Drug delivery system containing oxidized cholesterol

Abstract

Disclosed herein are lipid nanoparticles and compositions thereof. Exemplary nanoparticle compositions include an ionizable lipid, a phospholipid, a PEG-lipid, and cholesterol modified with a hydroxyl group near the D-sterol ring. The disclosed nanoparticle compositions can more preferentially target liver Kupffer cells and endothelial cells than hepatocytes that should be beneficial in the treatment of liver diseases in which functional Kupffer cells and endothelial cells are involved in disease pathogenesis.

Description

This application is a divisional application of Chinese patent application No. 202080010141.5, filed on January 15, 2020, with the invention name “Drug delivery system containing oxidized cholesterol”. Related application information

[0001] This application claims priority to U.S. Serial No. 62 / 793,671, filed on January 17, 2019. The aforementioned application is hereby incorporated by reference herein in its entirety. Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Grant Nos. 1RO1DE026941-01A1, T32GM008433, and T32EB021962 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical field of the invention

[0003] The present invention generally relates to drug delivery systems and methods of using the same. Background of the Invention

[0004] Using RNA to produce therapeutic proteins or modify gene expression is a promising method for treating diseases. Several advances have been made in local and systemic mRNA delivery (KA Hajj, KA Whitehead, Nature Reviews Materials, 2, 17056 (2017)). However, it is still challenging to deliver low-dose mRNA to many cell types in organs and tumor microenvironments. It is difficult to identify nanoparticles that can effectively deliver therapeutic RNA to cells in certain microenvironments, because nanoparticles tend to preferentially target specific cell types, for example, nanoparticles preferentially target hepatocytes in the liver. In addition, to a large extent, nanoparticles of specific cell types in the targeting microenvironment are difficult to design, because there is no high-throughput method to study nanoparticle mRNA delivery in vivo.

[0005] This general problem in nanomedicine has slowed the development of all RNA therapeutics because scientists are forced to perform high-throughput nanoparticle assays in vitro, even though cell cultures can be poor predictors of delivery within complex in vivo microenvironments (Paunovska, K. et al., Nano Letters, 18: 2148-2157 (2018)). In vivo mRNA delivery is affected by pulsatile blood flow, heterogeneous vasculature, and clearance by the kidneys, spleen, liver, lymph, and immune systems. Barcoding technology has quantitative lipid nanoparticle (LNP) biodistribution, which is necessary but not sufficient for cytoplasmic nucleic acid delivery. More specifically, less than 3% of the drugs that reach the target cells can escape into the cytoplasm, and the genes that change whether the nanoparticles escape into the endosome may vary with each cell type.

[0006] Therefore, more effective nanoparticles are needed to target specific cells in organs or tumor microenvironments.

[0007] It is an object of the present invention to provide compositions and methods for delivering therapeutic and prophylactic agents to various organ microenvironments.

[0008] Another object of the present invention is to provide nanoparticle compositions for treating and preventing diseases, including liver diseases. Summary of the invention

[0009] Compositions for delivering nucleic acids to specific cells or tissue microenvironments are provided. It has been found that oxidized cholesterol can change the tropism of nanoparticles. One embodiment provides nanoparticles containing oxidized cholesterol. Another embodiment provides lipid nanoparticles containing one or more oxidized cholesterols. Exemplary nanoparticle compositions include ionizable lipids, phospholipids, one or more PEG lipids and oxidized cholesterol. By using specific oxidized cholesterol, the tropism of the nanoparticles can be adjusted to the desired cell type or tissue microenvironment.

[0010] Another embodiment provides nanoparticles, such as lipid nanoparticles containing cholesterol modified with hydroxyl groups near the D-sterol ring, which can preferentially deliver cargo to non-hepatocytes in the liver. In one embodiment, the nanoparticle composition includes 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12), C 14 PEG 2000 or C 18 PEG 2000and oxidized cholesterol modified with a hydroxyl group near the D-sterol ring. The oxidized cholesterol can be 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 7β,25-dihydroxycholesterol, 27-hydroxycholesterol, 25-hydroxycholesterol, or a combination thereof.

[0011] Another embodiment provides a nanoparticle composition having about 30 mol % to about 80 mol % ionizable lipid, about 5 mol % to about 55 mol % cholesterol, about 10 mol % to about 35 mol % phospholipid, and about 0 mol % to about 20 mol % PEG-lipid.

[0012] Another embodiment provides a disclosed nanoparticle loaded with cargo. The cargo can be a therapeutic agent or a prophylactic agent. In one embodiment, the therapeutic agent is a nucleic acid, including but not limited to ssRNA, dsRNA, sgRNA, mRNA, shRNA, siRNA, miRNA, ssDNA, dsDNA, antisense DNA or a combination thereof. The nanoparticle can have a diameter of about 20nm to about 215nm. In one embodiment, the nanoparticle can have a diameter of about 50nm to about 100nm.

[0013] Another embodiment provides a pharmaceutical composition comprising one or more disclosed lipid nanoparticles and a pharmaceutically acceptable excipient.

[0014] Another embodiment provides a method of delivering a therapeutic or prophylactic agent to an individual in need thereof by administering to the individual one or more disclosed lipid nanoparticle compositions loaded with a therapeutic nucleic acid, such as a nucleic acid encoding a therapeutic protein, or an inhibitory nucleic acid or an enzyme nucleic acid. The method may also include administering a second therapeutic agent. In one embodiment, the nanoparticles preferentially deliver cargo to cells in the liver.

[0015] Another embodiment provides a method for preventing or treating liver disease in an individual in need thereof, by administering one or more disclosed lipid nanoparticles to the individual in an amount effective to prevent or treat the liver disease. The lipid nanoparticles can be administered to the individual at a dose of 0.05 mg / kg. The liver disease can be liver cancer, cirrhosis, nonalcoholic fatty liver disease, and alcohol-related liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A FIG1 is a diagram showing different sterol variants of cholesterol. FIG1B is the chemical structure of the ionizable marker cKK-E12. FIG1C is the chemical structure of cholesterol. FIG1D is the chemical structure of C 14 PEG 2000Figure 1E is the chemical structure of C 18 PEG 2000 FIG. 1F is the chemical structure of DOPE. FIG. 1G is a bar graph showing the formulation ratios of 10 different lipid nanoparticle designs. The graph shows the molar percentages of DOPE, lipid-PEG, cholesterol, and cKK-E12 in each formulation. The X-axis represents the formulation ratio, and the Y-axis represents the molar percentages of each component of the lipid nanoparticle. Figure 1H is a diagram showing the workflow for barcoding the disclosed nanoparticles. Fig. 1I is a diagram showing the workflow for testing the disclosed nanoparticles.

[0017] Figure 2A 2B is a bar graph showing the percentage of tdTomato-positive cells in 8 cell types from the liver and spleen. The X-axis represents the cell type, and the Y-axis represents the percentage of tdTomato+ cells. FIG. 2C is a scatter plot showing the hydrodynamic diameter of all administered lipid nanoparticles. FIG. 2D is a scatter plot showing the normalized delivery of all administered lipid nanoparticles for eight cell types from the liver and spleen. The X-axis represents the cell type, and the Y-axis represents the normalized delivery percentage. FIG. 2E shows the average normalized delivery of all administered lipid nanoparticles in the liver and spleen. Figure 2F is an unbiased Euclidean clustering of eight different cell types in two different tissues based on lipid nanoparticle delivery cluster cell type according to tissue. Asterisks indicate naked barcodes.

[0018] Figure 3AThe average normalized delivery of lipid nanoparticles classified by cholesterol type is shown. Fig. 3B shows the chemical structure of cholesterol with modification on the cholesterol tail. Fig. 3C is a dot plot showing the average normalized delivery of lipid nanoparticles (tails) with modified cholesterol on the cholesterol tail and lipid nanoparticles (rings / cholesterol) with cholesterol modified to the B ring in all 8 liver and spleen cell types. Fig. 3D is a bar graph showing the average normalized delivery of all eight liver and spleen cell types for lipid nanoparticles with different cholesterol (including 27-OH, 25-OH, 20α-OH, 7β-OH, 7β-OH, 7β-25-dihydroxy, 7-keto, 6-keto-5α-OH and cholesterol). The X-axis represents the type of cholesterol in the lipid nanoparticles, and the Y-axis represents the average normalized delivery of the whole animal. Fig. 3E is a diagram showing that cholesterol variants are enriched in the first 10% and the last 10% of lipid nanoparticles. FIG3F is a bar graph showing the fold enrichment in the top 10% of lipid nanoparticles, calculated by subtracting the enrichment in the bottom 10% of lipid nanoparticles from the enrichment in the top 10% of lipid nanoparticles. FIG3G is the chemical structure of the ionizable lipid cKK-E12. FIG3H is a bar graph showing the fold enrichment in the top 10% of lipid nanoparticles, calculated by subtracting the enrichment in the bottom 10% of lipid nanoparticles from the enrichment in the top 10% of lipid nanoparticles. 18 PEG 2000 Figure 3I is the chemical structure of DOPE. Figure 3J is the chemical structure of 20α-OH. Figure 3K is a graph showing the lipid nanoparticle mole percentage, diameter, polydispersity (PDI), and pKa of lipid nanoparticles for each component of the two formulations. Figure 3L-3O It is a bar graph showing the percentage of tdTomato-positive endothelial cells (Figure 3L), hepatocytes (Figure 3M), Kupffer cells (Figure 3N) and immune cells (Figure 3O) in mice administered 0.25 mg / kg or 50 mg / kg of LNP1 or InP2. The X-axis represents the lipid nanoparticle dose and the Y-axis represents the percentage of tdTomato+ cells.

[0019] Figure 4A is a representative autocorrelation curve for included and excluded lipid nanoparticles. The X-axis represents time (μs) and the Y-axis represents autocorrelation intensity. Figure 4B is a line graph showing the diameter distribution of included and excluded lipid nanoparticles. The X-axis represents diameter (nm) and the Y-axis represents mass percentage. Figure 4C shows the percentage of tdTomato positive cells for cell types (endothelial cells, E; immune cells, I; macrophages, M; others, O; and hematopoietic stem cells, HSC) isolated from the heart, lungs, kidneys, pancreas and bone marrow. The X-axis represents cell type and the Y-axis represents tdTomato+ cell percentage.

