2-dimethylaminoethyl)- )-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine 1-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9-Z,12Z)-heptadeca-9,13-dien-1-yloxy]propan-1-amine (octylCLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octylCLinDMA(2S)).

[0068] Ionizable lipids are a class of lipid molecules that are neutral and nonionic at physiological pH, but are protonated and positively charged at lower pH. Ionizable lipids can also form complexes with SA-containing entities, while promoting endosomal escape and reducing toxicity. Examples of commercially available ionizable lipids include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, DODAP, ALC-0315, SM-102, SS-OP, SS-EC, etc.

[0069] Cationic lipid can be added to the formulation in an amount of at least about 10% (molar percentage of total lipid in the composition) (including any PEG-lipid that can be added as explained in more detail below), preferably at least 20%, at least 30%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48% or at least 49%. For example, the content of the cationic lipid can be 25% to 75% (molar percentage of total lipid), preferably between 30% and 60%, more preferably between 45% and 55%.

[0070] PEG lipids

[0071] Polyethylene glycol (PEG) lipids can also be used. The term "PEG lipid" refers to a lipid modified by polyethylene glycol (PEG). The 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-diacyloxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. In some embodiments, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.

[0072] The PEG-lipid can be added to the formulation in an amount of at least about 1% (molar percentage of total lipids in the composition) (including any PEG-lipid that can be added as explained in more detail below), at least about 0.1% (molar percentage of total lipids in the composition) (including any PEG-lipid that can be added as explained in more detail below), preferably between 0.1-50%, 0.5-20% or 1-10%.

[0073] It should be noted that, in the present invention, the interpolation of PEG-lipid is optional. It is useful to construct LNP without PEG-lipid, because PEG-lipid may cause the formation of anti-PEG, which may cause allergic reaction, accelerate blood clearance and unnecessary immunogenicity. Ganglioside in LNP of the present invention can play the role of stabilizing nanoparticles, and prolong the circulation time of LNP in blood circulation, so that nanoparticles find its target site. Without being bound by any theory, it is believed that ganglioside resides on the surface of LNP to prevent hydrophilic particle surface, thereby avoiding being absorbed by MPS and prolonging circulation.

[0074] The hydrophilicity of the LNP of the present invention can also reduce the formation of protein coronaviruses, such as protein coronaviruses formed by apolipoproteins (e.g., Apo-E). Apo-E coronavirus has been thought to drive nanoparticles into the liver. Therefore, the LNP composition of the present invention can also be used to deliver therapeutic agents to tissues and organs outside the liver. Such "extrahepatic" organs include, but are not limited to, the spleen, kidneys, lungs, heart, bone marrow, lymph nodes, brain, skin, ears, and eyes.

[0075] In order to improve the stability, functionality and other performance characteristics of the LNP formulation, other chemical entities commonly used in lipid nanoparticle (LNP) formulations, such as structural lipids, cholesterol, phospholipids, etc., can be added to the nanoparticle formulation of the present invention.

[0076] For example, the cholesterol content may be between 5% and 60% (molar percentage of total lipids), preferably between 10% and 55%, and more preferably between 25% and 50%.

[0077] Phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In a preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC). The content of the phospholipid may be between 1% and 50% (molar percentage of total lipids), preferably between 2% and 40%, more preferably between 5% and 15%.

[0078] Active Agent

[0079] LNP described here can also further include an activating agent. The composition can include an API. The API can be encapsulated in the LNP. The amount of the API accounts for about 0.01% to about 50% (w / w) of the nanoparticle, or accounts for 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. The ratio of lipid to nucleic acid can be about 1 to 20, preferably 3 to 15, more preferably 5 to 10.

[0080] In some aspects, the active agent is preferably a drug (also referred to herein as an active pharmaceutical ingredient or API). However, depending on the method, non-therapeutic active agents may also be part of the particles. For example, agents that can be used for diagnosis, agriculture, cosmetics, personal products, household products, industrial chemicals, dyes, fluorescent agents or colorants, etc. may be included. Preferred active ingredients are oligonucleotides, nucleic acid molecules and their mimetics, such as DNA, RNA, PNA, siRNA, microRNA, oRNA, antisense, oligonucleotides, aptamers and combinations thereof. It should be understood that API can be replaced by non-therapeutic compounds, such as diagnostic agents, agricultural agents or chemical agents. Therefore, in each case where the term API is used, it should be understood that the term "active agent", including diagnostic agents, agricultural agents or chemical agents, can also be used to replace API. The terms "API" and "goods" are used interchangeably herein.

