Inhibition of endosomal escape detection to ameliorate the inflammatory side-effects of lipid nanoparticles

Abstract

Composition and methods for preventing or reducing inflammation resulting from nanoparticle administration to a subject are provided. In one embodiment a method of delivering an agent to a target organ of a subject, comprises administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.

Description

INHIBITION OF ENDOSOMAL ESCAPE DETECTION TO AMELIORATE THE INFLAMMATORY SIDE-EFFECTS OF LIPID NANOPARTICLESSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under HL 153510, HL160694, AH 66778 and HL 164594 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTINGThe sequence listing file, entitled “23-10450. PCT”, created December 12, 2024, being 7 kb is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTIONLipid nanoparticles (LNPs) emerged as a prominent drug delivery system for nucleic acid therapeutics and have paved the way for advanced vaccinations such as those against SARS-CoV-2 which historically received Emergency Use Authorization in 2020. In addition to vaccines, LNPs offer cell-type-specificity of expression or knockdown for a wide variety of target proteins. In 2018, the FDA approved the first LNP nucleic acid- loaded therapeutic (Partisiran; Alnylam, Inc) for the treatment of hereditary transthyretin- mediated amyloidosis.While LNPs are considered relatively safe carriers for RNA, several studies have elucidated the inflammatory side effects of RNA-LNPs under naive conditions whether administered intravenously, intradermally, intranasally, or intramuscularly. We have additionally demonstrated that this inflammation is amplified when LNPs are administered in models of pre-existing inflammation. While this immune activation could be advantageous for vaccines due to added adjuvanticity, it can be detrimental in other indications. These inflammatory side-effects therefore limit the use of RNA-LNP therapeutics in any condition with inflammation such as ARDS, heart attack, and stroke, and in the background of comorbid infection and inflammation. Nucleic acid-loaded LNPs may have a powerful effect in the critical care space to treat such diseases since the short time course of expression for LNP-delivered mRNA (~48 hours) is advantageous duringacute illness. There is therefore a need to identify the signaling pathways that contribute to LNP -induced inflammation and determine the optimal therapeutics to ameliorate this effect to maximize their therapeutic potential.There remains a need in the art for improved nanoparticle compositions and methods of effectively targeting nanoparticles to various target organs.SUMMARY OF THE INVENTIONIn one aspect, provided herein is a method of delivering an agent to a target organ of a subject. The method includes administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP) or liposome. In certain embodiments, the nanoparticle comprises the galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface. In certain embodiments, the nanoparticle comprises the galectin inhibitor encapsulated therein. In certain embodiments, the galectin inhibitor is an inhibitor of galectin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and / or 15.In another aspect, a composition is provided that includes a nanoparticle comprising an agent and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.In another aspect, a composition is provided that includes a nanoparticle comprising a galectin inhibitor encapsulated therein.In another aspect, a method of delivering an agent to a target organ of a subject is provided. The method includes administering to the subject a galectin inhibitor and a nanoparticle as described herein, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In yet another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and / or preservative, and a nanoparticle comprising an agent and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.In yet another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and / or preservative, and a nanoparticle comprising a galectin inhibitor encapsulated therein.In another aspect, a method of delivering an agent to a target organ of a subject is provided. The method includes administering to the subject a gal ectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In another aspect, provided herein is a method of delivering an agent to a target organ of a subject. The method includes administering to the subject an NFKB inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented. In certain embodiments, the nanoparticle is a lipid nanoparticle (LNP) or liposome. In certain embodiments, the nanoparticle comprises the galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface. In certain embodiments, the nanoparticle comprises the galectin inhibitor encapsulated therein.In another aspect, a composition is provided that includes a nanoparticle comprising an agent and an NFKB inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.In another aspect, a composition is provided that includes a nanoparticle comprising an NFKB inhibitor encapsulated therein.In another aspect, a method of delivering an agent to a target organ of a subject is provided. The method includes administering to the subject an NFKB inhibitor and a nanoparticle as described herein, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In yet another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and / or preservative, and a nanoparticle comprising an agent and an NFKB inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.In yet another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and / or preservative, and a nanoparticle comprising an NFKB inhibitor encapsulated therein.In another aspect, a method of delivering an agent to a target organ of a subject is provided. The method includes administering to the subject an NFKB inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In yet another aspect, a method of delivering an agent to a target organ of a subject is provided. In certain embodiments, the method includes administering to the subject an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3, and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In another aspect, a method of delivering an agent to a target organ of a subject is provided. In certain embodiments, the method includes administering to the subject an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3, and a nanoparticle comprising the agent and an siRNA against NFkB, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.In yet another aspect, a nanoparticle is provided, comprising an agent and an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3 conjugated to a lipid component of the nanoparticle’s outer surface. In yet another aspect, a nanoparticle is provided comprising an agent and an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3 encapsulated therein. In yet another aspect, a nanoparticle is provided formulated with an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3and encapsulating an agent. In certain embodiments, the nanoparticle further comprises an NFkB siRNA encapsulated therein.In one aspect, a method of treating ARDS in a subject in need thereof is provided. In certain embodiments, the method includes administering to the subject a galectin inhibitor and a nanoparticle comprising mRNA encoding IL- 10. In certain embodiments, the nanoparticle comprises an outer surface layer comprising an ESCRT-recruiting cationic lipid. In certain embodiments, the galectin inhibitor is Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, P-D-lactosyl-steroid, thiodigalactoside (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)-modified peptide, DB16, DB21, OTX008, PTX013, anti-galectin antibodies, galectin-specific aptamers, siRNA and shRNA-coding vectors, LLS30, or LLS2.In another aspect, a nanoparticle comprising an mRNA encoding IL-10 and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface is provided. In another aspect, a pharmaceutical composition is provided that contains thenanoparticle and a pharmaceutically acceptable carrier, excipient and / or preservative.In another aspect, a method of treating ARDS in a subject in need thereof is provided, the method comprising administering to the subject a nanoparticle comprising an outer surface layer comprising an ESCRT -recruiting cationic lipid, the nanoparticle comprising mRNA encoding IL- 10.In another aspect, a nanoparticle is provided that comprises an outer surface layer comprising an ESCRT -recruiting cationic lipid, the nanoparticle comprising mRNA encoding IL-10. In another aspect, a pharmaceutical composition is provided that contains the nanoparticle and a pharmaceutically acceptable carrier, excipient and / or preservative.In yet another aspect, a method of delivering an agent to a target organ of a subject is provided. In certain embodiments, the method comprises administering to the subject a nanoparticle comprising the agent, wherein the nanoparticle comprises an ESCRT - recruiting cationic lipid, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented. In certain embodiments, the method further includes administering a galectin inhibitor.In another aspect, a nanoparticle comprising an agent, an ESCRT-recruiting cationic lipid, and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface is provided.In yet another aspect, a method of delivering an agent to a target organ of a subject is provided. In certain embodiments, comprises administering to the subject a galectin inhibitor and the nanoparticle as described herein, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.Still other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.BRIEF DESCRIPTION OF THE DRAWINGSFIGs. 1 A-1E show that thiodigalactoside (TG) pre-treatment ameliorates mRNA lipid nanoparticle (LNP) induced inflammation in vitro, and in vivo when administered intratracheally (IT), and intravenously (IV). (A) In in vitro RAW 264.7 macrophages, thiodigalactoside pre-treatment attenuates the production of the pro-inflammatory cytokines IL-6, TNFa, IL- la, and the chemokine MCP-1. (B) When TG and LNPs are administered IV into mice, TG pre-treatment significantly reduces the plasmaconcentrations of IL-6, TNFa, IL- la, and MCP-1. (C) TG pre-treatment reduces the concentrations of IL-6, TNFa, and IL-la in the bronchoalveolar lavage (BAL) fluid when TG and LNPs are administered IT. (D) IV TG pre-treatment reduces the white blood cell (WBC) and neutrophil (NEU) count in whole blood. IT TG treatment reduces the BAL protein concentration (E) and leukocyte count in the BAL which are measures of lung capillary leak and leukocyte infiltration. In all experiments, TG was administered 1 hour before LNP treatment. In vitro, cytokines were measured 6 hours after LNP administration. In vivo, cytokines and BAL protein and leukocyte levels were measured 2 hours after LNP treatment while blood count was measured 6 hours after LNP treatment.FIGs. 2A-2E show that TG pre-treatment has positive effects on mRNA expression. (A) In in vitro RAW 264.7 macrophages, TG pre-treatment increases luciferase mRNA expression by > 2-fold. TG pre-treatment does not impede luciferase mRNA expression in the (B) lung and improves expression in the (C) liver and (D) spleen when TG and LNPs are injected IV. Similarly, expression in the (E) lung is not attenuated when TG and LNPs are administered IT. In all experiments, TG was administered 1 hour before LNPs, and luminescence measurements were taken 6 hours after LNP treatment.FIG. 3A is a cartoon of an active loading scheme we tried for JSH, as described in Example 2. FIG. 3B is a cartoon of aqueous vs lipid loading of the small molecule drugs into LNPs. FIG. 3C is a a graph showing calculated concentration of celastrol by UPLC measurement vs mole drug to lipid ratio loading for aqueous versus organic (lipid) loaded celastrol. FIG. 3D is a graph showing encapsulation efficiency of celastrol in the aqueous versus lipid phaseFIG. 