[0020] Figure 5Ais a graph showing the enrichment calculation used to obtain the enrichment (fold) difference values, the average normalized delivery for the whole animal (%), and the enrichment in the top 10% and bottom 10% of lipid nanoparticles. Figures 5B-5D The enrichment fold differences with respect to cholesterol mol % are shown. Figure 5E-5G The enrichment fold differences with respect to ionizable lipid mole % are shown. Figures 5H-5J The enrichment fold differences with respect to phospholipid mol % are shown. Figure 5K-5M It is a dot plot showing the normalized delivery of lipid nanoparticles relative to the average whole animal for all lipid nanoparticles (Fig. 5K), lipid nanoparticles containing only tail-modified cholesterol (Fig. 5L) and lipid nanoparticles containing epoxidized cholesterol (Fig. 5M). Fig. 5N and 5P are bar graphs showing the percentage of lipid nanoparticles formed based on the cholesterol variants (Fig. 5N) and cholesterol mole % (Fig. 5P) used. Fig. 5O and 5Q are dot plots showing the lipid nanoparticle diameters based on the cholesterol variants (Fig. 5O) and cholesterol mole % (Fig. 5Q) used.

[0021] Figure 6A-6B is a diagram showing the synthesis of CKK-E12. Figure 6C The structure of CKK-E12 was confirmed 1 H-NMR spectrum.

[0022] Figures 7A-7D It is a bar graph showing the body weight of mice injected with 1 mg / kg lipid nanoparticle assembly (Fig. 7A), LNP1 and LNP2 (Fig. 7B) containing cre mRNA at a dosage of 0.25 mg / kg, LNP1 and LNP2 containing cre mRNA at a dosage of 0.05 mg / kg and 0.01 mg / kg, and LNP1 (Fig. 7D) containing cre mRNA at a dosage of 0.05 mg / kg compared to the control. The X-axis represents the days after the intravenous injection of lipid nanoparticles, and the Y-axis represents the normalized mouse body weight. PBS injection is used as a control. Detailed description of the invention I. Definitions

[0023] It should be understood that the present disclosure is not limited to the compositions and methods described herein and the experimental conditions described, as they may vary. It should also be understood that the terminology used herein is only for the purpose of describing certain embodiments and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Any compositions, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The entire contents of all publications mentioned are incorporated herein by reference.

[0025] The use of the terms "a," "an," "the" and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0026] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0027] The use of the term "about" is intended to describe values ​​that are within a range of about + / -10% above or below the stated value; in other embodiments, the range of values ​​may be within a range of about + / -5% above or below the stated value; in other embodiments, the range of values ​​may be within a range of about + / -2% above or below the stated value; in other embodiments, the range of values ​​may be within a range of about + / -1% above or below the stated value. The foregoing ranges are intended to be clear from the context and no further limitation is implied. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise stated. The language in this specification should not be interpreted as indicating that any unclaimed element is essential to the practice of the invention.

[0028] As used herein, "RNA" refers to natural or non-naturally occurring ribonucleic acid. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem loop, a poly A sequence and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA in a mammalian cell, may produce the encoded polypeptide. RNA may be selected from a non-limiting group consisting of small interfering RNA (siRNA), micro RNA (miRNA), Dicer enzyme-substrate RNA (dsRNA), short hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9mRNA and mixtures thereof.

[0029] The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a string of at least three amino acids linked together by peptide bonds. A peptide may refer to a single peptide or a collection of peptides. A peptide may contain natural amino acids, non-natural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain), and / or amino acid analogs. In addition, one or more amino acids in a peptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, and the like. Modifications may include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0030] As used herein, "sterol" refers to an organic molecule that is a subgroup of steroids. Sterols are also known as sterols. Sterols are naturally present in plants (called phytosterols) and animals (called zoosterols).

[0031] As used herein, the terms "treat," "treating," "treatment," and "therapeutic use" refer to the elimination, alleviation, or amelioration of one or more symptoms of a disease or condition. As used herein, a "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate clinically relevant elimination, alleviation, or amelioration of such symptoms. An effect is clinically relevant if the magnitude of the effect is sufficient to affect the health or prognosis of the recipient individual. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease (e.g., delay or minimize the spread of cancer). A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0032] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a condition or disease before any symptoms of the condition or disease are detected. A "prophylactically effective" amount is an amount of a prophylactic agent sufficient to mediate such protection. A prophylactically effective amount can also refer to an amount of a prophylactic agent that provides a prophylactic benefit in the prevention of a disease.

[0033] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans, rodents such as mice and rats, and other laboratory animals.

[0034] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents.

[0035] As used herein, "Kupffer cells" refers to resident liver macrophages. Kupffer cells function as scavenger cells responsible for removing particulate matter from the portal circulation. Kupffer cells are also involved in a variety of liver pathologies, including but not limited to viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholestasis, activation or rejection of the liver during liver transplantation, and liver fibrosis.

[0036] As used herein, "liver microenvironment" refers to the normal cells, molecules and blood vessels surrounding and nourishing the liver. Exemplary cells of the liver include, but are not limited to, sinusoidal endothelial cells (SEC), Kupffer cells (KC), hepatic stellate cells (HSC); and hepatocytes, as well as various immune cells.

[0037] "Inhibitory nucleic acid" refers to a nucleic acid that inhibits the expression of a specific gene or mRNA. Inhibitory nucleic acids include, but are not limited to, dsDNA, ssDNA, antisense DNA, siRNA, shRNA, miRNA, tasiRNA, and rasiRNA. II. Lipid Nanoparticles

[0038] The effective targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids is a continuous challenge in the field of medicine. The relative instability and low cell permeability of nucleic acids make the specific delivery of nucleic acids difficult. It has been found that lipid nanoparticles with modified cholesterol variants can more effectively deliver nucleic acids to specific tissues in vivo. In one embodiment, lipid nanoparticles can be prepared by mixing nucleic acids with ionizable lipids, PEG-lipids, phospholipids and modified cholesterol. Exemplary lipid nanoparticle preparations may include ionizable lipid-like substances cKK-E12, PEG-lipids, 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE) and one or more cholesterol variants. In one embodiment, compared to hepatocytes, the disclosed lipid nanoparticles preferentially target other cells (i.e., Kupffer cells, endothelial cells, immune cells) of the liver microenvironment.

[0039] In one embodiment, lipid nanoparticle preparation comprises about 30mol% to about 80mol% ionizable lipid, about 10mol% to about 35mol% phospholipid, about 5mol% to about 55mol% modified cholesterol and about 0mol% to about 20mol% PEG-lipid. In another embodiment, lipid nanoparticle preparation comprises about 50mol% ionizable lipid, about 15mol% phospholipid, about 30mol% modified cholesterol and about 5mol% PEG-lipid. In another embodiment, lipid nanoparticle preparation comprises about 30mol% ionizable lipid, about 20mol% phospholipid, about 45mol% modified cholesterol and about 5mol% PEG-lipid.

[0040] The size of lipid nanoparticle can affect the distribution of nanoparticle.In one embodiment, lipid nanoparticle can have the diameter of about 20nm to about 215nm.The diameter that lipid nanoparticle can have is about 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 210nm or 215nm.In a preferred embodiment, the diameter of nanoparticle is 50nm to 100nm. A. Sterols

[0041] The disclosed lipid nanoparticles may include one or more sterols. In one embodiment, the sterol is cholesterol. Cholesterol may be modified, for example, oxidized. Unmodified cholesterol may be acted upon by an enzyme to form a variant with oxidized side chains or rings ( Figure 1A ). Cholesterol can be oxidized on the β-ring structure or on the hydrocarbon tail structure. Exemplary cholesterols considered for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, or combinations thereof. In one embodiment, cholesterol with oxidized side chains can enhance cargo delivery relative to other cholesterol variants. In a preferred embodiment, the modified cholesterol is 25-hydroxycholesterol (25-OH) or 20α-hydroxycholesterol (20α-OH).

[0042] Without being bound by any one theory, it is believed that lipid nanoparticles containing oxidized cholesterol can preferentially deliver cargo to other cells in the liver microenvironment, rather than being delivered only to hepatocytes as is characteristic of existing lipid nanoparticles. Cholesterol is transported in lipoproteins by using forward and reverse transport and transport to endothelial cells, hepatocytes, and macrophages, and may vary in vivo with cholesterol structure. In addition, lipid nanoparticles and lipoproteins have similar sizes and compositions, and therefore, lipid nanoparticles with cholesterol structures incorporated therein can be transported to cells similar to lipoproteins. B. Ionizable lipids

[0043] In one embodiment, the disclosed lipid nanoparticles include ionizable lipids. Ionizable lipids have a positive charge or a partial positive charge at physiological pH. Exemplary ionizable lipids include, but are not limited to, 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl carbonate acyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilmoleoyl-3-dimethylaminopropane (DLm-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethyl aminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilmoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaeonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylpropaneammonium (DODAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), dioctadecylamido gly cyocarboxysperrnine (DOGS), spermine cholesteryl carbamate (GL-67), biguanidinyl-spermidine-cholesterol (BGTC), 3β-(N—(N^N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), N-tert-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecyl ammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMR1E), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dimethoxybenzylamine (DMA), Oleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxy)propyl)-N-2-(spenninecarboxamido)ethyl)-N,N-dimethyltrifluoroacetic acid ammonium salt (DOSPA), 2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propylammonium bromide (GAP-DLRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE"), or a combination thereof. In a preferred embodiment, the ionizable lipid is cKK-E12. C. PEG-lipid

[0044] The lipid component of the disclosed nanoparticle composition can include one or more PEG or PEG-modified lipids. This substance can also be referred to as PEGylated lipids. The inclusion of PEGylated lipids can be used to enhance the in vitro colloidal stability and in vivo circulation time of lipid nanoparticles. PEG lipids are lipids modified with polyethylene glycol. In some embodiments, PEGization is reversible because the PEG moiety is gradually released in the blood circulation. Exemplary PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol and mixtures thereof. For example, PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.