[0081] Preparation of particles

[0082] The LNP can be prepared by coprecipitation of lipid and nucleic acid. Generally, the lipid (e.g., ganglioside) containing SA can be dissolved in an organic solvent preferably miscible with water, such as alcohol, together with other lipids. Any nucleic acid API can be dissolved in an aqueous medium or an organic solvent. Then the two solutions are mixed. Preferably, the aqueous solution and the organic solution are slowly (e.g., dropwise) mixed or uniformly mixed (e.g., by a microfluidic device) to produce nano precipitation. For example, a small amount of organic solution can be added to the aqueous phase under stirring.

[0083] The particles can also be prepared using preassembled devices (eg, T-mixers) or automated microfluidic devices, such as the NanoAssemblr from Precision Nanosystems and the automated nanoparticle system from Particle Works.

[0084] Exemplary water-miscible solvents include methanol, ethanol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0085] The solvent is then removed and / or the particles are collected, for example by evaporation, solvent exchange, centrifugation or filtration, dialysis, tangential flow filtration, and then dehydrated, for example, by concentration or lyophilization.

[0086] The aqueous solution may optionally include a surfactant, including an organic or inorganic pharmaceutical excipient; various polymers; oligomers; natural products; nonionic surfactants, cationic surfactants, zwitterionic surfactants or ionic surfactants; and mixtures thereof. The surfactant may include an acidic buffer such as citrate, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polysorbate (Tween series) surfactant, PEO-PPO-PEO (polyethylene oxide polypropylene oxide-polyethylene oxide) triblock copolymer (Pluronic series or Poloxamer series) surfactant, or tert-octylphenyl-polyethylene glycol (Triton X-100) surfactant or its salt, derivative, copolymer or mixture.

[0087] Particle size

[0088] The size of the nanoparticles is from about 1 nm to about 10 μm, preferably from about 10 nm to about 2 μm, more preferably from about 20 nm to about 1 μm, and most preferably from about 40 nm to about 500 nm. For example, the average size of the nanoparticles can be between about 50 and 900 nm, such as about 50, 75, 100, 300, 500, 700 or 900 nm.

[0089] As used herein, particle size can be determined by any conventional particle size measurement technique 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.

[0090] Pharmaceutical composition

[0091] One aspect of the present invention provides a pharmaceutical composition comprising a subject lipid nanoparticle and optionally comprising a pharmaceutically acceptable carrier or excipient. Preferably, these compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the particles of the present invention may be administered to a patient in need thereof in combination with one or more other therapeutic agents. For example, the additional therapeutic agent used for co-administration with the compounds of the present invention or included in a pharmaceutical composition having the compounds of the present invention may be an approved anti-inflammatory agent, an immunotherapeutic agent, or a chemotherapeutic agent, or the other therapeutic agent may be any of a variety of agents approved by the U.S. Food and Drug Administration. It should also be understood that some of the particles of the present invention may exist in free form, or, where appropriate, in the form of a pharmaceutically acceptable derivative thereof, for treatment.

[0092] Preferably, the pharmaceutical composition of the present invention also includes a pharmaceutically acceptable carrier, as described herein, including any and all solvents, diluents or other liquid carriers, dispersing aids or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. suitable for the desired specific dosage form. Various carriers for formulating pharmaceutical compositions and known techniques for their preparation are disclosed in Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980). Unless any conventional carrier medium is incompatible with the compounds of the present invention, such as producing any adverse biological effects or interacting with any other components of the pharmaceutical composition in other harmful ways, its use is considered to be within the scope of the present invention.

[0093] Some examples of substances that can be used 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 carboxymethylcellulose, ethylcellulose 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; corn oil and soybean oil; dihydroxy alcohols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, phosphate buffered solution, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants, adjuvants including but not limited to alum, MF59, AS04 and CPG, may also be present in the composition.

[0094] 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, livestock animal (horse, mule, cattle, bull, cow, sheep, goat, pig, camel, etc.), rodent (rabbit, hamster, mouse, rat, etc.) or pet (cat, dog).