4A is a graph showing luminescence measuring expression of the luciferase mRNA loaded into the LNPs using the previous loading method. 1 : 1 N50:mRNA complexation by weight to weight leads to a nearly 100 fold decrease in mRNA expression. FIG. 4B is a graph showing luminescence measuring expression of the luciferase mRNA loaded into the LNPs using the new method. 1 :0.25 N50:mRNA complexation with the remaining 0.75 mRNA being added in directly prior to LNP formation bring expression almost back to that of just mRNA loaded LNPs. To mitigate the loss of mRNA expression, the positively charged peptides were complexed with 1 / 4 of the mRNA for ten minutes and just before LNP synthesis the remainder of the mRNA wasadded leaving uncomplexed mRNA still able to express back to the level of mRNA alone LNPs.FIGs. 5A-5F show the results of an initial drug screening. FIGs. 5A-5D show cytokine production of free drug and 98N12 mRNA cotreatments on RAW macrophages FIG. 5A shows IL-6 heatmap. FIG. 5B shows TNFa heatmap. FIG. 5C shows IFN-b heatmap. FIG. 5D shows IL1 a heatmap. FIG. 5E is a cartoon of a loaded LNP. FIG. 5F is a graph showing drug loading encapsulation efficiency and loaded amount represented as mole drug to lipid ratio (D / L)FIGs. 6A-6P show the results of an initial drug screening. FIG. 6A-6B show cytokine production of macrophages treated with loaded drug in 98N12 mRNA LNPs. FIG. 6C-6D, 6I-6K show dose response for Carfilzomib loaded LNPs on macrophages. FIG. 6E-6H, 6L-6N show dose response for celastrol loaded LNPs on macrophages. FIG. 60 and 6P show celastrol loading is independent of concentration added.FIG. 7A shows drug loaded in the aqueous versus lipid phase of LNPs. FIG. 7B shows dose response of aqueous vs organic loaded celastrol on NFKB luciferase macrophage reporter line. FIG. 7C shows luminescence representing NFKB activation versus the encapsulated celastrol concentration calculated via UPLC.FIG. 8 A is a cartoon showing NFKB luciferase reporter line in RAW 264.7 Macrophages. FIG. 8B is a graph showing dose response of inflammatory 98N12 LNPs loaded with mCherry mRNA.FIGs. 9A-9J show the results of siRNA treatment on NFKB activation. FIG. 9A shows a treatment scheme for testing siRNA sml02 LNPs for p50 and p65 subunits of NFKB on NFKB macrophage reporter line luminescence representing NFKB activation at T=24 hours post siRNA treatment (9B), T=30 post 24 hours siRNA treatment and post 6 hours of 98N12 mCherry LNP treatment (9C), T=48 hours post siRNA treatment (9D), and T=54 post 24 hours siRNA treatment and post 6 hours of 98N12 mCherry LNP treatment (9E). FIG. 9F and 9G show that celastrol -loaded LNPs have a dose dependent affect on NFKB activity. FIG. 9H-9J show significant decrease of cytokine and chemokine expression in tissue after intradermal injection of LNPs containing celastrol.FIG. 10A shows a treatment scheme for ICH injury and VCAM targeted 4A3 siRNA LNPs. FIG. 1 OB is a graph showing the results of a foot fault test for mousebehavior. Left foot being the unaffected foot as it’s on the same side as the ICH injury and right foot being the affected foot.FIGs. 11 A-l IN demonstrate that LNPs induce inflammation in vitro in various cell types and in vivo across species when administered intratracheally (IT) or intravenously (IV). (A) Gross anatomical image of lungs from mice intratracheally instilled with 7.5pg of mRNA in cKK-E12 LNPs per mouse. LNPs were instilled intratracheally into mice and lungs were harvested 24h after. Effect of the dose of intratracheally instilled cKK-E12 LNPs on (B) protein levels and (C) leukocyte count in the bronchoalveolar lavage (BAL) fluid 24 post-LNP instillation. LNPs increase BAL protein and leukocytes in a dosedependent manner. (D) Gross anatomical image of ex vivo pig lungs instilled with cKK- E12 LNPs. LNPs or saline were incubated in the lungs for 3h at 37°C at a dose of 0.8mg of mRNA in LNPs. Compared to control ex vivo pig lungs instilled with saline, the BAL fluid extracted from LNP -treated lungs shows increased levels of (E) protein, (F) leukocytes, (G) IL-6, and (H) TNF-a. (I) Multiplex analysis of pro-inflammatory, antiinflammatory, and regulatory cytokine and chemokine concentrations (IL- la, IL-ip, IL-6, TNF-a, IL-17A, IFN-y, GM-CSF, IL-10, IL-27, IL-12p70, IL-23, IFN- , MCP-1) induced in the BAL fluid by intratracheally instilled cKK-E12 LNPs. LNPs were intratracheally instilled at a dose of 7.5pg and BAL fluid was extracted 2h later. (J) Cytokine and chemokine concentrations in the plasma after intravenous injection of cKK-E12 LNPs. LNPs were injected at a dose of 7.5pg of mRNA in LNPs per mouse and allowed to circulate for 2h. (K) Cytokine and chemokine concentrations induced in RAW 264.7 macrophages by cKK-E12 mRNA LNPs, cKK-E12 empty LNPs (formulated with no cargo), and cKK-E12 LNPs formulated with a negatively charged polymer, polystyrene sulfonate (PSS). Cells were treated with LNPs for 6h and the supernatant was collected for multiplex analysis. (L) Effect of cKK-E12 LNP dose on cell viability after 24h in RAW 264.7 macrophages and MLE-12 epithelial cells. Fraction of cell death induced by apoptosis, necrosis, or pyroptosis in (M) control RAW 264.7 macrophages and (N) RAW 264.7 macrophages treated with 400ng / ml of mRNA in LNPs 24h after treatment. After LNP treatment, cells were isolated and stained with various markers for apoptosis, necrosis, and pyroptosis for flow cytometry analysis.FIGs. 12A-12N demonstrate LNP-Induced inflammation but also expression of RNA cargo has a positive correlation with ionizable lipid endosomal escape capability.(A) Schematic of hypothesis: LNPs formulated with mild ionizable lipids have less efficient endosomal escape leading to lower mRNA expression and lower inflammation. LNPs formulated with potent ionizable lipids have the reverse effect. (B) Luciferase expression in RAW macrophages of 15 LNPs formulated with 15 different ionizable lipids 6h after treatment. The highest expressing ILs cKK-E12 and 4A3-SC8 (red bar) expressed >400-fold and >700-fold more than the lowest expressing IL 93-O17S. The LNP luciferase expression profile in vivo in the (C) liver and (D) spleen 6h after intravenous injection into mice follows the in vitro trend. Dlin-MC3-DMA LNPs< SM-102 LNPs < cKK-E12 LNPs < 4A3-SC8 LNPs. (E) Similarly, after intratracheal administration, 4A3- SC8 LNPs have a 3-fold higher luciferase expression in the lung than cKK-E12 LNPs 6h after intratracheal administration. In vitro (F) IL-6 and (G) TNF-a concentrations 6h after treatment with the 15 ionizable lipid LNP formulations in (B). In vitro (H) IL-6 and (I) TNF-a concentrations have a positive correlation with luciferase expression. LNPs formulated with the highest expressing ionizable lipids (cKK-E12, C 12-200, 98N12-5) are also the most inflammatory except for 4A3-SC8 which does not increase cytokine levels above control levels. This is illustrated in the linear regression fits excluding 4A3-SC8 LNPs (black trendlines, (H) R2 = 0.8662 and (I) R2 = 0.7703) versus that including 4A3- SC8 LNPs (red trendlines, (H) R2 = 0.3314 and (I) R2 = 0.2933). After IV LNP injection, the plasma concentrations of (J) IL-6 and (K) TNF-a follow the trend Dlin-MC3-DMA LNPs< SM-102 LNPs < cKK-E12 LNPs in line with the luciferase expression trend, except 4A3-SC8 LNPs which does not cause significant cytokine upregulation. Plasma was extracted 2h after LNP treatment. 6h after treatment of in vitro RAW macrophages with LNPs, (L) Dlin-MC3-DMA LNPs, which have low mRNA expression, do not increase pro-inflammatory cytokine (IL-la, IL-6, TNF-a, IL-27, IFN-P, MCP-1) concentrations above control levels, (M) cKK-E12 LNPs, which have high mRNA expression, significantly upregulate pro-inflammatory cytokines, and (N) high-expressing 4A3-SC8 LNPs do not upregulate cytokine levels above control.FIGs. 13A-13L demonstrate that escape of RNA payload from endosomes proportionally induces endosomal damage. (A) At 0.5h, Ih, and 6h post-treatment in RAW macrophages, LNPs are colocalized with endosomes. (B) Pearson’s coefficient values from (A). (C) Schematic showing the payload release from small and large endosomal ruptures. Small endosomal ruptures are only permeable to protons or lowmolecular weight molecules such as Acridine Orange (AO) while large endosomal ruptures additionally allow for the permeation of high molecular weight material such as mRNA. Control or cKK-E12 LNP -treated (D) RAW macrophages or (E) A549s were treated with AO which emits red fluorescence in acidic endosomes and green fluorescence in the nucleus or cytosol. LNP pre-treatment increases the green MFI and decreases the red MFI of AO in both cell types. (F) Fraction of intact and ruptured endosomes in control RAW macrophages or those treated with LNPs or the endosomal rupture agent LLOME, calculated by obtaining the ratio of the red MFI values in (C) to the green MFI values. While almost 100% of endosomes are intact in control cells, LNPs and LLOME rupture >50% and >90% of endosomes respectively. (G) Fraction of ruptured endosomes after treatment with 11 ionizable lipid LNP formulations or LLOME. (H) Schematic of the potential outcomes of damaged endosomes after LNP endocytosis. Large endosomal ruptures primarily recruit the sugar-binding proteins galectins which facilitate lysophagy and degradation. Small endosomal damages primarily recruit the ESCRT machinery which promote repair. RAW macrophages were treated LLOME or with LNPs formulated with MC3, 4A3-SC8, or cKK-E12, and stained with (I) Galectin-9 or (J) ALIX. The number of (K) Galectin-9 or (L) ALIX puncta per cell per image from (I) and (J). cKK-E12 LNPs lead to the highest levels of galectin recruitment while 4A3-SC8 LNPs most significantly recruit the ESCRT machinery.FIGs. 14A-14F Inhibition of endosomal escape detection with the galectin inhibitor thiodigalactoside (TG) ameliorates mRNA lipid nanoparticle (LNP) induced inflammation in vitro, and in vivo when administered intratracheally (IT), and intravenously (IV). (A) TG pre-treatment in RAW macrophages significantly reduces the number of galectin-9 puncta. (B) In in vitro RAW 264.7 macrophages, thiodigalactoside pre-treatment attenuates the production of the pro-inflammatory cytokines IL-6, TNFa, IL- la, and the chemokine MCP-1. (C) When TG and LNPs are administered IV into mice, TG pretreatment significantly reduces the plasma concentrations of IL-6, TNFa, IL- la, and MCP-1 and (D) reduces the white blood cell (WBC) and neutrophil (NEU) count in the plasma to control levels. (E) When TG and LNPs are administered IT into mice, TG pretreatment reduces the concentrations of IL-6, TNFa, and IL- la in the bronchoalveolar lavage (BAL) fluid and (F) BAL leukocyte count which is a measure of leukocyte infiltration. In all experiments, TG was administered 1 hour before LNP treatment. Invitro, cytokines were measured 6 hours after LNP administration. In vivo, cytokines and BAL protein and leukocyte levels were measured 2 hours after LNP treatment while blood count was measured 6 hours after LNP treatment.FIGs. 15A-15H demonstrate that inhibition of endosomal escape detection has positive effects on mRNA expression. (A) In in vitro RAW 264.7 macrophages, TG pretreatment increases