[0045] In a preferred embodiment, the PEG lipid is C 14 PEG 2000 or C 18 PEG 2000 . D. Phospholipids

[0046] The lipid component of the nanoparticle composition can include one or more phospholipids, such as one or more (poly) unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. Typically, the phospholipids can include a phospholipid portion and one or more fatty acid portions.

[0047] Phospholipid moiety can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. Fatty acid moiety can be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural species are also considered, which include natural species with modifications including branching, oxidation, cyclization and alkynes and replacement. For example, phospholipid can be functionalized with one or more alkynes (for example, wherein one or more double bonds are replaced by triple bond alkenyl groups) or cross-linked with the alkynes. Under suitable reaction conditions, alkynyl groups can experience copper-catalyzed cycloaddition when exposed to azide. Such reactions can be used to functionalize the lipid bilayer of the nanoparticle composition to facilitate membrane permeation or cellular recognition, or to conjugate the nanoparticle composition to useful components such as targeting moieties or imaging moieties (eg, dyes).

[0048] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-heneicosanoyl-sn-glycero-3-phosphocholine (DUPPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DUPPC). C), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (CI6LysoPC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl -sn-glyceryl-3-phosphocholine, 1,2-diphytanoyl-sn-glyceryl-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine, rac-(1-glycerol) sodium salt ( Phospholipids include phospholipids such as DOPG, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE). In a preferred embodiment, the phospholipid is DOPE. E. Goods

[0049] In one embodiment, the disclosed lipid nanoparticle compositions include a therapeutic or prophylactic agent for an individual. The therapeutic or prophylactic agent can be contained in the lipid nanoparticle as a cargo. Exemplary cargoes traditionally delivered via nanoparticles include, but are not limited to, chemotherapeutic agents, cytotoxic agents, radioactive ions, small molecules, proteins, polynucleotides, and nucleic acids.

[0050] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cristaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, The invention relates to apoptotic agents, for example, leucovorin, liposome doxorubicin, liposome daunomycin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, seratraplatin, streptozotocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine or a combination thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ (2) and a combination thereof.

[0051] Representative nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), RNAi inducers, RNAi agents, miRNA, antisense RNA, asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer enzyme-substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA) ribozymes, catalytic DNA, RNA inducing triple helix formation, aptamers, vectors, Cas9 / sgRNA and plasmid DNA. F. Pharmaceutical Compositions

[0052] The nanoparticle composition can be formulated in whole or in part as a pharmaceutical composition. A pharmaceutical composition can include one or more nanoparticle compositions. For example, a pharmaceutical composition can include one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein.

[0053] Pharmaceutical compositions comprising the disclosed lipid nanoparticles are provided. Pharmaceutical compositions containing nanoparticles can be administered parenterally (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous), transdermal (passive or using iontophoresis or electroporation) or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration, or using bioerodible inserts, and can be formulated into dosage forms suitable for each route of administration.

[0054] In some in vivo methods, the compositions disclosed herein are administered to an individual in a therapeutically effective amount. The terms "effective amount" or "therapeutically effective amount" as used herein refer to a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated or otherwise provide the desired pharmacological and / or physiological effects. The exact dose will vary according to a variety of factors, such as individual dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being performed.

[0055] For the disclosed nanoparticles, as further studies are conducted, information about appropriate dosage levels for treating various conditions in various patients will emerge, and the skilled person, taking into account the treatment background, age, and general health of the recipient, will be able to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment. For the disclosed nanoparticles, a dosage level of 0.01 to 5 mg / kg body weight per day is generally administered to mammals. More specifically, the preferred dosage of the disclosed nanoparticles is 0.05 to 0.25 mg / kg. Typically, for intravenous injection or infusion, the dosage can be lower.

[0056] In certain embodiments, the lipid nanoparticle composition is topically administered, for example, by direct injection into the site to be treated. Typically, injection causes an increased local concentration of the lipid nanoparticle composition that is greater than the concentration that can be achieved by systemic administration. The lipid nanoparticle composition can be combined with a matrix as described above to help produce an increased local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide outside the site to be treated. 1. Preparations for parenteral administration

[0057] In some embodiments, the compositions disclosed herein, including those containing lipid nanoparticles, are administered by parenteral injection in aqueous solution. The preparation can also be in the form of a suspension or emulsion. Generally, a pharmaceutical composition comprising an effective amount of lipid nanoparticles is provided, and it optionally comprises a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Such a composition optionally includes one or more of the following substances: diluent, sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate) with various buffer contents, pH and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium pyrosulfite) and preservatives (e.g., thimerosal, benzyl alcohol) and filler materials (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation can be lyophilized and redissolved / resuspended immediately prior to use. The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by adding a bactericide to the composition, by irradiating the composition, or by heating the composition. 2. Controlled delivery polymer matrix

[0058] Lipid nanoparticles disclosed herein can also be administered in the form of controlled release formulations. After implanting a polymer device (rod, cylinder, film, disk) or injecting (microparticles), a controlled release polymer device for long-term systemic release can be prepared. Matrix can be a form of microparticles such as microspheres, wherein the medicament is dispersed in a solid polymer matrix or microcapsules, wherein the core is a material different from the polymer shell, and the peptide is dispersed or suspended in the core, which can be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres and microcapsules are used interchangeably. Alternatively, polymers can be cast into nanometer to 4 cm thin pieces or films, powders produced by grinding or other standard techniques, or even gels such as hydrogels.

[0059] Non-biodegradable or biodegradable matrix can be used for delivering lipid nanoparticles, although biodegradable matrix is ​​preferred in some embodiments. These can be natural or synthetic polymers, although due to the better characterization of degradation and release curve, synthetic polymers are preferred in some embodiments. Select polymer based on the time period of expected release. In some cases, linear release may be the most useful, although pulse release or "large-scale release" can provide more effective results in other cases. Polymer can be in the form of hydrogel (usually absorbing up to about 90 % by weight of water), and can optionally be cross-linked with multivalent ions or polymers.

[0060] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any method developed for preparing microspheres for drug delivery, for example, as described in Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6: 275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35: 755-774 (1988).

[0061] The devices can be formulated for local release to treat the area of ​​implantation or injection, which typically delivers a dose much smaller than that used for treatment of the entire body - or systemic delivery. These can be implanted or injected subcutaneously into muscle, fat or swallowed. III. How to use

[0062] Disclosed herein are methods for delivering cargo to specific cells or organs using the disclosed lipid nanoparticles. In some embodiments, the nanoparticles can deliver therapeutic or preventive agents to specific cells or organs in individuals in need. In another embodiment, the disclosed lipid nanoparticles can be used to treat or prevent diseases in individuals in need. A. Methods for delivering cargo to cells and organs

[0063] Provided herein are methods of delivering therapeutic and / or prophylactic agents to individuals in need thereof.

[0064] In some embodiments, the disclosed lipid nanoparticle compositions can target specific types or categories of cells (e.g., cells of a specific organ or its system). For example, a nanoparticle composition comprising a therapeutic and / or prophylactic agent of interest can be specifically delivered to the liver, kidney, spleen, intestine, or lung of a mammal. Specific delivery to a specific category of cells, organs, or systems or groups thereof means that a higher proportion of the nanoparticle composition comprising a therapeutic and / or prophylactic agent is delivered to a destination of interest (e.g., tissue) relative to other destinations. In some embodiments, specific delivery can result in an increase in the amount of a therapeutic and / or prophylactic agent greater than 2 times, 5 times, 10 times, 15 times, or 20 times relative to each 1g of tissue at the target destination.

[0065] Another embodiment provides the targeted or specific delivery of mRNA encoding protein binding partners such as antibodies or their functional fragments, scaffold proteins or peptides, or receptors on the cell surface. mRNA can be used in addition or alternatively to direct the synthesis and extracellular localization of lipids, carbohydrates or other biological parts. Alternatively, other therapeutic agents and / or preventive agents or elements of the nanoparticle composition can be selected based on the affinity of the therapeutic agent and / or preventive agent or element to a specific receptor, so that the nanoparticle composition can more easily interact with the target cell population including the receptor. For example, ligands can include, but are not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, Fv fragments, single-chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent forms thereof; multivalent binding agents, including monospecific or bispecific antibodies, such as disulfide-stabilized Fv fragments, scFv tandems, bispecific antibodies, trispecific antibodies or tetraspecific antibodies; and aptamers, receptors and fusion proteins.

[0066] Targeted cells may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0067] In one embodiment, the disclosed lipid nanoparticles target cells of the liver microenvironment, excluding hepatocytes. These cells can be liver endothelial cells, Kupffer cells, and immune cells.

[0068] In another embodiment, the disclosed lipid nanoparticles target cells of the immune system. Exemplary immune cells include, but are not limited to, granulocytes, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, including cytotoxic T cells, T helper cells, and regulatory T cells. b. Methods of Modulating Immune Responses

[0069] The disclosed lipid nanoparticles can be used to modulate the immune response of an individual in need of such treatment. In one embodiment, the disclosed lipid nanoparticles target immune cells in the microenvironment of a diseased organ, an organ system, or an immune cell in a tumor. The immune cell can be a macrophage, more specifically a Kupffer cell. 1. Stimulation of immune response a. Treatment strategies

[0070] Methods of inducing or enhancing an immune response in an individual are provided. Generally, the method comprises administering to the individual an effective amount of a disclosed lipid nanoparticle or a pharmaceutical composition comprising a disclosed lipid nanoparticle. The immune response can be, for example, stimulating innate immunity at the site of a disease or tumor, more specifically, stimulating Kupffer cells. Kupffer cells are the first macrophage population of the body and come into contact with bacteria, bacterial endotoxins, and microbial debris that originate from the gastrointestinal tract and are transported to the liver via the portal vein.

[0071] Alternatively, lipid nanoparticles can stimulate signal transduction through immune cell receptors and promote or enhance immune responses.