[0095] In one embodiment, the method comprises administering a subject composition or pharmaceutical composition comprising subject microparticles or nanoparticles (e.g., carboxylated particles) by any suitable means or route, such as by oral administration, nasal administration, intravenous administration, intramuscular administration, ocular administration, transdermal administration, or subcutaneous administration. In a specific embodiment, the particles are administered nasally. In another embodiment, the particles are administered by intravenous injection. Example

[0096] Example 1. Lipid nanoparticles comprising Poly(A) and ganglioside GM3 Preparation

[0097] Ganglioside GM3 is dissolved in a solvent mixture consisting of 1 part ethanol and 3 parts methanol. Other lipids are dissolved in ethanol. MC3, cholesterol, DSPC and GM3 are mixed in a molar ratio of 50:38.5:10:1.5. Poly (A) (MW700,000-3,500,000, chain length 2,100-10,000 nucleotides) is dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer is 7.5ml, and the volume of the lipid solution is 2.5ml. The two solutions are loaded into two syringes installed on a syringe pump, and then the lipid solution at a flow rate of 8ml / min and the Poly (A) solution at a flow rate of 24ml / min are introduced into a T-type mixer to achieve an N:P ratio of 5.79. The Z average particle size of the resulting lipid nanoparticles is 93.7nm, and the zeta potential is -11.9mV.

[0098] Example 2. Preparation of lipid nanoparticles comprising Poly(A) and ganglioside GM3

[0099] Ganglioside GM3 is dissolved in a mixed solvent consisting of 1 part ethanol and 3 parts methanol. Other lipids are dissolved in ethanol. MC3, cholesterol, DSPC and GM3 are mixed in a molar ratio of 48.5:37.3:9.7:4.5. Poly (A) (MW700,000-3,500,000, chain length 2,100-10,000 nucleotides) is dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer is 7.5ml, and the volume of the lipid solution is 2.5ml. The two solutions are loaded into two syringes installed on a syringe pump, and then the lipid solution at a flow rate of 8ml / min and the Poly (A) solution at a flow rate of 24ml / min are introduced into a T-type mixer to achieve an N:P ratio of 5.79. The Z average particle size of the resulting lipid nanoparticles is 122.3nm, and the zeta potential is -14.6mV.

[0100] Example 3. Preparation of lipid nanoparticles comprising Poly(A), PEG-lipid and ganglioside GM3

[0101] Ganglioside GM3 is dissolved in a mixed solvent consisting of 1 part of ethanol and 3 parts of methanol. Other lipids are dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000 and GM3 are mixed at a molar ratio of 49.2:37.9:9.8:1.5:1.5. Poly (A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) is dissolved in 10mM citrate buffer (pH 3), and its N: P ratio is 5.79. The volume of the citrate buffer is 7.5ml, and the volume of the lipid solution is 2.5ml. The two solutions are loaded into two syringes installed on a syringe pump, and then the lipid solution at a flow rate of 8ml / min and the Poly (A) solution at a flow rate of 24ml / min are introduced into a T-type mixer to achieve an N: P ratio of 5.79. Next, LNP is dialyzed with PBS for 12 hours. The obtained lipid nanoparticles were found to have a Z-average particle size of 65.7 nm and a zeta potential of -2.54 mV.

[0102] Example 4. Preparation of lipid nanoparticles comprising Poly(A), PEG-lipid and ganglioside GD3

[0103] Ganglioside GM3 was dissolved in a mixed solvent consisting of 1 part ethanol and 3 parts methanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000 and GD3 were mixed at a molar ratio of 49.2:37.9:9.8:1.5:1.5. Poly (A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) was dissolved in 10mM citrate buffer (pH 3). Both solutions were introduced into a T-type mixer, and the resulting LNPs were dialyzed with PBS for 12 hours.

[0104] Example 5. Preparation of lipid nanoparticles comprising mRNA, PEG-lipid and ganglioside GD3

[0105] Ganglioside GM3 was dissolved in a mixed solvent consisting of 1 part ethanol and 3 parts methanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000 and GD3 were mixed in an appropriate molar ratio. GFP mRNA was dissolved in an aqueous buffer. Both solutions were introduced into a T-type mixer, and the resulting LNPs were dialyzed with PBS for 12 hours.

[0106] Example 6. Comprising siRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 3 mol% gangliosides Preparation of GD3 lipid nanoparticles

[0107] Dissolve ganglioside GD3 in methanol. Dissolve other lipids in ethanol. Mix MC3, cholesterol, DSPC, PEG2000, and GD3 in a molar ratio of 49:37.5:9:1.5:3.TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of citrate buffer was 3.0 ml and the volume of lipid solution was 1.0 ml. Both solutions were loaded into a 5 mL Luer-lok syringe mounted on a syringe pump and then introduced into a T-type mixer at a flow rate of 5 ml / min for lipid solution and 15 ml / min for RNA solution to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 130.3 nm with a zeta potential of -5.75 mV. The encapsulation efficiency determined by Ribogreen assay was 90.8%.