luciferase mRNA expression by > 2-fold. (B) TG pre-treatment does not attenuate expression in the lung when TG and LNPs are administered IT. TG pretreatment improves expression in the (C) liver and spleen when TG and LNPs are injected IV. In all experiments, TG was administered 1 hour before LNPs, and luminescence measurements were taken 6 hours after LNP treatment. (D) Timeline of treatments. (E) IL- 10 4A3-SC8 and TG + IL-10 cKK-E12 LNPs induced upregulation of IL-10 by ~40-fold and ~60-fold respectively in the BAL fluid. IL-10 4A3-SC8 LNPs and TG + IL-10 cKK- E12 LNPs completely abrogated (F) leukocyte infiltration (BAL leukocyte count) and (G) capillary leak into the alveolar space (BAL protein levels). (H) Summary schematic showing the two strategies for ameliorating LNP-induced inflammation while maintaining high mRNA expression namely utilizing ESCRT -recruiting ionizable lipids and inhibiting galectins.FIGs. 16A-16D demonstrate that cKK-E12 PECAM LNPs exacerbate inflammation in a nebulized-LPS model of ARDS. cKK-E12 PECAM LNPs dose- dependently (FIG. 16A) increase BAL protein levels, (FIG. 16B) decrease blood platelet count, and (FIG. 16C) decrease blood lymphocyte count and increase neutrophil count. (FIG. 16D) cKK-E12 PECAM LNPs increase the plasma levels of pro-inflammatory cytokines by up to three orders of magnitude compared to nebulized-LPS controls. For A- C, LNPs were injected intravenously 2 hours after nebulized-LPS injury and mice were sacrificed 22 hours after LNP administration. For D, LNPs were injected intravenously 2 hours after nebulized-LPS injury and mice were sacrificed 2 hours after LNP administration.FIGs. 17A-17D demonstrate that various inhibitors failed to ameliorate exacerbated inflammation induced by LNPs in models of inflammatory lung pathology. (FIG. 17A) Dexamethasone palmitate loaded into LNPs, (FIG. 17B) 15-minute pretreatment with MCC950 (lOmg / kg), (FIG. 17C) 2-hour pre-treatment with IL-IRa (lOpg / mouse), and (FIG. 17D) 2-hour pre-treatment with IL-6 antibodies (lOOpg / mouse) didnot significantly ameliorate the exacerbated capillary leak into the alveolar space (measured by BAL protein levels), lymphopenia, and neutrophilia induced by LNPs. FIG. 17A, C and D were carried out in the nebulized-LPS model while B was conducted in a hyperoxia model of ARDS achieved by exposing mice to >90% oxygen for 72 hours. For nebulized LPS experiments, LNPs were injected intravenously 2 hours post injury and mice were sacrificed 22 hours after LNP administration. For hyperoxia experiments, LNPs were administered after the 72-hour hyperoxia injury period for a circulation time of 24 hours while mice were maintained under hyperoxia.FIGs. 18A-18F demonstrate that non-inflammatory LNPs loaded with IL- 10 ameliorate mouse ARDS phenotypes. (FIG. 18 A) Timeline of treatments. (FIG. 18B) IL- 10 4A3-SC8 and TG + IL-10 cKK-E12 LNPs induced upregulation of IL-10 by ~40-fold and ~60-fold respectively in the BAL fluid. (FIG. 18C) IL-10 4A3-SC8 LNPs and TG + IL-10 cKK-E12 LNPs completely abrogated (FIG. 18C) leukocyte infiltration (BAL leukocyte count) and (FIG. 18D) capillary leak into the alveolar space (BAL protein levels). (FIG. 18E) IL-10 4A3-SC8 LNPs and (FIG. 18F) IL-10 cKK-E12 LNPs reduce the BAL concentration of pro-inflammatory cytokines.FIGs. 19A-19B demonstrate that TG pre-treatment does not affect ARDS phenotypes. TG administered in the nebulized LPS model does not affect (FIG. 19A) BAL protein levels and (FIG. 19B) BAL leukocyte count. TG was injected intravenously into mice at a dose of lOOpg / mouse 1 hour before nebulized LPS injury and mice were sacrificed 4 hours after injury.FIGs. 20A-20E demonstrate dose-dependent toxicity of intratracheally administered LNPs. cKK-E12 LNPs were instilled intratracheally into mice at doses of 2.5 pg - lOpg of mRNA in LNPs and 24 hours later, the blood and BAL fluid was harvested and analyzed. In mice, cKK-E12 LNPs dose-dependently (FIG. 20A) Induce weight loss, (FIG. 20B) decrease white blood cell count, (FIG. 20C) increase neutrophil count, (FIG. 20D) decrease lymphocyte count, and (FIG. 20E) decrease platelet count.FIGs. 21 A-21B demonstrate that cytokines are upregulated by intratracheally administered LNPs and LNPs in MLE-12 cells. cKK-E12 LNPs were instilled intratracheally into mice at a of 7.5pg of mRNA in LNPs and 2 hours later, the cytokines in the BAL fluid were analyzed and compared to BAL cytokine levels of mice administered with nebulized-LPS 4 hours post-injury (peak of cytokine expression) (FIG.21 A). Mice that had been instilled with LNPs had generally higher cytokine levels in the BAL compared to nebulized-LPS mice. MLE-12 cells were treated with cKK-E12 LNPs and supernatant was analyzed 4 hours later (FIG. 2 IB). LNP treatment did not increase cytokine levels above control levels although severe MLE-12 cell death was observed.FIGs. 22A-22D demonstrate LNP uptake in A549GFP1-10 and RAW264.7 cells. To evaluate in vitro LNP uptake, A549GFP1-10 and RAW264.7 cells were treated with 18: 1 TopFluor PE AF594-labeled LNPs (400 ng / mL, 6h). (A) Histogram and mean fluorescence intensity of LNP -treated A549GFP1-10 cells. (B) Relative ratio of LNP - intemalized cell populations. (C) LNP MFI and (D) uptake % in RAW264.7 cells 6 hours post LNP incubation.FIGs. 23A-23F demonstrate cell viability in RAW264.7 cells. To evaluate in vitro LNP toxicity, RAW264.7 cells were treated with either MC3, SM-102, 4A3-SC8, or cKK- E12 LNPs (A-D) or LLOMe, a lysosomotropic agent (E, F). After 6 hours, cck8 assay was performed to determine cell viability relative to untreated control. cKK-E12 LNPs led to the most significant levels of cell death amongst the LNP formulations tested.FIGs. 24A-24B demonstrate cell viability in A549 cells. To evaluate in vitro LNP toxicity, A549 cells were treated with either (A) cKK-E12 or (B) 4A3-SC8 LNPs. After 6 hours, cck8 assay was performed to determine cell viability relative to untreated control. cKK-E12 LNPs led to the most significant levels of cell death amongst the LNP formulations tested.FIGs. 25A-25F demonstrate that LNP-Induced inflammation but also expression of RNA cargo has a positive correlation with ionizable lipid endosomal escape capability. (A) IL- la and (B) MCP-1 concentrations 6h after treatment with 15 ionizable lipid LNP formulations in RAW macrophages. In vitro (C) IL-1 a and (D) MCP-1 concentrations have a positive correlation with luciferase expression. However, 4A3-SC8 LNPs do not increase cytokine levels above control. Graphs show the linear regression fits excluding 4A3-SC8 LNPs (black trendlines, (C) R2 = 0.7711 and (D) R2 = 0.8190) versus that including 4A3-SC8 LNPs (red trendlines, (C) R2 = 0.2726 and (D) R2 = 0.3318). After IV LNP injection, the plasma concentrations of (E) IL-la and (F) MCP-1 follow the trend Dlin-MC3-DMA LNPs< SM-102 LNPs < cKK-E12 LNPs in line with the luciferase expression trend, except 4A3-SC8 LNPs which does not cause significant cytokine upregulation.FIGs. 26A-26B demonstrate human monocyte-derived macrophage (hMDM) NF- KB response to LNP treatment. (A) Representative images of NF-KB nuclear translocation in hMDMs treated with vehicle (1 X PBS), cKK-E12 LNPs, 4A3-SC8 LNPs, or LPS (1 ng / mL) for 90 mins. Nuclei are stained with DAPI (blue), cytoplasm with cell mask deep red (CMDR, red). NF-KB p65 (green) is highlighted by white arrows. (B) Fold change in the nuclear translocation ratio (nuclear NF-kB / cytoplasmic NF-kB) in control and treated conditions. hMDM treated with lOOOng / mL cKK-E12 LNPs showed significantly increased NF-kB nuclear translocation while 4A3-SC8 LNPs did not alter NF-kB translocation compared to control levels.FIGs. 27A-27C demonstrate that LNP-Induced inflammation correlates with RNA expression with a library of lipidoids with one-carbon differences in tail structures. (A) 306 series lipidoids with different carbon tail lengths were synthesized as previously described31 and LNPs were formulated using designated mol fractions. (B) Luciferase expression of 306-06, 306-08, 306-09 and 306-011 LNPs in RAW 264.7 macrophages (400ng / ml mRNA in LNPs, 6 hours). (C) Luciferase expression from (B) correlates with TNF-a upregulation.FIGs. 28 demonstrates SM-102 LNP dose response in AW264.7 cells. To evaluate the extent of SM-102 LNP inflammation, RAW264.7 cells were treated with various doses of SM-102 LNPs (400-3000 ng / mL). 6 hours post treatment, supernatant was collected and used for cytokine quantification.FIGs. 29A-29E demonstrate LLOME-induced cytokine expression in RAW264.7 cells. To evaluate the extent of inflammation induced by LLOME compared to cKK-E12 LNPs, RAW264.7 cells were treated with various doses of LLOME (0.01063-6.8mM) or cKK-E12 LNPs (400ng / ml). 6 hours post-treatment, supernatant was collected and used for cytokine quantification. LLOME upregulates mostly the same cytokines as cKK-E12 LNPs.FIGs. 30A-30C demonstrate colocalization of LNP and endo-lysosomes. Cells were treated with 18: 1 TopFluor PE AF594-labeled cKK-E12 LNPs (400 ng / mL) for different durations. (A-B) Representative confocal images of endocytosed LNP in A549GFP1-10 cells (A) and Pearson’s coefficients (B). (C) Representative confocal images in Raw264.7 cells. Endo-lysosomes were labeled with LysoTracker DeepRed.Nuclei were stained by Hoechst. Line scans show the colocalization of LNPs (green) and endo-lysosomes (red) by pixel. Scale bars: 10 pm (A) and 5 pm (C).FIGs. 31A-31B show acridine orange imaging in Raw264.7 cells. (A) Representative confocal images of LNP -treated cells. Scale bar: 20 pm. (B) Quantitative analysis of endo-lysosome damage. Data were normalized to control group. Red = acridine orange in endosomes and green = acridine orange in nucleus / cytosol.FIGs. 32A-32B demonstrate time-dependent endo-lysosome damage in LNP- treated Raw264.7 cells. (A) Fraction of intact endolysosomes and (B) ruptured endolysosomes after treatment with different LNP formulations at different timepoints. After LNP treatment, cells were stained with acridine orange to test endo-lyososome acidic changes by flow cytometry. Data were normalized to control group.FIGs. 33A-33C demonstrate endosomal escape efficiency measured using a split- GFP assay. A549 GFP1-10 cells were used as split-GFP reporters, which express a nonfluorescent cytosolic fragment of the GFP chromophore (GFPl-lO)while the other non-fluorescent peptide fragment SI 1 was loaded into LNPs. Once LNP -loaded SI 1 is released from endosomes into the cytosol, the complementation of SI 1 and GFP1-10 leads to GFP fluorescence. (A) Encapsulated split-GFP in LNP was confirmed by running LNPs through a size exclusion chromatograph. A549GFP1-10 cells were treated with free SI 1 or SI 1 -loaded LNPs for 6 h and then collected for flow cytometry. (B) GFP MFI and (C) fraction of GFP positive cells after treatment with free SI 1, lipofectamine+Sl 1, LLOME + Si l, and LNP-loaded SI L Endosomal escape efficiency generally follows the trend MC3 LNPs < C12-200 LNPs < SM-102 LNPs < cKK-E12 LNPs < 4A3-SC8 LNPs.FIGs. 34 demonstrates galectin recruitment in A549GFP1-10 cells. After treated with 4A3-SC8 and ckk-E12 LNPs, the intracellular recruitment of gal-1, gal-3, gal-8 and gal-9 were imaged by immunofluorescence and puncta formation was quantified by Imaged. LLOMe was used as a positive control to induce severe endolysosome damage. Scale bar: 10 pm.FIGs. 35A-B show Alix puncta in Raw264.7 cells. (A) Confocal images and line scan of ALIX puncta in LNP -treated cells. Scale bar: 10 pm. 4A3-SC8 LNP -treated cells have significantly more ALIX puncta than cKK-E12 LNP -treated cells. (B) The