[0072] In some embodiments, lipid nanoparticles can be used to block inhibitory immune cells to the tumor microenvironment. In another embodiment, lipid nanoparticles can be used to inhibit, reduce or block tumor metastasis. In some embodiments, lipid nanoparticles can reduce or inhibit the activity of immunosuppressive macrophages, reduce the production of cytokines (such as the production of IL-12 by M1 macrophages), reduce the differentiation of macrophages, reduce the number of macrophages, reduce the ratio of macrophages within the immune cell population, or reduce the survival of macrophages.

[0073] The method can be used in vivo or in vitro to suppress, reduce or block inhibitory immune responses, thereby having a stimulating therapeutic effect. In some embodiments, lipid nanoparticles are directly applied to individuals. In some embodiments, lipid nanoparticles are contacted with cells (e.g., immune cells) in vitro, and the treated cells are applied to individuals (e.g., adoptive transfer). Lipid nanoparticles can make stronger immune responses possible. The disclosed composition can be used to stimulate or enhance immune responses involving T cells by suppressing, reducing or blocking inhibitory immune signal transduction through T cell receptors. b. Individuals to be treated i. Cancer treatment

[0074] The disclosed compositions and methods can be used to treat cancer. Typically, lipid nanoparticles are used to stimulate or enhance an individual's immune response to cancer by administering a certain amount of lipid nanoparticles to an individual. The method can alleviate one or more symptoms of cancer. In one embodiment, the disclosed lipid nanoparticles inhibit, block or reduce the number of macrophages at the tumor site, thereby reducing tumor angiogenesis and inhibiting anti-tumor immune cells.

[0075] Cancer cells acquire a set of characteristic functional capabilities through various mechanisms during their development. This ability includes escaping apoptosis, self-sufficiency of growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, unlimited replication potential and continuous angiogenesis. The term "cancer cell" means including precancerous cancer cells and malignant cancer cells. In some embodiments, cancer refers to benign tumors that have maintained local positioning. In other embodiments, cancer refers to malignant tumors that have invaded and destroyed adjacent body structures and spread to distant sites. In other embodiments, cancer is related to specific cancer antigens (e.g., pan-cancer antigens (KS1 / 4), ovarian cancer antigens (CA125), prostate-specific antigens (PSA), carcinoembryonic antigens (CEA), CD19, CD20, HER2 / neu, etc.).

[0076] The methods and compositions disclosed herein can be used to treat or prevent a variety of cancers or other abnormal proliferative diseases, including, but not limited to, the following: cancers, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer; including squamous cell carcinoma; hematopoietic tumors of the lymphoid lineage, including leukemias, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of the myeloid lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma.

[0077] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions (e.g., familial adenomatous polyposis and myelodysplastic syndrome). In specific embodiments, malignancies or abnormal changes in proliferation (e.g., metaplasia and dysplasia) or hyperproliferative conditions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus are treated or prevented by the methods and compositions. In other specific embodiments, sarcomas, melanomas, or leukemias are treated or prevented by the methods and compositions.

[0078] Specific cancers and related conditions that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, leukemias, including, but not limited to, acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic leukemia complications, chronic leukemias such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, Large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, such as but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as but not limited to osteosarcoma (bone sarcoma), osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (angiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumor, acoustic neurotheca, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pineoblastoma, primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, myxoid breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease and inflammatory breast cancer; adrenal cancer, including, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vasodilatory peptide tumor, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancer, including, but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancer, including, but not limited to, eye melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;Cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous cell carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, mycosis fungoides (polypoid) carcinoma, ulcerative carcinoma, superficial spreading carcinoma, diffuse spreading carcinoma, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, including but not limited to adenocarcinoma; bile duct cancer, including but not limited to papillary carcinoma, nodular carcinoma and diffuse carcinoma; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; testicular cancer testicular cancer, including but not limited to reproductive tumors, seminoma, anaplastic testicular cancer, classical (typical) testicular cancer, spermatocytoma, non-seminoma, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor), prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma and rhabdomyosarcoma; penile cancer; oral cancer, including but not limited to squamous cell carcinoma; basal cell carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and warts; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal carcinoma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor; bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinoma (for a review of such conditions, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). ; ii. Treatment of infection

[0079] The disclosed lipid nanoparticles can be used to treat infections and infectious diseases. Typically, lipid nanoparticles are used to stimulate or enhance an individual's immune response to infection. The disclosed lipid nanoparticles can stimulate macrophages, more specifically Kupffer cells of the liver. In one embodiment, the lipid nanoparticles inhibit, reduce or block inhibitory immune signal transduction. In another embodiment, the lipid nanoparticles induce, promote or enhance an immune response by inducing, promoting or enhancing signal transduction through immune cell receptors. The method can alleviate one or more symptoms of infection.

[0080] The infection or disease may be caused by bacteria, viruses, protozoa, helminths or other microbial pathogens that enter cells and are attacked, ie, by cytotoxic T lymphocytes.

[0081] Infection or disease can be acute or chronic. Acute infection is usually an infection of short duration. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Therefore, in some embodiments, the method includes increasing the immunostimulatory response to acute infection.

[0082] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacteria.

[0083] In some embodiments, the disclosed compositions are used to treat chronic infections, e.g., infections in which T cell exhaustion or T cell anergy has occurred, resulting in the infection persisting in the host for a prolonged period of time.

[0084] Exemplary infections to be treated are chronic infections caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes viruses, Epstein-Barr virus or human papillomavirus.

[0085] Because bacterial infections are primarily cleared by macrophages, an increase in macrophage activity is therapeutically useful in situations where more rapid or complete clearance of infectious bacterial agents is beneficial to an animal or human subject. Thus, the disclosed compositions can be administered for the treatment of local or systemic bacterial infections.

[0086] Representative infections that can be treated include, but are not limited to, infections caused by microorganisms including, but are not limited to, Actinomycetes, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobacter, Chromobacterium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter, Haemophilus, Haemophilus influenzae type B (HIB), Mycobacterium, Legionella, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma , Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochlorococcus, Proteus, Pseudomonas, Phosphospira, Rickettsia, Salmonella, Shigella, Helicobacter, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

[0087] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria, such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix (Schenckia schenckii), Blastomyces (Blastomyces dermatitidis), Paracoccidioides (Paracosporidioidomyces brasiliensis), Coccidioides (Coccidioides immitis) and Histoplasma (Histoplasma capsulatum), Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba histolytica, Bagworm, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia or Trypanosoma, etc. 2. Suppression of immune response a. Treatment strategies

[0088] Methods for reducing or suppressing an immune response in an individual are provided. Generally, the method comprises administering to the individual an effective amount of the disclosed lipid nanoparticles, or contacting cells with antigens ex vivo with these nanoparticles. The immune response can be, for example, promoting or enhancing an inhibitory immune response. In one embodiment, the disclosed compositions reduce, block or reduce the activity of M1 macrophages or M1 macrophage signaling and reduce the production of cytokines such as IL-12 to provide an immunosuppressive response.

[0089] In another embodiment, the lipid nanoparticles promote an inhibitory immune response by inducing, promoting or enhancing signal transduction through immune cell receptors.

[0090] The method can be used in vivo or in vitro as a therapeutic application for suppressing immune response. Therefore, in some embodiments, lipid nanoparticles are directly applied to individuals. In some embodiments, lipid nanoparticles are contacted with cells (e.g., immune cells) in vitro, and the treated cells are applied to individuals (e.g., adoptive transfer). Generally, the disclosed lipid nanoparticles can be used to treat individuals suffering from or susceptible to any disease or illness, and the individual's immune system applies an overactive or inappropriate immune response to the disease or illness. Medicaments can make less strong immune responses possible. The disclosed compositions can be used to reduce or suppress immune responses involving T cells. b. Inflammatory response

[0091] The disclosed lipid nanoparticles can be used to treat inflammation. Typically, by administering a certain amount of the disclosed lipid nanoparticles to an individual, the medicament is used to reduce or suppress an immune response in the individual. The method can reduce one or more symptoms of inflammation. Inflammation can be acute, chronic or persistent inflammation.

[0092] In some embodiments, lipid nanoparticles slow down the immune system. For example, the medicament can be used to control excessive inflammatory responses that cause damage to healthy tissues. Therefore, in some embodiments, the medicament is administered to individuals who experience excessive inflammatory responses. In this case, controlling excessive immune responses is beneficial to individuals. c. Inflammatory and autoimmune diseases / disorders

[0093] The disclosed lipid nanoparticles can also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases / disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue syndrome immunodeficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, De Ge's disease, dermatomyositis, dermatomyositis-juvenile, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyalgia Myositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0094] In some embodiments, the inflammatory or autoimmune disease is caused by a pathogen or is the result of an infection. C. Methods of treating liver disease

[0095] The disclosed methods and compositions can be used to reduce, decrease, prevent or otherwise limit the onset, development, progression, signs or symptoms of liver diseases and disorders. Dysfunctional liver endothelial cells, Kupffer cells and other cells in the liver microenvironment cause more disease than hepatocytes. Historically, nanoparticles tend to preferentially target hepatocytes rather than other cells in the liver microenvironment.

[0096] Without being bound by any one theory, it is believed that the cholesterol modifications disclosed herein allow lipid nanoparticles to more effectively deliver cargo to cells in the liver microenvironment in vivo.

[0097] In one embodiment, the disclosed nanoparticles can be used for protein replacement therapy within the liver microenvironment.

[0098] Lipid nanoparticles can be administered locally or systemically in an effective amount to cause local or systemic reactions, for example, to treat or prevent liver disease. In a preferred embodiment, one or more lipid nanoparticles are administered once a week at a dosage of 0.05-0.25 mg / kg body weight. In another embodiment, nanoparticles are delivered twice a month, once a month, or at a lower frequency. Exemplary liver diseases and disorders that can be treated by lipid nanoparticles are provided below. 1. Liver cancer

[0099] The disclosed methods and compositions can be used to reduce, decrease, prevent or otherwise limit the onset, development, progression, signs or symptoms of liver cancer. In some embodiments, the liver cancer to be treated is asymptomatic liver cancer. In some embodiments, liver cancer has been identified by detecting diagnostic markers associated with the onset, development or progression of liver cancer. In further embodiments, risk factors for developing liver cancer are used as a mechanism to identify individuals who may benefit from preventive treatment with the disclosed methods and compositions.