[0108] Example 7. Comprising siRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 5 mol% gangliosides Preparation of GD3 lipid nanoparticles

[0109] Dissolve ganglioside GD3 in methanol. Dissolve other lipids in ethanol. Mix MC3, cholesterol, DSPC, PEG2000, and GD3 in a molar ratio of 49:36.5:8:1.5:5. TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 9.0 ml and the volume of the lipid solution was 3.0 ml. Both solutions were loaded into a 10 mL Luer-lok syringe mounted on a syringe pump and then introduced into a T-type mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 101.9 nm and the zeta potential was -32.56 mV. The encapsulation efficiency determined by the Ribogreen assay was 92.8%.

[0110] Example 8. Comprising siRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 10 mol% gangliosides Preparation of GD3 lipid nanoparticles

[0111] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000 and GD3 were mixed in a molar ratio of 45:35:8.5:1.5:10. TMGAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 9.0 ml and the volume of the lipid solution was 3.0 ml. Both solutions were loaded into a 10 mL Luer-lok syringe mounted on a syringe pump and then introduced into a T-type mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles were found to have a Z-average particle size of 119.9 nm and a zeta potential of -35.68 mV.

[0112] Example 9. Lipids containing siRNA, ionizable lipid MC3, cholesterol, phospholipids and 5 mol% ganglioside GD3 Preparation of high-quality nanoparticles

[0113] Dissolve ganglioside GD3 in methanol. Dissolve all other lipids in ethanol. Mix MC3, cholesterol, DSPC, and GD3 in a molar ratio of 49:37:9:5. TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-lok syringe mounted on a syringe pump, and then introduced into a T-type mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was mixed with 2.5 volumes of PBS solution and dialyzed against PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 184.9 nm with a zeta potential of -32.55 mV. The encapsulation efficiency determined by Ribogreen assay was 95.0%.

[0114] Example 10. Lipid nanostructured products comprising ionizable lipid MC3, cholesterol, phospholipids and 5 mol% ganglioside GD3 Preparation of particles

[0115] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 49:37:9:5 to prepare a blank 10mM citrate buffer (pH3) in which no RNA was dissolved. The volume of the citrate buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were respectively loaded into a 10ml Luer-lok syringe installed on a syringe pump, and then the lipid solution at a flow rate of 8ml / min and the buffer solution at a flow rate of 24ml / min were introduced into a T-type mixer. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles were found to have a Z-average particle size of 116.8 nm.

[0116] Example 11. Comprising mRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 3 mol% gangliosides Lipid Nanoparticles of GD3

[0117] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000 and GD3 were mixed in a molar ratio of 49:37.5:9:1.5:3. GFP-mRNA was dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution at a flow rate of 8ml / min and the RNA solution at a flow rate of 24ml / min were introduced into a T-type mixer to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 81.5 nm and the zeta potential was -33.46 mV. The encapsulation efficiency determined by the Ribogreen assay was 92.5%.

[0118] Example 12. Comprising mRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 5 mol% gangliosides Preparation of GD3 lipid nanoparticles

[0119] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 48:36.5:9:1.5:5. GFP-mRNA was dissolved in 10mM citric acid buffer (pH3). The volume of the citric acid buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 8ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 24ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 106.9 nm with a zeta potential of -33.08 mV. The encapsulation efficiency determined by the Ribogreen assay was 92.6%.

[0120] Example 13. Comprising mRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 10 mol% gangliosides Preparation of GD3 lipid nanoparticles

[0121] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 45:35:8.5:1.5:10. GFP-mRNA was dissolved in 10mM citric acid buffer (pH3). The volume of the citric acid buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 8ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 24ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 101.5 nm with a zeta potential of -35.22 mV. The encapsulation efficiency determined by the Ribogreen assay was 85.3%.

[0122] Example 14. Lipids containing mRNA, ionizable lipid MC3, cholesterol, phospholipids and 10 mol% ganglioside GD3 Preparation of high-quality nanoparticles

[0123] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. The MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 46:35:9:10. GFP-mRNA was dissolved in 10mM citric acid buffer (pH3). The volume of the citric acid buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 8ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 24ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 40ml of PBS solution, stirred for one hour, and then dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles had a Z-average particle size of 164.0 nm and a zeta potential of -50.03 mV. The encapsulation efficiency determined by the Ribogreen assay was 86.0%.