number of ALIX puncta per cell per image 0.5h, Ih, and 6h post-treatment with 4A3-SC8 or cKK- E12 LNPs.FIGs. 36A-36F demonstrate TG pre-treatment ameliorates LNP-induced inflammation. (A) TG treatment does not affect cell viability in MLE-12 cells after 24 hours at doses ranging from lOpg / ml -2500pg / ml. (B) TG pre-treatment reduces the plasma IL-6 levels induced by C12-200 LNPs. TG was intravenously injected 1 hour before C 12-200 LNPs and LNPs were allowed to circulate for 2 hours. (C) Intratracheal TG reduces BAL leukocyte levels at longer timepoints. TG was instilled 1 hour before cKK-E12 LNP intratracheal instillation and BAL was harvested 24 hours later LNPs. At different doses, 1 hour TG pre-treatment reduces pro-inflammatory cytokine levels upregulated by LNPs (D) intravenously (7.5 pg mRNA in LNP / mouse, 2h), (E) intratracheally (7.5 g mRNA in LNP / mouse, 2h), and (F) in RAW macrophages (400ng / ml LNP, 6h).FIGS. 37A-37D demonstrate that cKK-E12 PEC AM LNPs exacerbate inflammation in a nebulized-LPS model of ARDS. In a mouse model of ARDS achieved by administering nebulized-LPS, cKK-E12 LNPs, conjugated to Platelet Endothelial Cell Adhesion Molecule (PEC AM) antibodies to target the lung, dose-dependently (A) increase BAL protein levels, (B) decrease blood platelet count, and (C) decrease blood lymphocyte count and increase neutrophil count. (D) cKK-E12 PEC AM LNPs increase the plasma levels of pro-inflammatory cytokines by up to three orders of magnitude compared to nebulized-LPS vehicle controls. For A-C, LNPs were injected intravenously 2 hours after nebulized-LPS injury and mice were sacrificed 22 hours after LNP administration. For D, LNPs were injected intravenously 2 hours after nebulized-LPS injury and mice were sacrificed 2 hours after LNP administration. Abbrv: white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and neutrophil (NEU).FIGs. 38A-38D demonstrate that various inhibitors failed to ameliorate exacerbated inflammation induced by LNPs in models of inflammatory lung pathology. (A) Dexamethasone palmitate loaded into LNPs, (B) 15-minute pre-treatment with MCC950 (lOmg / kg), (C) 2-hour pre-treatment with IL-IRa (lOpg / mouse), and (D) 2- hour pre-treatment with IL-6 antibodies (lOOpg / mouse) did not significantly amerliorate the exacerbated capillary leak into the alveolar space (measured by BAL protein levels), lymphopenia, and neutrophilia induced by LNPs. A, C and D were carried out in the nebulized-LPS model while B was conducted in a hyperoxia model of ARDS achieved by exposing mice to >90% oxygen for 72 hours. For nebulized LPS experiments, LNPs wereinjected intravenously 2 hours post injury and mice were sacrificed 22 hours after LNP administration. For hyperoxia experiments, LNPs were administered after the 72-hour hyperoxia injury period for a circulation time of 24 hours during which mice were maintained under hyperoxia. Abbrv: white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and neutrophil (NEU).FIGs. 39A-39D demonstrate that LNP-induced inflammation exacerbation persists in various knockout mice. In LNP -treated ASC knockout mice injured with nebulized- LPS, (A) BAL protein and (B) BAL leukocyte levels are not changed and remain elevated compared to wild - type mice injected with LNPs. (C,D) Nebulized- LPS-injured, CCR2 and caspase-3 knockout mice have similarly elevated BAL protein and leukocyte levels as wildtype mice following LNP injection. MyD88 knockout mice show reduced BAL leukocyte levels compared to wild-type LNP - injected mice and vehicle control mice (nebulized -LPS only). However, MyD88 is vital in the LPS signaling pathway. In all experiments, nebulized-LPS was administered to mice and 4 hours later, PECAM-cKK- E12 LNPs were injected for a circulation period of 20 hours.FIGs. 40A-40B show IL-10 mRNA-targeted LNPs in Neb-LPS model of ARDS. To assess the therapeutic efficacy of IL-10 mRNA-LNPs, nebulized LPS was administered to mice along with 7.5pg of mRNA in LNPs of mRNA in LNPs. LNPs were surface- modified with PECAM targeting antibody and administered intravenously. (A) IL-10 4A3- SC8 LNPs and (B) TG+ IL-10 cKK-E12 LNPs reduce the BAL concentration of pro- inflammatory cytokines. For cKK-E12 treatment group, lOOpg of thiodigalactoside (TG) was administered 1 hour prior to nebulization. 4 hours post nebulized LPS injury and LNP injection, BAL was collected and used for cytokine quantification.FIGs. 41 A-41B demonstrate that 4A3-SC8 PECAM LNPs loaded with IL-10 mRNA ameliorate ARDS phenotypes at longer timepoints. 4A3-SC8 LNPs loaded with IL-10 (7.5pg of mRNA in LNPs) and conjugated to PECAM (A) do not alter total white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and neutrophil (NEU) counts in the blood compared to control levels and (B) ameliorate leukocyte infiltration into the alveolar space 24 hours after intravenous injection. 4A3-SC8 PECAM LNPs loaded with a model luciferase cargo did not ameliorate ARDS phenotypes but did not exacerbate existing inflammation.FIGs. 42A-42B demonstrate that TG pre-treatment does not affect ARDS phenotypes. TG administered in the nebulized LPS model does not affect (A) BAL protein levels and (B) BAL leukocyte count. TG was injected intravenously into mice at a dose of lOOpg / mouse 1 hour before nebulized LPS injury and mice were sacrificed 4 hours after injury.FIG. 43 is a table showing ionizable lipid classification and LNP Size and PDI. All LNP formulations consisted of 50% ionizable lipid, 38.5% cholesterol, 10% DOPE, and 1.5% DMG-PEG2K. Ionizable lipids are categorized into 4 groups indicated by colored dots. Representative structures are also shown on the right.DETAILED DESCRIPTIONProvided herein are nanoparticle compositions and methods that reduce or prevent inflammation in a subject. As described herein, toxicity associated with delivery of cationic nanoparticles, particularly in the lungs, was not previously reported. The present inventors developed several approaches to avoid deleterious effects, including inflammation, resulting from nanoparticle delivery to a target organ.We hypothesized that the inflammatory responses of LNPs result from sensing of endosomal escape of nucleic acid cargo. In this process, LNP ionizable lipids facilitate the rupture of the endosome to release RNA into the cytosol for translation and expression. For this reason, we show that more potent ionizable lipids that have a higher RNA expression capacity are more inflammatory due to more severe endosomal damage. Cells sense endosomal damage with sugar-binding lectins, known as galectins, which detect the exposure of glycans to the cytoplasm upon membrane damage. Galectins facilitate cellular membrane damage responses which initiate and modulate downstream inflammatory effects. Specifically, it has been shown that galectins 1, 3, 8, and 9 are primarily recruited to the damaged endosome after LNP endosomal escape. Here, we demonstrate that treatment with the pan-galectin inhibitor thiodigalactoside ameliorates LNP -induced inflammation. When we administer intratracheally before treatment with RNA-LNPs, thiodigalactoside reduces protein and leukocyte amounts in the bronchoalveolar lavage of mice to negative control levels.LNPs induce strong pro-inflammatory cytokine and chemokine responses and increase inflammatory forms of cell death. The inflammation has been attributed to theendosomal escape of the RNA cargo, creating endosomal holes detected by the sugar binding proteins, galectins. While we have shown that inhibition of galectin reduces LNP associated inflammation, galectins are only the most upstream damage sensors and their inhibition is not completely efficacious. Endosomal damage also leads to a leakage of toxic components such as cathepsins, which triggers inflammasome activation and further downstream inflammation. Transcription factors such as NF-KB and STAT3 are also activated leading to the generation of proinflammatory cytokines. Thus, there still is a need to understand all these possible inflammation pathways activated by LNPs to completely attenuate LNP-associated inflammation.We tested various drugs that inhibit all the aforementioned pathways in vitro (RAW 264.7 Macrophages) using mRNA-LNPs formulated with the ionizable lipid 98N12-5. These inhibitors include mitogen-activated protein kinase kinase (MEK) inhibitors (Trametinib, U0126), caspase inhibitors (Wedel olactone, Z-VAD-OMe-FMK), galectin inhibitors (Olitigaltin, OTX008), cathepsin inhibitors (E64), etc. We also tested siRNAs against proinflammatory cytokines, and oligodeoxynucleotide (ODN) against various transcription factors such as NF-KB and STAT3.Out of all these potential candidates, we showed that drugs Trametinib, U0126, and Olitigaltin, significantly inhibited cytokine production while maintaining cell viability and mRNA expression. The inhibition of MEK with Trametinib decreased IL-6 production by fivefold and increased mRNA expression by two-fold, without a significant impact on cell viability. Another MEK inhibitor, U0126, had a tenfold decrease in IL-6 production and two-fold increase in mRNA expression. The inhibition of galectin with Olitigaltin also decreased IL-6 production by two-fold, while maintaining similar mRNA expression levels. These results have caused us to explore further MEK, caspase, inflammasome, galectin, cathepsin, and other inflammatory pathway inhibitors.We further demonstrate that NFKB is an excellent target to inhibit the carrier and cargo LNP associated inflammation due to its critical role in innate immune response. NFKB is a central mediator of pro-inflammatory gene induction including cytokines such as IFN-b and IL. Therefore, many different inflammation pathways result in the activation of NFKB, including pathways we have shown to be important in LAI such as galectin recognition after endosomal escape, inflammasome activation, and caspases. Additionally, NFKB is activated in case of carrier-based LAI. We have shown cGAS-STING pathway isthe major pathway activated in DNA-LNPs based inflammation. STING activation leads to downstream activation of NFKB and upregulation of proinflammatory cytokines such as IFN-b and IL-Therefore, NFKB is an excellent candidate to inhibit both carrier and cargo LAI.There are hundreds of NFKB inhibitors reported in literature; however, as with any immune system pathways, systemic inhibition can lead to dangerous side effects including greater susceptibility to infections. Therefore, it is critical to find NFKB inhibitors capable of being loaded into LNPs to reduce systemic immunosuppression.The research described herein is focused on solving inflammation associated with delivery of lipid nanoparticles. This inflammation can be classified into two types: LNP- associated inflammation (LAI), stemming from the LNP themselves, and cargo-associated inflammation, which is a particular concern with DNA cargoes.Described herein are compositions and methods that reduce or eliminate LAI. Among other benefits, these compositions and methods allow delivery of LNPs to “sensitive” patients otherwise incapable of handling the side effects of LNPs. A primary goal of our research is that the compositions will not induce systemic immunosuppression. By only delivering the inhibitor to cells where LNPs are being delivered it will allow for resolution of LAI without systemically suppressing the immune system. This is essential for patients especially those who have suffered from diseases or injuries that open them up to the threat of infections. Notably, for patients that suffer from strokes, nearly 1 / 3 of them develop pneumonia so any