[0100] In one embodiment, the disclosed lipid nanoparticles can deliver chemotherapeutic agents to the liver to treat liver cancer and / or reduce tumor burden. In another embodiment, the disclosed lipid nanoparticles can deliver apoptotic agents to the liver to treat liver cancer and / or reduce tumor burden. 2. Cirrhosis

[0101] The disclosed methods and compositions can be used to reduce, decrease, prevent, or otherwise limit the onset, development, progression, signs, or symptoms of cirrhosis.

[0102] Cirrhosis of the liver is a disease in which liver cells are damaged and replaced by scar tissue. Factors that lead to cirrhosis of the liver include, but are not limited to, infectious diseases, alcohol abuse, recreational drug abuse, and non-fatty liver disease. Cirrhosis of the liver is associated with the development of liver cancer. It has been recognized that chronic infection with hepatitis C virus (HCV) is an increased risk of hepatocellular carcinoma (HCC). About 20% of HCV-infected individuals suffer from disease that progresses to cirrhosis of the liver, and about 40% of these patients develop HCC after an average of 10-15 years. Cirrhosis of the liver can also be caused by infection with hepatitis B virus (HBV).

[0103] In one embodiment, the disclosed lipid nanoparticles can deliver therapeutic agents to the liver to treat symptoms of cirrhosis, or to halt the progression of the disease. 3. Fatty liver disease

[0104] The disclosed methods and compositions can be used to reduce, decrease, prevent or otherwise limit the onset, development, progression, signs or symptoms of fatty liver disease.Fatty liver disease can be a result of alcohol abuse, which is the leading cause of cirrhosis in the United States.

[0105] Fatty liver disease can also be non-alcoholic fatty liver disease. Cirrhosis can be caused by non-alcoholic fatty liver disease, which is a fatty liver disease that develops in people who consume little or no alcohol. Non-alcoholic fatty liver disease is common in obese people. People with this type of disease, known as non-alcoholic steatohepatitis (NASH), may go on to develop cirrhosis. Type 2 diabetes is associated with an increased risk of liver cancer, particularly in patients who also have other risk factors such as alcoholism and / or chronic viral hepatitis. This risk may increase because people with type 2 diabetes tend to be overweight or obese, which in turn may cause liver problems.

[0106] In some embodiments, the disclosed lipid nanoparticles are used prophylactically. Thus, the lipid nanoparticles can be administered daily in the absence of symptoms or markers of liver disease to promote general liver health and prevent the development of liver disease in individuals at risk of liver disease. IV. Combination Therapy

[0107] The disclosed lipid nanoparticles can be administered to an individual in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the lipid nanoparticles and additional therapeutic agents are administered separately but simultaneously. The lipid nanoparticles and additional therapeutic agents can also be administered as part of the same composition. In other embodiments, the lipid nanoparticles and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regimen.

[0108] The first therapeutic agent may be administered to an individual 1, 2, 3, 4, 5, 6 or more hours or 1, 2, 3, 4, 5, 6, 7 or more days prior to administration of the second therapeutic agent. In some embodiments, one or more doses of the first agent may be administered to an individual every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 or 48 days prior to the first administration of the second agent. The lipid nanoparticle may be the first therapeutic agent or the second therapeutic agent.

[0109] The lipid nanoparticles and additional therapeutic agents can be administered as part of a treatment regimen. For example, if the first therapeutic agent can be administered to an individual every 4 days, the second therapeutic agent can be administered on the first, second, third, or fourth day, or a combination thereof. The first therapeutic agent or the second therapeutic agent can be repeatedly administered throughout the treatment regimen.

[0110] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral agents (particularly protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic agents (helminths, protozoa), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized, single chain and chimeric antibodies), antigens and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind to Toll-like receptors (including but not limited to CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and other molecules that inactivate or downregulate inhibitory or regulatory T cells.

[0111] The additional therapeutic agent is selected based on the condition, disorder or disease to be treated.For example, an immunomodulatory agent can be co-administered with one or more additional agents that act to enhance or promote an immune response or to reduce or inhibit an immune response. A. Increase immune response 1. Antimicrobial agents

[0112] For example, the disclosed lipid nanoparticles can play a preventive or prophylactic role in the treatment and prevention of the above-mentioned diseases and in the case of severe traumatic injuries such as severe burns, open fractures, accidental amputations or other wounds. Therefore, the disclosed lipid nanoparticles can be administered to individuals in combination with antimicrobial agents such as antibiotics, antifungals, antivirals, antiparasitics or essential oils.

[0113] In some embodiments, lipid nanoparticles and / or antimicrobial agents are administered to individuals upon admission to further prevent bacterial, fungal or viral complications. Antibiotics can target pathogens and lipid nanoparticles can stimulate the immune system to provide an enhanced response to further treat or prevent infection or disease. 2. Chemotherapeutic agents

[0114] The disclosed lipid nanoparticles can be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cretasone, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, subacute Folic acid, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozotocin, tegafururacil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine or a combination thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ (2) and a combination thereof. 3. Other immunomodulators a. PD-1 antagonists

[0115] In some embodiments, lipid nanoparticles are co-administered with PD-1 antagonists. Programmed death-1 (PD-1) is a member of the CD28 receptor family that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces the intensity and / or duration of T cell proliferation and / or T cell responses. Suitable PD-1 antagonists are described in U.S. Patents Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated herein by reference in their entirety, and include binding to PD-1 ligands and blocking PD-1 ligands to interfere with or inhibit the ligand from binding to PD-1 receptors, or directly binding to PD-1 receptors and blocking PD-1 receptors without inducing a compound or agent for inhibitory signal transduction by PD-1 receptors.

[0116] In some embodiments, the PD-1 receptor antagonist directly binds to the PD-1 receptor without triggering inhibitory signal transduction, and also binds to the ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are weakened by the negative signals transmitted by the PD-1 signal transduction, and a stronger immune response can be achieved.

[0117] It is believed that PD-1 signaling is driven by binding to PD-1 ligands (e.g., B7-H1 or B7-DC), which are in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent PD-1 and TCR from being co-attached on the T cell membrane are also useful PD-1 antagonists.

[0118] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to a ligand of PD-1 or PD-1 itself, particularly when co-ligation of PD-1 with the TCR is not followed by such binding, thereby not triggering inhibitory signaling through the PD-1 receptor.

[0119] Other PD-1 antagonists contemplated by the methods of the invention include antibodies and other antibodies that bind to PD-1 or a ligand of PD-1.

[0120] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos.: 7,332,582; 7,488,802; 7,521,051; 7,524,498; 7,563,869; 7,981,416; 8,088,905; 8,287,856; 8,580,247; 8,728,474; 8,779,105; 9,067,999; 9,073,994; 9,084,776; 9,205,148; 9,358,289; 9,387,247; 9,492,539; 9,492,540, which are incorporated herein by reference in their entirety.

[0121] See also Berger et al., Clin. Cancer Res., 14:3044-3051 (2008).

[0122] Exemplary anti-B7-H1 (also known as anti-PD-L1) antibodies include, but are not limited to, those described in the following U.S. Patent Nos.: 8383796, 9102725, 9273135, 9393301, and 9580507, all of which are specifically incorporated herein by reference in their entireties.

[0123] For anti-B7-DC (also known as anti-PD-L2) antibodies, see U.S. Patent Nos.: 7,411,051, 7,052,694, 7,390,888, 8188238, and 9255147, the entire contents of which are specifically incorporated herein by reference.

[0124] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologues and variants of these polypeptides, and active fragments of any of the foregoing, and fusion proteins incorporating any of these. In some embodiments, the fusion protein includes a soluble portion of B7-DC coupled to the Fc portion of an antibody (e.g., human IgG), and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0125] The PD-1 antagonist may also be a fragment of mammalian B7-H1, such as from a mouse or primate, such as a human, wherein the fragment binds to PD-1 and blocks PD-1 but does not cause inhibitory signal transduction through PD-1. The fragment may also be part of a fusion protein, such as an Ig fusion protein.

[0126] Other useful polypeptide PD-1 antagonists include those that bind to ligands of the PD-1 receptor. These include PD-1 receptor proteins or soluble fragments thereof, which can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction. B7-H1 also shows binding to protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of PD-1 proteins, which include mutations that increase binding to natural ligands, such as A99L mutations (Molnar et al., PNAS, 105: 10483-10488 (2008)). B7-1 or its soluble fragments can bind to B7-H1 ligands and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction, which is also useful.

[0127] PD-1 and B7-H1 antisense nucleic acids, both DNA and RNA, and siRNA molecules can also be PD-1 antagonists. This antisense molecule prevents the expression of PD-1 on T cells and the production of T cell ligands such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (e.g., a length of about 21 nucleotides, which is specific to genes encoding PD-1 or encoding PD-1 ligands, and the oligonucleotides can be easily commercially available) (see Cubillos-Ruiz et al., J. Clin. Invest. 119 (8): 2231-2244 (2009), which is easily taken up by cells expressing PD-1 and PD-1 ligands, and reduces the expression of these receptors and ligands to achieve a reduction in inhibitory signal transduction in T cells, thereby activating T cells. b. CTLA4 antagonists

[0128] Other molecules used to mediate the role of T cells in immune responses are also considered to be additional therapeutic agents. In some embodiments, the molecule is an antagonist of CTLA4, such as an antagonist anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies contemplated for use in the methods of the present invention include antibodies described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0129] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may be, for example, 0.1 to 100 mg / kg, or 1 to 50 mg / kg, or 10 to 20 mg / kg. Suitable doses for human subjects may be 5 to 15 mg / kg, with 10 mg / kg of an antibody (e.g., human anti-PD-1 antibody) being a specific embodiment.