[0124] Example 15. A method comprising siRNA, ionizable lipid SM-102, cholesterol, phospholipids, PEG-lipids and 5 mol% neurotransmitters Preparation of GD3 Lipid Nanoparticles

[0125] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. SM-102, cholesterol, DSPC, DMG-PEG2000 and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Anti-GAPDH siRNA was dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0ml, and the volume of the lipid solution was 1.0ml. The two solutions were loaded into a 5mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 5ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 15ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 74.9 nm with a zeta potential of -25.12 mV. The encapsulation efficiency determined by Ribogreen assay was 71.1%.

[0126] Example 16. A method comprising siRNA, ionizable lipid ALC-0315, cholesterol, phospholipids, PEG-lipids and 5 mol% narcotics Lipid Nanoparticles of Ganglioside GD3

[0127] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. ALC-0315, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Anti-GAPDH siRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 5 ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 15 ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The Z-average particle size of the resulting lipid nanoparticles was found to be 73.6 nm with a zeta potential of -30.88 mV. The encapsulation efficiency determined by Ribogreen assay was 35.1%.

[0128] Example 17. Preparation of a siRNA, ionizable lipid MC3, PEG-lipid and 5% Ganglioside GD3 Lipid Nanoparticles

[0129] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000 and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 1.5 ml and the volume of the lipid solution was 0.5 ml. The cargo solution was loaded into a 3 mL Luer-lok syringe and the lipid solution was loaded into a 1 mL Luer-lok syringe, which was mounted on a Nanoassembly Ingite (PrecisionNanosystems, Vancouver, Canada). The flow rate of the lipid solution was set to 8 ml / min and the flow rate of the RNA solution was set to 24 ml / min to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles were found to have a Z-average particle size of 90.3 nm and a zeta potential of -25.57 mV

[0130] Example 18. Fluorescently labeled GFP-mRNA, ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and Preparation of 3 mol% ganglioside GD3 lipid nanoparticles

[0131] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000 and GD3 were mixed in a molar ratio of 49:37.5:9:1.5:3. Cy5-labeled GFP-mRNA (EZ Cap Cy5 EGFP mRNA from APExBIO) was dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5ml, and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 8ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 24ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 20ml of PBS and then dialyzed in PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles were found to have a Z-average particle size of 101.0 nm, a PDI of 0.164, and a zeta potential of -23.7 mV. The encapsulation efficiency determined by the Ribogreen assay was 84.9%.

[0132] Example 19. Fluorescently labeled GFP-mRNA, ionizable lipid MC3, cholesterol, phospholipids and 10 mol% narcotics Preparation of GD3 Lipid Nanoparticles

[0133] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC and GD3 were mixed in a molar ratio of 46:35:9:10. Cy5-labeled GFP-mRNA (EZ Cap Cy5EGFP mRNA from APExBIO) was dissolved in 10mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5ml and the volume of the lipid solution was 2.5ml. The two solutions were loaded into a 10ml Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 8ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 24ml / min to achieve an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 20ml of PBS and then dialyzed with PBS for 6 hours using Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage at -20°C with 5% w / v sucrose. The resulting lipid nanoparticles were found to have a Z-average particle size of 226.3 nm, a PDI of 0.251, and a zeta potential of -43.58 mV. The encapsulation efficiency determined by the Ribogreen assay was 95.8%.

[0134] Example 20. Comprising siRNA, ionizable SS-OP, cholesterol, phospholipids and 10 mol% ganglioside GD3 Preparation of lipid nanoparticles

[0135] Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. SS-OP (purchased from NOF Corporation), cholesterol, DSPC and GD3 were mixed in a molar ratio of 46:35:9:10. TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml and the volume of the lipid solution was 1.0 ml. Both solutions were loaded into a 5 mL Luer-lok syringe mounted on a syringe pump, and then the lipid solution was introduced into a T-type mixer at a flow rate of 5 ml / min, and the RNA solution was introduced into the T-type mixer at a flow rate of 15 ml / min to achieve a 6:1 N:P ratio. After mixing, the nanoparticle suspension was mixed with 30 ml of PBS and used Ultra centrifugal filter devices were concentrated at 2000 ref and prepared for storage with 5% w / v sucrose at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 253.6 nm, a PDI of 0.157, and a zeta potential of -43.59 mV.