immunosuppression of the lungs can increase this likelihood. Therefore, it is necessary to use both quick acting and long term inhibitory methods of NFKB.Small molecules are useful for quick inhibition of their targets but have a few major drawbacks. First, small molecule drugs are infamous for being “dirty” meaning they inhibit other things outside of the target of interest. This can greatly increase off target side effects. Second, rapid clearance of small molecules means that it’s only possible to have short term inhibition of inflammation. While this provides a benefit for resolving LAI, for other therapeutic uses including stroke and other inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, long-term inhibition of inflammation is needed. Further, loading into LNPs is not straightforward with smallmolecule drugs. Every small molecule can have very different loading capabilities therefore the loading process has to be optimized for each drug individually.Since small molecules are not capable of long-term inhibition, we looked to other inhibitory methods to obtain this benefit. siRNAs are capable of long-term inhibition lasting up to 7 days; however, it takes a while for them to knockdown the protein of interest. In the case of proteins with low constitutive expression and rapid transcription upon a signal, siRNAs do not take long to act as they’re able to bind to mRNA and prevent transcription from taking place. For proteins that are constitutively expressed, it takes until the protein is degraded for them to become effective. In the case of NFKB its half-life is around 12-24 hours meaning it takes around 24 hours to begin to see an inhibitory impact on NFKB activation. Because of this delay in NFKB inhibition by siRNAs, it is not suited for quick inhibition of NFKB. A benefit to these siRNAs is that they are extremely specific for their target of interest, therefore they do not have the same off-target side effects associated with other inhibitory methods like small molecule inhibitors. In addition, siRNAs are incredibly easy to load into LNPs and varying the target for inhibition does not affect their loadability meaning the loading does not have to be reoptimized each time. siRNAs are also very well studied and so far there are six FDA approved siRNA therapeutics. This shows that they are safe for human therapeutics and makes it easier to get other siRNAs through the FDA in the future.Here, in one embodiment, we utilize small molecule inhibitor celastrol for quick acting inhibition and siRNAs for the p65 subunit in the NFKB complex for long term and specific inhibition. In one embodiment, the siRNA has the sequence GGGAUGAGAUCUUCUUGCU (SEQ ID NO: 1). Two candidates are described that are capable of loading into LNPs and decreasing LNP associated inflammation in vitro as measured by secreted cytokines. They are a small molecule inhibitor known as celastrol and an NFKB nuclear localization sequence binding peptide referred to as N50. Celastrol is a cholesterol mimetic capable of inhibiting NFKB activation through several different pathways including inhibiting IKK activation, inhibition of IKB degradation, and antioxidant activity. N50 is a nuclear localization sequence that when added into cells prevents the translocation of activated NFKB into the nucleus, therefore preventing the upregulation of pro-inflammatory cytokines. Here we show loading of N50 and celastrol do not impact mRNA loading or particle stability. We demonstrate that celastrol and N50ameliorate both carrier and cargo inflammatory effects. When treated in vitro (RAW 264.7 Macrophages) mRNA celastrol LNPs, mRNA N50 LNPs, DNA celastrol LNPs, and DNA N50 LNPs reduced secreted inflammatory cytokines (IFNb and IL6) back to baseline levels.We and others have shown that in addition to the inflammation induced by LNPs under naive conditions, LNPs exacerbate pre-existing inflammation by orders of magnitude, whether in the diseased organ or an organ with a comorbid inflammationl. This phenomenon makes the use of inflammatory LNPs prohibitive in patients with conditions such as ARDS, stroke, and heart attack which form a large fraction of potential LNP indications.We found a branched-tail, biodegradable ionizable lipid, 4A3-SC8, that potently expresses mRNA but does not induce inflammation. Our studies therefore revealed an ionizable lipid with desirable purposes for therapeutic applications of LNPs (high expresser but non-inflammatory).The primary sensors of large, severe endosomal ruptures (>100nm) are sugar- binding lectins, known as galectins, which detect the exposure of glycans to the cytosol upon membrane damagel4,15. Galectins then modulate downstream inflammatory responses resulting from the prolonged exposure of toxic endosomal content into the cytosol and facilitate the process of autophagy to degrade irreparably damaged organelles. When endosomal ruptures are small and less severe (<100nm), ESCRT (endosomal sorting complex required for transport) proteins are recruited to repair endolysosomal membranes by recognizing and sealing membrane disruptions. This limits inflammation by maintaining cellular homeostasis and preventing cytoplasmic leakagel6-19.We first found that LNPs formulated with multiple ionizable lipids - even the less potent ones - cause endosomal damage that is significant enough to cause endosomes to lose their pH gradient. We then showed that LNPs formulated with 4A3-SC8 create intermediate-sized holes that are repaired by ESCRT proteins and can potently express mRNA without inducing inflammation. Further, we demonstrated that inhibition of large endosomal damage sensing by inhibiting galectins ameliorates LNP-induced inflammation in vitro, and in vivo across multiple routes of delivery. Finally, we show that galectin inhibition or the use of LNPs designed to create ESCRT-recruiting endosomal holes are able to not only avoid LNP-induced exacerbation of inflammation, but when loaded with atherapeutic mRNA, the LNPs ameliorate a highly inflammatory disease model. These findings show that LNP -induced membrane damage both produces both RNA endosomal escape and inflammation, but such endosomal damage can be controlled to provide high- expressing, non-inflammatory LNPs.We also demonstrate that preventing the sensing of severe endosomal damage by inhibiting galectins ameliorates LNP -induced inflammation. Pre-treatment with the pan- galectin inhibitor thiodigalactoside (TG) ameliorates LNP -induced inflammation in vitro and in vivo with both intravenous and intratracheal delivery. TG also increases mRNA expression by up to 2-fold. Galectins have numerous functions that are dependent on the type of galectin, the galectin location (intracellular vs. extracellular), and the cell type. In this study, we have harnessed the role that galectins play in sensing severe endosomal ruptures to determine the degree of damage induced by LNPs formulated with various ionizable lipids. However, our results demonstrate for the first time that galectins have a net pro-inflammatory effect following LNP-induced endosomal damage. This may be due to several mechanisms that warrant further investigation. Galectins have been shown to promote NLRP3 inflammasome activation, particularly in macrophages50,51.The investigation of the signaling pathways underlying LNP-induced inflammation resulted in a non-inflammatory LNP formulations that do not exacerbate existing inflammation, leading to a therapeutic for ARDS. The principles behind the development of these non-inflammatory LNP formulations can therefore be harnessed to expand the therapeutic potential of RNA-LNPs and can be applied to other inflammatory conditions such as heart attack and stroke.As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” nanoparticle can mean a single nanoparticle or a multiplicity of nanoparticles.Various embodiments in the specification are presented using “comprising” language, which is inclusive of other components or method steps. When “comprising” is used, it is to be understood that related embodiments include descriptions using the “consisting of’ terminology, which excludes other components or method steps, and “consisting essentially of’ terminology, which excludes any components or method steps that substantially change the nature of the embodiment or invention.As used herein, the term “about” refers to a variant of ±10% from the reference integer and values therebetween. For example, “about” 100 nanometers (nm), includes±10 nm (i.e., 90 - 110 nm), which includes the integers 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110). The term “about” is inclusive of all values within the range including both integers and fractions.Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.By “nanoparticle” or “NP” (also referred to as “nanocarrier” or “NC”) as used herein is meant a particle that is typically between about 1 to about 500 nm across the largest dimension of the structure. Nanoparticles include nanogels, solid colloidal nanoparticles, magnetic nanoparticles, noble metal nanoparticles, semiconductor nanoparticles, multimodal nanoparticles, composite nanoparticles, and other nanoparticles typically used for biomedical applications (see, e.g., Blanco- Andujar et al. Annu. Rep. Prog. Chem., Sect. A, 2010, 106, 553-568, incorporated by reference herein). A nanoparticle may be spherical, but is not required to be spherical. Regardless of the shape of the nanoparticle, the exterior of the nanoparticle should be capable of comprising at least one agent, therapeutic, diagnostic, imaging, or otherwise. Inclusive in the definition of nanoparticle are particles that are at least 20, at least 40, at least 60, at least 80, at least100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least240, at least 260, at least 280, or at least 300 nm across their largest dimension. In certain embodiments, also included are particles of at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least520, at least 540, at least 560, at least 580, at least 600, at least 620, at least 640, at least660, at least 680, or at least 700nm. In yet other embodiment, also included are particles having diameters of at least 720, at least 740, at least 760, at least 780, at least 800, at least 820, at least 840, at least 860, at least 880, at least 900, at least 920, at least 940, at least 960, at least 980, or up to about 1000 nm across their largest dimension. In yet further embodiments, the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension. All numbers and fractions between any two of these numbersare also included. The size of a nanoparticle can be represented, for example, by the z- average as determined by dynamic light scattering.In certain embodiments, the nanoparticle is a liposome. By “liposome” as used herein is meant a material a microscopic spherical particle formed by a lipid bilayer enclosing an aqueous compartment. In certain embodiments, the nanoparticle is a hydrogel nanoparticle (also referred to as a nanogel or NG). In certain embodiments, the nanoparticle is a dendrimer. In yet other embodiments, the nanoparticle is a polymersome, a hybrid carrier combining artificial and natural components, or a protein multimolecular composition (e.g., ferritin or albumin aggregates).By “hydrogel nanoparticle” or “nanogel” as used herein is mean a polymeric material having a hydrophilic structure which renders it capable of holding amounts of selected drug compounds in their three-dimensional networks, the resulting particle having nanoparticle dimensions. Macroscopic dextran hydrogels have shown Young’s moduli of ~10-50kPa in the literature. See, e.g., Hwang MR, Kim JO, Lee JH, Kim YI, Kim JH, Chang SW, Jin SG, Kim JA, Lyoo WS, Han SS, Ku SK,Yong CS, Choi HG. Gentamicin- Loaded Wound Dressing with Polyvinyl Alcohol / Dextran Hydrogel: Gel Characterization and / / ? vivo Healing Evaluation. AAPS PharmSciTech 2010; 11 (3): 1092- 1103. In certain embodiments, the NG is a lysozyme-dextran nanogel (also referred to as LDNG). In certain embodiments, the LDNG is a synthetic construct of lysozyme and dextran. In other embodiments, the nanogel is made of chitosan or chitin, pullulan, hyaluronic acid, PEG, pluronics (e.g. F127), poly(acrylic acid) or poly(acrylate), poly(oligo(ethylene glycol)methyl ether methacrylate), polyethylene oxide), polyethylenimine, poly(caprolactone), and poly(N-isopropylacrylamide), among other options encompassing a wide range of hydrophilic polymers capable of chemical modifications enabling incorporation in a nanoparticle. See, e.g., Eckmann DM, Composto RJ, Tsourkas A, Muzykantov VR. Nanogel Carrier Design for Targeted Drug Delivery. J Mater Chem B Mater Biol Med 2015;2(46):8085-8097; and Ahmed EM, Mar. 2015, “Hydrogel: Preparation, Characterization, and Applications: A Review”, J. Adv. Res., 6(2): 105-121, among other publications in the art. In certain embodiments described herein, nanogel particles containing a drug and associated with an RBC behave best in this form of drug delivery.In certain embodiments, the nanoparticle employed herein is a lipid nanoparticle (“LNP”). LNPs useful herein are known in the art. As used herein, LNPs are comprised of cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP -forming composition. Fenton et al, Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent in vivo mRNA Delivery, Adv Mater. 