[0130] Specific examples of anti-CTLA4 antibodies for use in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg / kg, and Tremelimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135-137 (2010), published online December 2009.

[0131] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277: 7363-7368 (2002). Such small organic compounds can be administered alone or in combination with anti-CTLA4 antibodies to reduce inhibitory signal transduction of T cells. 4. Synergist

[0132] In some embodiments, the additional therapeutic agent includes a potentiator. Potentiators may increase the efficacy of the immune response upregulator through more than one mechanism, although the exact mechanism of action is not necessary for the broad practice of the invention.

[0133] In some embodiments, the potentiator is cyclophosphamide. or ) is an oxazahosphorine drug, and analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide (trofosfamide; Ixoten); and pharmaceutically acceptable salts, solvates, prodrugs and metabolites thereof (U.S. Patent Application 20070202077, the entirety of which is incorporated herein). Ifosfamide It is a structural analog of cyclophosphamide, and its mechanism of action is believed to be the same or substantially similar to that of cyclophosphamide. Perphosphamide (4-hydroperoxycyclophosphamide) and chlorhexylcyclophosphamide are also alkylating agents that are structurally related to cyclophosphamide. For example, perphosphamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorine derivatives have been designed and evaluated in an attempt to improve selectivity and response while reducing host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007; 13 (9): 963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphamide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldehyde phosphoramide perhydrothiazine) and NSC 613060 (aldehyde phosphoramide thiazolidine). Mafosfamide is an oxazaphosphorabenzene analog that is a chemically stable 4-thioethanesulfonate salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphamide mustard (an alkylated metabolite of IFO) is glycosidically linked to a β-D-glucose molecule. Additional cyclophosphamide analogs are described in U.S. Pat. No. 5,190,929, entitled "Cyclophosphamide Analogs Useful as Antineoplastic Agents," which is incorporated herein by reference in its entirety.

[0134] Although CTX itself is non-toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and, at higher doses, chain breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes because lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and circulating cells are most sensitive to DNA alkylating agents.

[0135] Low doses of CTX (<200 mg / kg) can have immunostimulatory effects, including stimulating antitumor immune responses in human and mouse cancer models (Brode & Cooke Crit Rev. Immunol. 28: 109-126 (2008)). These low doses are subtherapeutic and do not have direct antitumor activity. In contrast, high doses of CTX inhibit antitumor responses. Several mechanisms can explain the role of CTX in enhancing antitumor immune responses: (a) depletion of CD4+CD25+FoxP3+Treg (and specific proliferation of Treg, which can be particularly inhibited), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), leading to inhibition of tumor cell growth; (d) mobilization and expansion of CD11b+Gr-1+MDSC. These primary effects have many secondary effects; for example, after Treg depletion, macrophages produce more IFN-γ and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

[0136] Treg depletion is often cited as a mechanism by which CTX enhances antitumor immune responses. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment on day 9 gave a 75% cure rate. Transferring purified Tregs on day 12 almost completely suppressed the CTX response (van der Most et al. Cancer Immunol. Immunother. 58: 1219-1228 (2009). Similar results were observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+Tregs after CTX pretreatment eliminated the therapeutic response to the vaccine (Taieb, JJ Immunol. 176: 2722-2729 (2006)).

[0137] Many human clinical trials have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47: 1-12 (1998)).

[0138] The optimal dose of CTX to enhance anti-tumor immune responses is a dose that reduces total T cell counts by reducing Treg levels to below the normal range but is subtherapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[0139] In human clinical trials using CTX as an immunopotentiator, a dose of 300 mg / m2 is generally used. For an average male (6ft, 170 pounds (78 kg), with a body surface area of ​​1.98 m2), 300 mg / m2 is 8 mg / kg, or 624 mg of total protein. In a mouse cancer model, efficacy was observed in a dose range of 15-150 mg / kg, which involved 0.45-4.5 mg of total protein in 30 g mice (Machiels et al. Cancer Res. 61: 3689-3697 (2001), Hengst et al Cancer Res. 41: 2163-2167 (1981), Hengst Cancer Res. 40: 2135-2141 (1980)).

[0140] For larger mammals, such as primates, such as humans, patients, such mg / m2 dosages may be used, but unit doses administered at limited time intervals may also be used. Such unit doses may be administered daily for a limited period of time, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days, all of which are specifically contemplated by the present invention. The same regimen may be applied to the other synergists described herein.

[0141] In other embodiments, the potentiator is an agent that decreases the activity and / or number of regulatory T lymphocytes (T-regs), such as sunitinib Anti-TGFβ or imatinib ( The treatment regimen may also include the administration of an adjuvant.

[0142] Useful potentiators also include mitotic inhibitors such as paclitaxel, aromatase inhibitors (e.g., letrozole) and angiogenesis inhibitors (VEGF inhibitors, such as Avastin, VEGF-Trap) (see, e.g., Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory Tcells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15; 12(22): 6808-16.), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists. B. Reduce immune response 1. Immunosuppressants

[0143] In some embodiments, an immune response or inflammatory / autoimmune disease / disorder is treated by administering to an individual the disclosed lipid nanoparticles and a second agent that is an immunosuppressant. Immunosuppressants include, but are not limited to, antibodies against other lymphocyte surface markers (e.g., CD40, α-4 integrin) or antibodies against cytokines, fusion proteins (e.g., CTLA-4-Ig ( (abatacept)), TNFR-Ig( (etanercept), TNF-α blockers such as Enbrel (etanercept), Remicade (infliximab), Cimzia (certolizumab), and Humira (adalimumab), cyclophosphamide (CTX) (i.e., Revimmune TM ), methotrexate (MTX) (i.e., ), belimumab (i.e. ) or other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds or steroids), antiproliferative agents, cytotoxic agents or other compounds that may contribute to immunosuppression.

[0144] The therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). The CTLA-4-Ig fusion protein competes with the co-stimulatory receptor CD28 on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, thereby inhibiting T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein called Belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y), which significantly increase its affinity for CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

[0145] In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Revimmune TM Cyclophosphamide (CTX), also known as cytophosphane, is a nitrogen mustard alkylating agent from the oxazophorine group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the main drug for the treatment of diffuse proliferative glomerulonephritis in patients with renal lupus.

[0146] The therapeutic agent may be administered in an amount effective to reduce blood or serum levels of anti-double-stranded DNA (anti-ds DNA) autoantibodies and / or reduce proteinuria in a patient in need thereof.

[0147] In another embodiment, the therapeutic agent increases the amount of adenosine in serum, see, e.g., WO 08 / 147482. For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent that increases CD73 expression (e.g., a cytokine or a monoclonal antibody or a small molecule), see, e.g., WO04 / 084933. In another embodiment, the therapeutic agent is interferon-β.

[0148] The therapeutic agent can be a small molecule that inhibits or reduces the differentiation, proliferation, activity and / or cytokine production and / or secretion of Th1, Th17, Th22 and / or other cells, which secrete or cause other cells to secrete inflammatory molecules, including but not limited to IL-1β, TNF-α, TGF-β, IFN-γ, IL-18IL-17, IL-6, IL-23, IL-22, IL-21 and MMPs. In another embodiment, the therapeutic agent is a small molecule that interacts with Treg, enhances Treg activity, promotes or enhances Treg secretion of IL-10, increases the number of Treg, increases the inhibitory ability of Treg or a combination thereof.

[0149] In some embodiments, the composition increases Treg activity or production. Exemplary Treg enhancers include, but are not limited to, glucocorticoids fluticasone, salmeterol, antibodies to IL-12, IFN-γ, and IL-4; vitamin D3 and dexamethasone, and combinations thereof.

[0150] In some embodiments, the therapeutic agent is an antibody, for example, blocking the function of an antibody directed against a pro-inflammatory molecule such as IL-6, IL-23, IL-22, or IL-21.

[0151] As used herein, the term "rapamycin compound" includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds that are believed to have the same mechanism of action as rapamycin (e.g., inhibiting cytokine function). The phrase "rapamycin compound" includes compounds with structural similarity to rapamycin, such as compounds with similar macrocyclic structures that have been modified to enhance their therapeutic effectiveness. Exemplary rapamycin compounds are known in the art (see, e.g., WO95122972, WO 95116691, WO 95104738, US Patent Nos. 6,015,809; 5,989,591; US ​​Patent Nos. 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

[0152] The expression "FK506-like compounds" includes FK506 and FK506 derivatives and analogs, such as compounds having structural similarity to FK506, such as compounds having similar macrocyclic structures, which have been modified to enhance their therapeutic effectiveness. Examples of FK506-like compounds include, for example, those described in WO00101385. In some embodiments, the expression "rapamycin compound" as used herein does not include FK506-like compounds. 2. Anti-inflammatory drugs

[0153] Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent may be a non-steroidal, a steroidal, or a combination thereof. One embodiment provides an oral composition containing about 1% (w / w) to about 5% (w / w), typically about 2.5% (w / w), of an anti-inflammatory agent. Representative examples of non-steroidal anti-inflammatory agents include, but are not limited to, oxicams, such as piroxicam, isoxicams, tenoxicam, sudoxicam; salicylates, such as aspirin, disalicylic acid, pyraclostrobin, choline magnesium trisalicylate, safapryn, solprin, diflunisal, and fendusal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxacic acid, furofenac, tiopine, zidometacin, acemetacin, fentiazolic acid, zomepiracic acid, clomiphene citrate ... The present invention relates to the nonsteroidal anti-inflammatory agents of the present invention, for example, lindanac, oxipinac, felbinac and ketorolac; fenamates such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozine; pranoprofen, miprofen, thioxaprofen, suprofen, aminoprofen and thiaprofen; pyrazoles such as phenylbutazone, oxyphenylbutazone, feprazone, azapropazone and trimeprin. Mixtures of these nonsteroidal anti-inflammatory agents can also be used.