[0136] Example 21: In vitro knockdown of primary human NK cells and macrophages by siRNA using LNPs containing 3% GD3 GAPDH in mitochondria and CD8+ T cells

[0137] Lipid nanoparticles including Silencer™ GAPDH siRNA (ThermoFisher), ionizable lipid MC3, cholesterol, phospholipids, PEG-lipids and 3 mol% ganglioside GD3 were prepared as described in Example 6. The prepared lipid nanoparticles were incubated with primary human NK cells, CD8+T cells and macrophages at 30 nM and 90 nM concentrations of siRNA. Samples were collected at 12 hours and 48 hours, respectively, and qPCR analysis of GAPDH gene expression was performed, and the data were normalized to ActinB gene expression. The specific experimental conditions are described as follows:

[0138] A. Cell Culture

[0139] a. Primary human CD8+ T cells PBC-1041, NK cells PB56C-2, M1 macrophages PBM1C-MON-3

[0140] b. OptiMEM I Reduced Serum Medium with Pen-Strep

[0141] i.10U / mL recombinant human IL-2 acts on CD8 T cells

[0142] ii. 100U / mL recombinant human IL-2 and 25U / ml recombinant human IL-15

[0143] For NK cells

[0144] iii.25ng / mL recombinant human GM-CSF acts on macrophages

[0145] c. In 1x 10 5 / mL x 100uL / well (1x 10 4 Cells were seeded at 4% CO / well and incubated overnight at 37°C / 5% CO

[0146] B. Nanoparticles and Control Conditions

[0147] a. siRNA alone, untreated, and liposomes (Lipofectamine)

[0148] +siRNA

[0149] b. Standard LNP, GD3 3% LNP, GD3 5% LNP, GD3 5% LNP without PEG, PSA DOTAP liposomes

[0150] C. Treatment and Dosage

[0151] a. Treatment time is 12 hours and 48 hours

[0152] b. 30nM and 90nM siRNA

[0153] c. Prepare NP suspension at 2 times the desired final concentration in the above culture medium and add 100uL to each well

[0154] dN = 3 replicates, 2 technical replicates

[0155] D.qPCR method

[0156] a. Measure the expression levels of GAPDH and ActinB, using β-actin as a reference gene to evaluate GAPDH knockout

[0157] b. 2-step qPCR method

[0158] E. Analysis

[0159] a. Normalized data for siRNA alone treatment

[0160] b.2^(-ΔΔC T ) for relative fold change

[0161] F. Results

[0162] a. Table summarizing the average relative fold change in GAPDH gene expression in each cell type

[0163]

[0164] While the invention has been described with reference to particular features and 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 as encompassed by the appended claims.

Claims

1. A lipid nanoparticle composition comprising, optionally, a nucleic acid and at least one ganglioside, wherein the average particle size of the lipid nanoparticle is 10-800 nm, preferably 30-200 nm.

2. The composition according to claim 1, wherein The lipid nanoparticles include cationic or ionizable lipids, and optionally phospholipids, cholesterol and PEG- Lipids.

3. The composition according to claim 1, wherein The molar percentage of the ganglioside in the total lipid composition is 0.1-50%, 0.5-20% or 1-10%.

4. The composition according to claim 2, wherein The gangliosides comprise 1, 2, 3 or 4 sialic acid units.

5. The composition according to claim 2, wherein The gangliosides comprise more than 4 sialic acid units.

6. The composition according to claim 1, wherein The lipid nanoparticles enhance cell transfection and targeting.

7. The composition according to claim 6, wherein The cells include immune cells and / or tumor cells.

8. The composition according to claim 7, wherein The cells are immune cells.

9. The composition according to claim 8, wherein The cells are T cells, NK cells and macrophages.

10. The composition according to claim 9, wherein The cells are NK cells.

11. The composition according to claim 9, wherein The cells are macrophages.

12. The composition according to claim 1, wherein The nucleic acid is DNA, MRA, siRNA, microRNA, or an aptamer or a gene editing agent such as CRISPR-cas9.

13. A method of administering a nucleic acid to a subject in need thereof, comprising administering the composition of claim 1 to the subject.

14. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the composition of claim 1.

15. The method according to claim 14, wherein: The disease is cancer.

16. The method according to claim 14, wherein: The nucleic acid is an anticancer agent or an immunotherapeutic agent.

17. The method according to claim 14, wherein: The disease is an autoimmune disease.

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