2016 Apr 20; 28(15): 2939-2943, which is incorporated herein by reference.The various components of the LNP-forming composition may be selected based on the desired target, cargo, size, etc. For example, previous studies have shown that polymeric nanoparticles made of low molecular weight polyamines and lipids can deliver nucleic acids to endothelial cells with high efficiency. Dahlman, et al, In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight, Nat Nanotechnol. 2014 Aug; 9(8): 648-655, which is incorporated herein by reference in its entirety.The LNP-forming composition includes an ionizable lipid or lipid-like material. As exemplified herein, in one embodiment, the ionizable lipid is C12-200. In another embodiment, the ionizable lipid is CKK-E12. In another embodiment, the ionizable lipid is 5A2-SC8. In another embodiment, the ionizable lipid is BAMEA-O16B. In another embodiment, the ionizable lipid is 3060io. In another embodiment, the ionizable lipid is 7C1. See, Love et al, Lipid-like materials for low-dose, in vivo gene silencing, Proceedings of the National Academy of Sciences Feb 2010, 107 (5) 1864-1869; Dong et al, Lipopeptides and selective siRNA delivery, Proceedings of the National Academy of Sciences Mar 2014, 111 (11) 3955-3960; Cheng et al, Dendrimer-Based Lipid Nanoparticles Deliver Therapeutic FAH mRNA to Normalize Liver Function and Extend Survival in a Mouse Model of Hepatorenal Tyrosinemia Type I, Advanced Materials, 30(52) (Dec 2018); Liu et al, Fast and Efficient CRISPR / Cas9 Genome Editing In Vivo Enabled by Bioreducible Lipid and Messenger RNA Nanoparticles, Advanced Materials, 31(33), Aug 2019; and Hajj et al, Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH, Small, 15(6) (Feb 2019),each of which are incorporated herein by reference. Other ionizable lipids are known in the art and are useful herein.In certain embodiments, an ESCRT -recruiting cationic lipid is used. As discussed herein, in certain instances, ESCRT proteins are recruited to repair endolysosomal membranes by recognizing and sealing membrane disruptions. We determined that the use of certain cationic lipids in LNP production produces the correct size endosomal ruptures upon administration. These lipids are termed ESCRT -recruiting cationic lipids. In certain embodiments, the ESCRT -recruiting cationic lipid is 4A3-SC8. In certain embodiments, the ESCRT -recruiting cationic lipid is cKK-E12 5A4-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 3A4-SC8. In certain embodiments, the ESCRT - recruiting cationic lipid is 4A1-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 5A2-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O16B. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O12B. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O18B. Other ionizable lipids are known in the art and are useful herein.Nanoparticles may be prepared using any method known in the art. For example, particulate formulations can be formed by methods as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive, magnetic, organic, and other nanoparticles have been described.In certain embodiments, nanoparticles are prepared by the nanoprecipitation process or spray drying. Conditions used in preparing particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness,” shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the therapeutic agent to be delivered and / or the composition of the polymer matrix.Methods for making nanoparticles for delivery of encapsulated agents are described in the literature (see, e.g., Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J.Control. Release, 5: 13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; all of which are incorporated herein by reference).In certain embodiments, the nanoparticle is associated with one or more targeting moieties. By the term “targeting moiety” as used herein, is meant a molecule, including an antibody, a fragment thereof or an antibody fusion protein, which is capable of specifically binding to another molecule. A targeting moiety may be an antibody, an aptamer, a nucleic acid, a peptide, a carbohydrate (sugar), a lipid, a vitamin, a toxin, a component of a microorganism, a hormone, a receptor ligand, and or any derivative thereof. If the targeting moiety is an antibody, it may be a monoclonal antibody, a humanized antibody, a synthetic antibody, a heavy chain antibody, and a biologically active fragment of an antibody, wherein the biologically active fragment is a Fab fragment, a F(ab')2 fragment, and a Fv fragment.As used herein, the term “galectin inhibitor” is any agent that reduces the expression, activity, or binding of any of galectin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and / or 15. In certain embodiments, the galectin inhibitor is a pan-galectin inhibitor. In other embodiments, the galectin inhibitor is a galectin 1 inhibitor. In other embodiments, the galectin inhibitor is a galectin 3 inhibitor. In other embodiments, the galectin inhibitor is a galectin 8 inhibitor. In other embodiments, the galectin inhibitor is a galectin 9 inhibitor.The galectin inhibitor can be any currently known or as yet to be described. Galectin inhibitors include, without limitation, Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, P-D-lactosyl-steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)-modified peptide, DB16, DB21, OTX008, PTX013, anti -galectin antibodies, galectin-specific aptamers, siRNA and shRNA-coding vectors, LLS30, and LLS2. Known Galectin inhibitors that are antibodies / peptides include G3-C12 (Galectin-3 binding peptide), Anginex (Galectin -1 binding peptide), Galectin 8- IN-1 (Galectin 8 inhibitor), and Galectin 3-IN-l (Galectin 3 inhibitor).By the term “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibodyor antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.The antibody may be a naturally occurring antibody or may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like). The antibody may comprise at least one purification tag. In a particular embodiment, the framework antibody is an antibody fragment. The term “antibody fragment” includes a portion of an antibody that is an antigen binding fragment or single chains thereof. An antibody fragment can be a synthetically or genetically engineered polypeptide. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those in the art, and the fragments can be screened for utility in the same manner as whole antibodies. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment.As used herein, “specifically binding,” “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. Forexample, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, typically, more noncovalent bonds between the binding molecules or binding partners.The antibodies utilized herein may be further modified. For example, the antibodies may be humanized. In a particular embodiment, the antibodies (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and / or variable heavy domain of the antibodies of the instant invention may be inserted into another antibody construct. Methods for recombinantly producing antibodies are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available.In certain embodiments, the nanoparticle compositions provided herein include a second targeting moiety that is selected for delivery to, e.g., a target organ or tissue, such as antibody that bind to the endothelium (e.g., antibodies targeting endothelial proteins including PECAM-1, ICAM-1, VCAM, ACE, transferrin receptor, tissue factor / platelet tissue factor / factor III, and others) or antibodies that bind to other targeted cells. The targeting moiety may bind to at least one of a cell surface protein, carbohydrate, or lipid. In some embodiments, the targeting moiety binds to a cell adhesion molecule (CAM). The CAM may be intercellular adhesion molecule (ICAM), platelet-endothelial cell adhesion molecule (PEC AM), activated leukocyte cell adhesion molecule (ALCAM), B- lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesion molecule (VCAM), mucosal vascular addressin cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, P- selectin, and basigin. In certain embodiments, the targeting moiety binds specifically to a CAM selected from ICAM, PECAM, or VECAM. In certain embodiments, the nanoparticles include a second targeting moiety specific for ICAM-1 and are targeted to pulmonary endothelial cells and white blood cells in the lungs (since ICAM-1 is expressed on both cell types). In yet other embodiments, the targeting moiety binds specifically to ACE, transferrin receptor, or a suitable endothelial target known in the art (See e.g., Simone et al. Cell Tissue Res. 2009 Jan; 335(1): 283-300, which is incorporated herein by reference). In other embodiments, the targeting moiety binds to a cell surface moleculeassociated with classical endocytosis. Preferably, the cell surface molecule associated with classical endocytosis is one of mannose-6-phosphate receptor and transferrin receptor.In certain embodiments, the nanoparticle is pre-loaded with an agent (also referred to as a “cargo” or a “drug”). In certain embodiments, the agent is a drug. The drug may be encapsulated in the inner volume and / or bound to the surface of nanoparticles. In one embodiment, the NP loading is high capacity, e.g., the mass of the drug is >5% the mass of the NP. In one embodiment the selected nanoparticle contains a single drug component. In another embodiment, the selected nanoparticle is loaded with multiple drug components.The term “drug” as used herein is meant any therapeutic, prophylactic, or diagnostic compound or reagent that is contained within the flexible nanoparticles described herein. In one embodiment, the drug is a water-miscible compound. In another embodiment, the drug is an anti -rejection drug. Anti -rejection drugs include agents such as alemtuzumab, tacrolimus, and other