[0154] Representative examples of steroidal anti-inflammatory drugs include, but are not limited to, corticosteroids such as hydrocortisone, hydroxy-triamcinolone, alpha-methyldexamethasone, dexamethasone-phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, deoxycorticosterone acetate, dexamethasone, diclosone, diflusone diacetate, diflucortolone valerate, fluadrenolone, fluclosan acetonide, fludrocortisone, flumethasone pivalate, fluocortolone acetonide, fluocortolone acetate, flucortine butyl ester, fluocortine acetonide, fluocortine butyl ester ... butylester), fluocortolone, fluprednylidene acetate, flurandrenolide, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosonediacetate, fluradrenolone, fludrocortisone, difluorosonediacetate, fluradrenolone acetone compounds, medrysone, ancifar, ancifit, betamethasone and its other esters, cloprednisone, cloprednisone acetate, clocortelone, clescinolone, diclosone, diflurprednate, flucloronide, flunisolide, fluorometholone, fluperone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone aminoester, methylprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and mixtures thereof. Example method: Synthesis of CKK-E12:

[0155] Compound 1 (20 g, 41.9 mmol) was added to a 100 ml flask and trifluoroacetic acid (42 mL) was slowly added at 0 ° C, and then stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure and then the crude product dissolved in DMF (5 mL) was added dropwise to pyridine (300 mL) at 0 ° C. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure to obtain a white solid, which was washed with ethyl acetate to obtain compound 2 (8.4 g, 13.04 mmol, 31% yield) (Figure 6B). Pd / C (10 wt.%, 3.0 g) was added to a solution of compound 2 in acetic acid / CH2Cl2 (150 / 150 mL). The black suspension was degassed with hydrogen for 5 minutes and stirred overnight at room temperature under a hydrogen atmosphere. The reaction mixture was filtered through diatomaceous earth and washed with MeOH. The combined filtrate was concentrated to obtain a crude yellow viscous oil. The oil was solidified by adding ethyl acetate and washed with ethyl acetate to produce compound 3 ( Figure 6C). Triethylamine (0.12 mL, 0.88 mmol) was added to a solution of compound 3 (84 mg, 0.22 mmol) and 1,2-epoxydodecane (247 mg, 1.34 mmol) in EtOH (2 mL). After stirring at room temperature for 30 minutes, the reaction mixture was then irradiated at 150 ° C for 5 hours in a microwave reactor. The crude residue was purified by flash column chromatography. After the version with four alkyl tails was purified using flash column chromatography, the 1 H-NMR confirmed the chemical structure ( Figure 6C ). Nanoparticle formulations:

[0156] Nanoparticles were prepared in a microfluidic device by mixing Cre mRNA, DNA, ionizable lipids, PEG, and cholesterol as previously described (Poisson, et al., Journal of Hepatology, 66:212 (2017)). The experimental design can be found in Figure 1H Nanoparticles were prepared with these components in variable molar ratios. Nucleic acids (e.g., DNA barcodes, mRNA) were diluted in 10 mM citrate buffer (Teknova) and loaded into a syringe (Hamilton Company). The materials that make up the nanoparticles (CKK-E12, cholesterol, PEG, DOPE) were diluted in ethanol and loaded into a second syringe. The citrate phase and the ethanol phase were mixed together in a microfluidic device using a syringe pump. DNA barcoding:

[0157] Methods for screening and formulating nanoparticles using DNA barcoding are described in PCT / US2018 / 058171. Each chemically different LNP is formulated to carry its own different DNA barcode. For example, LNP1 carries Cre mRNA and DNA barcode 1, while chemically different LNP2 carries Cre mRNA and DNA barcode 2. The DNA barcodes are rationally designed to have universal primer sites and specific 8-nucleotide barcode sequences, similar to those previously described (Paunovaska, K., et al. Nano Lett, 18: 2148 (2018)). To ensure equal amplification of each sequence, we include universal forward and reverse primer regions on all barcodes. The 8nt sequence can generate more than 4 8 (65,536) different barcodes. Nanoparticle Characterization:

[0158] LNP hydrodynamic diameter is measured using dynamic light scattering (DLS). LNP is diluted to a concentration of ~0.06 μg / mL in sterile 1X PBS and analyzed. If they meet 3 criteria: diameter>20nm, diameter<215nm, and only have an autocorrelation function of 1 inflection point, LNP is included. The particles that meet these criteria are merged and dialyzed in 1X phosphate buffered saline (PBS, Invitrogen) and aseptically filtered with a 0.22 μm filter. Animal Experimentation:

[0159] All animal experiments were performed in accordance with the Georgia Institute of Technology'sInstitutional Animal Care and Use Committee (IACUC). C57BL / 6J (#000664) and Ai14 LSL-Tomato (#007914) mice were purchased from Jackson Laboratory. In all experiments, mice were 5-8 weeks old and N=3-4 mice / group, injected intravenously through the lateral tail vein. Nanoparticle concentrations were determined using NanoDrop (Thermo Scientific). The weights of all animals are included in Figures 7A-7C. Cell isolation and staining:

[0160] In all cases, mice were killed and immediately perfused with 20mL of 1XPBS by right atrium 3 days after LNP administration. Organs were separated immediately after perfusion. Tissue was cut and then placed in a digestive enzyme solution with collagenase type I (Sigma Aldrich), collagenase XI (Sigma Aldrich) and hyaluronidase (Sigma Aldrich) at 37°C and 750rpm for 45 minutes. Digestive enzymes for heart and spleen include collagenase IV (Sigma Aldrich). Digested tissue was passed through a 70 μm filter and lysed red blood cells. Cells were dyed to identify specific cell populations, and sorted using BD FacsFusion cell sorter. The antibody clones used for staining were: anti-CD31 (390, BioLegend), anti-CD45.2 (104, BioLegend), anti-CD11b (M1 / 70, BioLegend), anti-CD68 (FA-11, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD19 (6D5, BioLegend), anti-CD34 (SA376A4, BioLegend). Table 1 shows the FACS markers used for each cell and tissue type. Table 1. FACS markers for each cell type Cell Type Markers organize Hepatocytes (H) CD45-CD31- V Kupffer cells (K) CD45+CD31-CD68+ V Endothelial cells (E) CD45-CD31+ V, H, L, K, P, M Immune cells (l) CD45+CD31-CD11b- / 68- V, H, L, K, P Others (O) CD45-CD31- H, L, K, P.M. HSCs CD31-CD45+CD34+ M Macrophages (M) CD45+CD11b+ S, H, L, K, P, M T cells (T) CD45+CD19-CD3+ S B cells (B) CD45+CD3-CD19+ S Immune cells (I) CD45+CD19-CD3-CD11b- S PCR amplification:

[0161] All samples were amplified and prepared for sequencing using a nested PCR protocol. More specifically, 1 μL of each primer (10 μM reverse / forward) was added to 5 μL of Kapa HiFi2X reaction mixture (master mix), 2 μL sterile H2O, and 1 μL DNA template. The second PCR added NexteraXT chemistry, indexing, and i5 / i7 adapter regions, and used the product from 'PCR 1' as a template. Deep Sequencing:

[0162] Illumina deep sequencing was performed on an Illumina MiniSeqTM using the standard protocol proposed by Illumina. Sequencing was performed in a Georgia Tech Molecular Evolutioncore. Data normalization:

[0163] The counts per particle were normalized to the barcoded LNP mixture injected into mice as we previously described (Paunovaska, K., et al. Nano Lett, 18:2148 (2018)). Data Analysis:

[0164] Sequencing results were processed using a custom python-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized using an R script before further analysis. Statistical analysis was performed using GraphPad Prism 7. Unless otherwise stated, data were plotted as mean ± standard error. Example 1. Synthesis of a library of rationally designed nanoparticles containing oxidized cholesterol. result:

[0165] As quality control, analyze the size of each independent LNP.Only when the hydrodynamic diameter of LNP is 20nm to 215nm and their autocorrelation curve comprises an inflection point, just merge LNP (Fig. 4 A-4B).86 of the LNPs prepared in 125 meet these standards and merge.As a control, the diameter of all 86 LNPs is compared with the diameter of the LNP solution merged, and observe that they are similar (Fig. 2 C).This shows that the LNP merged after mixing does not gather.Add naked DNA barcode as negative control, because naked DNA is not easy to enter cells.After separating cells and carrying out NGS, determine that as expected, the frequency that naked DNA is delivered to cells is lower than all DNA barcodes ( Figure 2D-2E ).

[0166] Mice were sacrificed 72 hours after injection with the combined LNPs. This time point allows cells to express tdTomato ( Figure 2A ). The liver, spleen, heart, kidney, pancreas, lung, and bone marrow were then isolated. Using fluorescence-activated cell sorting (FACS), 28 different tdTomato+ cell types were isolated ( Figure 2A ). Comparing the percentages of tdTomato+ cell types in different organs, cells in the liver tended to be targeted more than cells in other organs (Figure 2B). The organ with the second highest percentage of tdTomato+ cells was the spleen. The remaining 5 organs had negligible delivery (Figure 4C). The barcode sequencing data were clustered using an unbiased Euclidean algorithm. This bioinformatics technique is often applied to gene expression data and can analyze nanoparticle barcode data. Euclidean clustering revealed that the 4 liver cell types were more closely "clustered" together than the spleen cell types ( Figure 2F ). We found that the percentages of tdTomato+ hepatic endothelial cells, hepatic immune cells, and Kupffer cells were much higher than those of tdTomato+ hepatocytes (Figure 2B). Example 2. Modified cholesterol can alter nanoparticle delivery in vivo method:

[0167] Unmodified cholesterol is acted upon by enzymes to form variants with oxidized side chains or rings ( Figure 1A To investigate whether these modifications alter LNP targeting, 125 LNPs ( Figure 1B-1GTo minimize changes in components other than cholesterol, LNPs were made from the ionizable lipid-like material cKK-E12, two well-validated PEG-lipids, the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and one of nine different cholesterol variants ( Figure 1B-1G ).