drugs currently delivered systemically post-transplant to prevent rejection. In another embodiment, the drug is an anti-inflammatory agent. Antiinflammatory agents include corticosteroids, methotrexate, mycophenolate mofetil, azathioprine and other agents intended to limit inflammation. In another embodiment, the drug is an anti -edema agent, e.g., albuterol. In still another embodiment the drug is a compound that prevent ischemia-reperfusion injury, such as N-acetylcysteine, allopurinol, L-arginine, among known agents. In certain embodiments, the drug includes combinations of agents that act on multiple cell types, such as albuterol (acting on epithelial ENac to pump out alveolar fluid), dexamethasone (enhances endothelial barrier function and decreases neutrophil activity), and palifermin (enhances repair and regeneration of alveoli).In certain embodiments, the nanoparticle includes an agent that is an RNA molecule. The RNA molecule may express, for example, a therapeutic protein.The RNA molecule may express, for example, a therapeutic protein. In certain embodiments, the RNA molecule expresses IL10.Interleukin- 10 (IL 10) is a major immune regulatory cytokine that acts on many cells of the immune system where it has profound anti-inflammatory functions, limiting excessive tissue disruption caused by inflammation. The canonical sequence of hILlO can be found, e.g., at Genbank AAA63207.1, and reproduced below: SEQ ID NO: 2MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNA representative mRNA encoding SEQ ID NO: 2 can be found, e.g., at Genbank M57627.1 and is reproduced below (SEQ ID NO: 3)1 aaaccacaag acagacttgc aaaagaaggc atgcacagct cagcactgct ctgttgcctg61 gtcctcctga ctggggtgag ggccagccca ggccagggca cccagtctga gaacagctgc121 acccacttcc caggcaacct gcctaacatg cttcgagatc tccgagatgc cttcagcaga181 gtgaagactt tctttcaaat gaaggatcag ctggacaact tgttgttaaa ggagtccttg241 ctggaggact ttaagggtta cctgggttgc caagccttgt ctgagatgat ccagttttac301 ctggaggagg tgatgcccca agctgagaac caagacccag acatcaaggc gcatgtgaac361 tccctggggg agaacctgaa gaccctcagg ctgaggctac ggcgctgtca tcgatttctt421 ccctgtgaaa acaagagcaa ggccgtggag caggtgaaga atgcctttaa taagctccaa481 gagaaaggca tctacaaagc catgagtgag tttgacatct tcatcaacta catagaagcc541 tacatgacaa tgaagatacg aaactgagac atcagggtgg cgactctata gactctagga601 cataaattag aggtctccaa aatcggatct ggggctctgg gatagctgac ccagcccctt661 gagaaacctt attgtacctc tcttatagaa tatttattac ctctgatacc tcaaccccca721 tttctattta tttactgagc ttctctgtga acgatttaga aagaagccca atattataat781 ttttttcaat atttattatt ttcacctgtt tttaagctgt ttccataggg tgacacacta841 tggtatttga gtgttttaag ataaattata agttacataa gggaggaaaa aaaatgttct901 ttggggagcc aacagaagct tccattccaa gcctgaccac gctttctagc tgttgagctg961 ttttccctga cctccctcta atttatcttg tctctgggct tggggcttcc taactgctac1021 aaatactctt aggaagagaa accagggagc ccctttgatg attaattcac cttccagtgt1081 ctcggaggga ttcccctaac ctcattcccc aaccacttca ttcttgaaag ctgtggccag1141 cttgttattt ataacaacct aaatttggtt ctaggccggg cgcggtggct cacgcctgta1201 atcccagcac tttgggaggc tgaggcgggt ggatcacttg aggtcaggag ttcctaacca1261 gcctggtcaa catggtgaaa ccccgtctct actaaaaata caaaaattag ccgggcatgg1321 tggcgcgcac ctgtaatccc agctacttgg gaggctgagg caagagaatt gcttgaaccc1381 aggagatgga agttgcagtg agctgatatc atgcccctgt actccagcct gggtgacaga 1441 gcaagactct gtctcaaaaa aataaaaata aaaataaatt tggttctaat agaactcagt1501 tttaactaga atttattcaa ttcctctggg aatgttacat tgtttgtctg tcttcatagc1561 agattttaat tttgaataaa taaatgtatc ttattcacat cThe mRNA encoding IL 10 may be any sequence that encodes the IL 10 protein (e.g., the sequence of SEQ ID NO: 2) or a sequence sharing at least 90% identity with an IL10 protein (including splice variants and isoforms of SEQ ID NO: 2). In certain embodiments, the mRNA encodes a protein having a sequence at least 90% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 91% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 92% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 93% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 94% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 95% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 96% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 97% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 98% identical to SEQ ID NO: 2. In certain embodiments, the mRNA encodes a protein having a sequence at least 99% identical to SEQ ID NO: 2.In certain embodiments, the mRNA has a sequence at least 80% identical to SEQ ID NO: 3. In certain embodiments, the mRNA has a sequence at least 85% identical to SEQ ID NO: 3. In certain embodiments, the mRNA has a sequence at least 90% identical to SEQ ID NO: 3. In certain embodiments, the mRNA has a sequence at least 95% identical to SEQ ID NO: 3. In certain embodiments, the mRNA has a sequence at least 99% identical to SEQ ID NO: 3. In certain embodiments, the mRNA is codon optimized for expression in a mammalian cell, e.g., a human cell.In still another embodiment, the agent is an imaging agent. An “imaging agent” as used herein is a compound that has one or more properties that permit its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds incorporating a labeled entity that permits detection. Among suitable imaging agents are molecules containing radionuclides that are amenable to SPECT or PET imaging (e.g., Indium-I l l for SPECT imaging); molecules containing moi eties that provide contrast for CT imaging (e.g., gold nanoparticles oriodinated contrast agents); molecules containing moieties that provide contrast for MRI imaging (e.g., gadolinium); nano- or micro-scale complexes that provide contrast for ultrasound imaging (e.g., microbubbles filled with gas). A “detectable label” is a marker used for detection or imaging. Examples of such labels include: a radiolabel, a fluorophore, a chromophore, or an affinity tag. In one embodiment, the label is a radiolabel used for medical imaging, for example tc99m or iodine- 123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen- 15, oxygen- 17, gadolinium, manganese, iron, etc.In another aspect, a suitable nanoparticle for use in the methods herein is miscible with endothelial glycocalyx (e.g., carbohydrates on nanogel interdigitate with endothelial cells).In certain embodiments, the nanoparticle of the invention includes other components, such as one or more anti-platelet agents, glycoprotein inhibitors, fibrinolytic agents, thrombolytic agents, antibodies, or small molecule drugs. Non-limiting examples may include streptokinase, urokinase, or tissue plasminogen.The terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more entities, means that the entities are physically associated or connected with one another, either directly or via one or more additional entities that serves as a linking agent, to form a structure that is sufficiently stable so that the entities remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated.As used herein, the terms “exterior” or “outer surface layer” with reference to a nanoparticle refers to the surface of the nanoparticle that contacts blood plasma when the nanoparticle is administered to a subject.The term “expression” is used herein in its broadest meaning and comprises the production of RNA, of protein, or of both RNA and protein. With respect to RNA, the term “expression” or “translation” relates in particular to the production of peptides or proteins.The term “antibody fragment” as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as e.g. single chain Fab, scFv, and multispecific antibodies formed from antibody fragments. The “single chain Fab” format is described, e.g., in Hust M. et al. BMC Biotechnol. 2007 Mar 8;7: 14. scFvV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs.As used herein, “specifically binding,” “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. For example, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, typically, more noncovalent bonds between the binding molecules or binding partners.“Patient” or “subject” or “individual” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In certain embodiments, the subject is a human.The term “therapeutically effective amount” or “effective amount” refers to an amount agent that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate a condition or disease.CompositionsProvided herein are nanoparticle compositions that are modified to reduce or prevent inflammation following their delivery to a subject. These compositions include one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3.In one embodiment, the composition includes a galectin inhibitor. In certain embodiments, the nanoparticle is associated with a galectin inhibitor on its exterior surface. In other embodiments, the nanoparticle is formulated with the galectin inhibitor.The galectin inhibitor can be any known in the art, including those described herein. In certain embodiments, the galectin inhibitor is a pan-galectin inhibitor, optionally TDG. In other embodiments, the inhibitor is a galectin- 1 inhibitor. In other embodiments, the inhibitor is a galectin-3 inhibitor. In other embodiments, the inhibitor is a galectin-8 inhibitor. In other embodiments, the inhibitor is a galectin-9 inhibitor.In one embodiment, the composition includes a galectin inhibitor. Galectin inhibitors include, without limitation, Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Olitigaltin, Davanat® (GM- CT-01), GB1107, P-D-lactosyl-steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)-modified peptide, DB16, DB21, OTX008, PTX013, anti -galectin antibodies, galectin-specific aptamers, siRNA and shRNA-coding vectors, LLS30, and LLS2. See Table 1 for a list of galectin inhibitors useful herein. Known Galectin inhibitors that are antibodies / peptides include G3-C12 (Galectin-3 binding peptide), Anginex (Galectin -1 binding peptide), Galectin 8-IN-l (Galectin 8 inhibitor), and Galectin 3-IN-l (Galectin 3 inhibitor).In one embodiment, the composition includes an NFKB inhibitor. NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. As used herein, the term “NFKB inhibitor” is an agent that reduces the expression, activity, or binding of NFKB, inhibits cytokine expression, and is able to load into LNPs. In one embodiment, the NFKB inhibitor is celastrol. In another embodiment, the NFKB is a NFKB nuclear localization sequence binding peptide referred to as N50 (SEQ ID NO: 4: VQRKRQKLMP).In certain embodiments, the nanoparticle is associated with a NFKB inhibitor on its exterior surface. In other embodiments, the nanoparticle is formulated with the NFKB inhibitor.The NFKB inhibitor includes those known in the art, including those described herein that are capable of loading into LNPs and decreasing LNP associated inflammation in vitro as measured by secreted cytokines. In one embodiment, the inhibitor is a small molecule inhibitor known as celastrol. In another embodiment, the inhibitor is an NFKBnuclear localization sequence binding peptide referred to as N50. Celastrol is a cholesterol mimetic capable of inhibiting NFKB activation through several different pathways including inhibiting IKK activation, inhibition of IKB degradation, and antioxidant activity. N50 is a nuclear localization sequence that when added into cells prevents the translocation of activated NFKB into the nucleus, therefore preventing the upregulation of pro-inflammatory cytokines. In one embodiment, the inhibitor is a small molecule inhibitor known as carfilzomib.In one embodiment, the composition includes a mitogen-activated protein kinase kinase (MEK, also caed MAPKK) inhibitor. MEK inhibitors are commonly used to interfere with the MAPKZERK signaling pathway, which is involved in cell