[0168] LNPs are formed by mixing the contents together in a microfluidic device. Each LNP carries a unique DNA barcode, which acts as a tag for that LNP, as well as Cre mRNA, which signals the delivery of a functional mRNA ( Figure 1H ). Combine the stable LNPs together ( Fig. 1I ) and administered to Ai14 mice at a total nucleic acid dose of 1.0 mg / kg. Ai14 mice contain a Lox-Stop-Lox-tdTomato construct under the control of a CAG promoter; therefore, if: (1) Cre mRNA is delivered to the cytoplasm, (2) Cre mRNA is translated into Cre protein, (3) Cre protein translocates from the cytoplasm to the nucleus, and (4) Cre protein edits the genome by removing the 'Stop' between the Lox sites, then cells in Ai14 mice become tdTomato+. Therefore, by isolating tdTomato+ cell types using fluorescence-activated cell sorting (FACS) and using next-generation sequencing (NGS) to quantify the barcodes therein, LNPS located in cells where functional mRNA delivery occurs can be identified ( Fig. 1I NGS sequencing data are quantified as “normalized delivery,” similar to counts per million in RNA-seq ( Fig. 1I ). result:

[0169] To quantify the effect of cholesterol structure on total spleen and liver delivery, we calculated normalized delivery for all eight cell types we sequenced (4 in spleen and 4 in liver) ( Figure 3A ). Side chain oxidized cholesterol variants tended to enhance delivery relative to other cholesterol variants (Figures 3B-3C). In particular, LNPs formulated with 25-hydroxycholesterol (25-OH) and 20α-hydroxycholesterol (20α-OH) resulted in higher normalized delivery across all 8 cell types (Figure 3D). To supplement these calculations, it was determined which cholesterol variants were enriched in the top 10% of LNPs (Figure 3E). The enrichment in the bottom 10% of LNPs was then calculated and subtracted from the enrichment in the top 10% (Figure 3B-3C). Figure 3E-3F ). This identifies how likely it is that a given cholesterol variant will be found in the best and worst performing LNPs. Figure 5AAs additional controls, the same two analyses were performed - normalized delivery and enrichment across all eight cell types - cholesterol molar percentage, ionizable lipid molar percentage, and phospholipid molar percentage ( Figures 5B-5D ). No significant trends were observed.

[0170] These data indicate that LNP cholesterol chemistry is an important factor in LNP targeting. However, it does not take into account LNP size. It was previously found that for hydrodynamic diameters of about 20nm to about 200nm, there was no relationship between nanoparticle size and delivery. In order to study how size changes delivery in this experiment, for all LNPs (Figure 5K), LNPs with tail-oxidized cholesterol (Figure 5L), and LNPs with epoxidized cholesterol (Figure 5M), normalized delivery was calculated to see if it varied with LNP size; no correlation was found. The percentage of formulated LNPs that met the inclusion criteria and the average size of stable LNPs based on cholesterol structure were calculated as a function of cholesterol structure. No significant differences were found ( Figure 5N-5O The same analysis as a function of cholesterol mole percentage yielded the same conclusions ( Figure 5P-5Q ). Therefore, there is no evidence that the size between 20 nm and 215 nm affects LNP delivery.

[0171] To this end, the first three LNP candidates identified were formulated with Cre mRNA. Of the three LNPs formulated, one LNP containing 25-OH cholesterol was inconsistently formulated and excluded. The remaining two LNPs, which contained 20α-OH cholesterol (Figures 3G-3K), formed stable LNPs and were administered to Ai14 mice at a total mRNA dose of 0.25 mg / kg. Encouragingly, both LNPs recapitulated the results from earlier screening ( Figure 3L-3O ). After 0.25 mg / kg injection, both LNP1 and LNP2 strongly targeted cells in the microenvironment. As predicted by the screening, hepatocytes were targeted much less efficiently. Encouraged by the strong delivery at 0.25 mg / kg, LNP1 and LNP2 were injected at a dose of 0.05 mg / kg. Again, strong delivery was observed ( Figure 3L-3O ). Although LNP1 and LNP2 performed equally well at 0.25 mg / kg by saturating the Cre-based system, LNP1 outperformed LNP2 at a lower dose of 0.05 mg / kg. LNP1 did not cause weight loss in mice in any experiment; LNP2 did cause weight loss in mice at 0.25 mg / kg (Figures 7A-7C).

[0172] Systemic delivery of therapeutic RNA to hepatocytes has led to FDA-approved drugs (Rizk, M and Tuzmen, S., Pharmacogenomics and Personalized Medicine, 10: 267 (2017)). Delivery to non-hepatocytes remains more challenging. Therefore, an unbiased high-throughput method for studying how LNPs deliver RNA in vivo can accelerate the discovery of nanoparticles with new tropisms. Here, it is reported that LNPs containing oxidized cholesterol can more effectively deliver mRNA to cells in the liver microenvironment than hepatocytes. It is worth noting that the robust delivery occurred at 0.05 mg / kg, which is lower than the dosage regimen for siRNA therapy in humans. Given the importance of hepatic endothelial cells (Poisson, et al., Journal of Hepatology, 66:212 (2017)) and Kupffer cells (Kolios, G., et al., World Journal of Gastroenterology, 12:7413 (2006)) in disease, these data suggest that additional advances in delivery could ultimately lead to protein replacement therapies within the liver microenvironment.

[0173] Although in the foregoing specification, the present invention has been described with respect to certain embodiments thereof and many details have been presented for the purpose of illustrative illustration, it is obvious to those skilled in the art that the present invention is directed to other embodiments and that certain details described herein may be varied considerably without departing from the basic principles of the invention.

[0174] All references cited herein are incorporated by reference in their entirety. The present invention may be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention, and therefore, reference should be made to the appended claims, rather than to the foregoing description, to indicate the scope of the present invention.

Claims

1. A lipid nanoparticle composition comprising: Ionizable lipids, Phospholipids, Polyethylene glycol (PEG), and Oxidized cholesterol, wherein the oxidized cholesterol is selected from the group consisting of 20α-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7β-hydroxycholesterol, and combinations thereof.

2. The lipid nanoparticle composition of claim 1, wherein the ionizable lipid is 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (CKK-E12).

3. The lipid nanoparticle composition of any one of claims 1-2, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

4. The lipid nanoparticle composition of any one of claims 1 to 3, wherein the PEG is C 14 PEG 2000 or C 18 PEG 2000 .

5. The lipid nanoparticle composition of any one of claims 1-4, wherein the oxidized cholesterol is 20α-hydroxycholesterol.

6. The lipid nanoparticle composition of any one of claims 1-4, wherein the oxidized cholesterol is 25-hydroxycholesterol.

7. The lipid nanoparticle composition of any one of claims 1-4, wherein the oxidized cholesterol is 7β-hydroxycholesterol.

8. The lipid nanoparticle composition of any one of claims 1-4, wherein the oxidized cholesterol is 27-hydroxycholesterol.

9. The lipid nanoparticle composition of any one of claims 1-8, wherein the composition comprises about 30 mol% to about 80 mol% ionizable lipids, about 5 mol% to about 55 mol% oxidized cholesterol, about 10 mol% to about 35 mol% phospholipids, and about 5 mol% to about 20 mol% PEG-lipids.

10. The lipid nanoparticle composition of any one of claims 1-9, further comprising a therapeutic or prophylactic agent.

11. The lipid nanoparticle composition of claim 10, wherein the therapeutic or prophylactic agent is encapsulated within the lipid nanoparticle as a cargo.

12. The lipid nanoparticle composition of claim 10, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

13. The lipid nanoparticle composition of claim 12, wherein the RNA is messenger RNA (mRNA).

14. The lipid nanoparticle composition of any one of claims 1-13, wherein the nanoparticle has a diameter of about 20 nm to about 215 nm.

15. A pharmaceutical composition comprising the lipid nanoparticle composition of any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

16. Use of the lipid nanoparticle composition of any one of claims 10-14 in the preparation of a medicament for delivering a therapeutic or prophylactic agent to an individual in need thereof.

17. The use of claim 16, further comprising administering a second therapeutic agent.

18. The use of claim 16, wherein the nanoparticles preferentially deliver the cargo to cells in the liver.

19. The use according to claim 18, wherein the liver cells include Kupffer cells and endothelial cells.

20. Use of the lipid nanoparticle composition of any one of claims 1-14 in the preparation of a medicament for preventing or treating a liver disease in an individual in need thereof, wherein the medicament is administered to the individual in an amount effective to prevent or treat the liver disease.

21. The use of claim 20, wherein the amount of the lipid nanoparticle composition administered to the individual is 0.05 mg / kg.

22. The use according to claim 21, wherein the liver disease is selected from liver cancer, liver cirrhosis, non-alcoholic fatty liver disease and alcohol-related liver disease.

23. Use of the lipid nanoparticle composition of any one of claims 10-14 in the preparation of a medicament for delivering an effective amount of a therapeutic or prophylactic agent to Kupffer cells of an individual in need thereof.

24. Use of the lipid nanoparticle composition of any one of claims 10-14 in the preparation of a medicament for delivering an effective amount of a therapeutic or prophylactic agent to liver endothelial cells of an individual in need thereof.

25. Use of the lipid nanoparticle composition of any one of claims 10-14 in the preparation of a medicament for delivering an effective amount of a therapeutic or prophylactic agent to the immune cells of an individual in need thereof.

26. A lipid nanoparticle composition comprising: Ionizable lipids, Phospholipids, Polyethylene glycol (PEG), and 20α-Hydroxycholesterol.

27. A lipid nanoparticle composition comprising: Ionizable lipids, Phospholipids, Polyethylene glycol (PEG), and 25-Hydroxycholesterol.

28. A lipid nanoparticle composition comprising: Ionizable lipids, Phospholipids, Polyethylene glycol (PEG), and 27-Hydroxycholesterol.

29. A lipid nanoparticle composition comprising: Ionizable lipids, Phospholipids, Polyethylene glycol (PEG), and 7β-Hydroxycholesterol.

30. A lipid nanoparticle composition comprising: cKK-E12, DOPE, C 18 PEG 2000 and 20α-Hydroxycholesterol.

31. A lipid nanoparticle composition comprising: cKK-E12, DOPE, C 18 PEG 2000 and 25-Hydroxycholesterol.

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