growth, differentiation, and survival. In one embodiment, the MEK inhibitor is trametinib. In one embodiment, the MEK inhibitor is cobimetinib. In one embodiment, the MEK inhibitor is selumetinib. In one embodiment, the MEK inhibitor is binimetinib. In one embodiment, the MEK inhibitor is PD0325901. In one embodiment, the MEK inhibitor is U0126. In one embodiment, the MEK inhibitor is selumetinib. In one embodiment, the MEK inhibitor is CI-1040.In one embodiment, the composition includes a caspase inhibitor. In one embodiment, the caspase inhibitor is Z-VAD-FMK (benzyloxy carbonyl-Val-Ala- Asp(OMe)-fluoromethylketone), a broad-spectrum caspase inhibitor commonly used in research to inhibit multiple caspases and block apoptosis. In one embodiment, the caspase inhibitor is wedolactone, an organic chemical compound classified as a coumestan that occurs in Eclipta alba (false daisy) and in Wedelia calendulacea. In one embodiment, the caspase inhibitor is Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]- methyl ketone), a more potent and less toxic pan-caspase inhibitor compared to Z-VAD- FMK, commonly used in research to block apoptosis. In one embodiment, the caspase inhibitor is Emricasan (IDN-6556), a broad-spectrum caspase inhibitor developed for treating chronic liver conditions like cirrhosis and hepatitis by inhibiting apoptosis and inflammation. In one embodiment, the caspase inhibitor is VX-765, a selective inhibitor of caspase- 1 that has been investigated for anti-inflammatory properties, particularly in the treatment of epilepsy and other inflammatory conditions. In one embodiment, the caspase inhibitor is IDN-1965, a broad-spectrum caspase inhibitor initially developed as a research tool for inhibiting caspases involved in apoptosis, with potential applications inneurodegenerative diseases. In one embodiment, the caspase inhibitor is Z-DEVD-FMK (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a selective caspase-3 and caspase-7 inhibitor, commonly used in research to study apoptotic pathways. In one embodiment, the caspase inhibitor is Z-IETD-FMK (benzyloxy carbonyl-He-Glu-Thr-Asp- fluoromethylketone), a selective inhibitor of caspase-8, often used in research on the extrinsic apoptotic pathway. In one embodiment, the caspase inhibitor is Z-LEHD-FMK (benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone), a selective caspase-9 inhibitor used in studies focusing on the intrinsic (mitochondrial) apoptotic pathway. In one embodiment, the caspase inhibitor is Belnacasan (VX-765 prodrug), designed to be converted into its active form in vivo, and it has been explored for treating conditions involving excessive inflammation by inhibiting caspase- 1. In one embodiment, the caspase inhibitor is Z-VAD-CHO, a reversible caspase inhibitor often used in early-stage apoptosis research.In one embodiment, the composition includes a cathepsin inhibitor. In one embodiment, the cathepsin inhibitor is E-64 (Epoxysuccinyl-L-leucylamido(4- guanidino)butane), a broad-spectrum cysteine protease inhibitor commonly used to inhibit cathepsins B, H, and L in research. In one embodiment, the cathepsin inhibitor is Leupeptin, which inhibits serine and cysteine proteases, including cathepsins B and L, through competitive inhibition at the enzyme’s active site. In one embodiment, the cathepsin inhibitor is Pepstatin A, a potent inhibitor of aspartic proteases like cathepsin D, by binding to the enzyme and blocking its proteolytic function. In one embodiment, the cathepsin inhibitor is K777 (KI 1777), an irreversible inhibitor of cathepsin B, developed for parasitic infections, which covalently modifies the active site cysteine residue. In one embodiment, the cathepsin inhibitor is CA-074, a specific and irreversible inhibitor of cathepsin B used in research to study its role in disease. In one embodiment, the cathepsin inhibitor is Z-FF-FMK (benzyloxy carbonyl-Phe-Phe-fluoromethylketone), an irreversible inhibitor selective for cathepsins B and L. In one embodiment, the cathepsin inhibitor is CTS-1027, a reversible inhibitor developed for treating inflammatory diseases, specifically inhibiting cathepsin K. In one embodiment, the cathepsin inhibitor is Odanacatib, a selective and reversible cathepsin K inhibitor developed for the treatment of osteoporosis by reducing bone degradation. In one embodiment, the cathepsin inhibitor is VBY-825, a broad-spectrum reversible inhibitor targeting cathepsins B, L, S, and V, investigated fortreating liver fibrosis and cancer. In one embodiment, the cathepsin inhibitor is CLIK-148, an irreversible inhibitor selective for cathepsin L, used in studies on cancer and other diseases. In one embodiment, the cathepsin inhibitor is Balicatib, a selective cathepsin K inhibitor originally developed for the treatment of osteoporosis by preventing bone resorption. In one embodiment, the cathepsin inhibitor is Z-Phe-Tyr(tBu)- diazomethylketone, an irreversible inhibitor selective for cathepsin B, covalently modifying its active site.In certain embodiments, the composition includes one or more siRNA against MEK, caspase, galectin, cathepsin, NF-KB or STAT3. siRNA (short interfering RNA) is a class of small, double-stranded RNA molecules, typically 20-25 nucleotides in length, that play a key role in the process of RNA interference (RNAi). siRNAs are used to silence or knock down the expression of specific genes by degrading messenger RNA (mRNA) molecules that correspond to the gene of interest. Using the known mRNA sequences of any of MEK, caspase, galectin, cathepsin, NF-KB or STAT3, siRNA may be designed using techniques known in the art. A table of exemplary mRNA accession numbers is provided below.Name GenbankIn certain embodiments, the nanoparticle is associated with a galectin inhibitor on its exterior surface. In other embodiments, the nanoparticle is formulated with the galectin inhibitor. In certain embodiments, the LNP comprises an ESCRT -recruiting ionizable lipid.The term “lipid nanoparticle”, also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids). In certainembodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In certain embodiments, an mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles.In the context of the present disclosure, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle.An LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.In certain embodiments, the LNP comprises one or more ionizable cationic lipids as described herein, cholesterol, a helper phospholipid, and a polyethylene glycol- modified lipid.As mentioned, the LNP comprises an ionizable cationic lipid. The cationic lipid is preferably ionizable, i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming aparticle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.In certain embodiments, an ESCRT -recruiting cationic lipid is used. As discussed herein, in certain instances, ESCRT proteins are recruited to repair endolysosomal membranes by recognizing and sealing membrane disruptions. We determined that the use of certain cationic lipids in LNP production produces the correct size endosomal ruptures upon administration. These lipids are termed ESCRT -recruiting cationic lipids. In certain embodiments, the ESCRT -recruiting cationic lipid is 4A3-SC8. In certain embodiments, the ESCRT -recruiting cationic lipid is cKK-E12 5A4-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 3A4-SC8. In certain embodiments, the ESCRT - recruiting cationic lipid is 4A1-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 5A2-SC8. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O16B. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O12B. In certain embodiments, the ESCRT-recruiting cationic lipid is 306-O18B. Other ionizable lipids are known in the art and are useful herein.In certain embodiments, the LNP may comprise any further cationic or ionizable lipid, i.e., any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl- N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2, 3 di oleoyloxy )propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3- (N — (N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3- dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl- 3 -dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMR.IE).Other useful lipids include, without limitation, 98N12-5, C12-200, PLGA, PEG, PEG-DMG, PEGylated lipids, amino alcohol lipids, and KL22.Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.);LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l- (2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).In certain embodiments, the further cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in W02012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoley oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy- 3 morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy- 3 dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3- (N,Ndilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 4- (dimethylamino)-butanoic acid, (1 OZ, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- l-yl-10,13- nonadecadien-l-yl ester (DLin-MC3-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12- octadecadien-l-yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA). See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, and US 8,853,377B2, which are incorporated by reference.In certain embodiments, LNP formulation is performed using routine procedures comprising cholesterol, ionizable lipid, helper lipid, PEG-lipid and polymer forming a lipid bilayer around encapsulated mRNA (Kowalski et al., 2019, Mol. Ther. 27(4):710- 728). In some embodiments, LNP comprises a cationic lipid (i.e. N-[l-(2,3- di oleoyloxy )propyl]-N,N,N-trimethylammonium chloride (DOTMA), or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP)) with helper lipid DOPE. In some embodiments,LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine- based ionizable lipids (cKK-E12). In some embodiments, polymer comprises a polyethyleneimine (PEI), or a poly(P-amino)esters (PBAEs). In some embodiments, the LNP comprises C14-4 / DOPE / Chol / PEG-lipid. See, Rybakova Y., Kowalski P. S., Huang Y., Gonzalez J. T., Heartlein M. W., DeRosa F., et al. . (2019). mRNA delivery for therapeutic anti-HER2 antibody expression in vivo. Mol. Ther. 27, 1415-1423 which is incorporated by reference. See also, e.g., WO2014 / 089486, US 2018 / 0353616A1, US2013 / 0037977A1, WO2015 / 074085 Al, US9670152B2, and US 8,853,377B2, which are incorporated by reference.In certain embodiments, where celastrol is utilized as an NFkB inhibitor, Celastrol in the lipid phase replaces a portion of the cholesterol normally added resulting in a five lipid component LNP rather than a typical four component LNP (Ionizable lipid, Cholesterol, DOPE, and DMG-PEG). In certain embodiments, celastrol is also comprised in the aqueous phase. Celastrol loaded in the mRNA phase was provided in a drug to lipid molar ratio equivalent to the amounts added in the lipid phase (e.g., about 40: 1). FIGs. IB and 1C.Certain LNPs useful herein include those that are described in WO 2021 / 077066 and WO 2021 / 055892, each of which is incorporated herein by reference in its entirety. LNP formulations may be varied to enhance delivery. For example, the type and ionizable lipid:mRNA ratio, molar ratio of ionizable lipid, phosopholipid, cholesterol, and PEG- lipid, etc. may be varied. In one embodiment, the LNP is one described by Kauffman, K. J.; Dorkin, J. R.; Yang, J. H; Heartlein, M. W .; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G., Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano letters 2015, 15 (11), 7300-7306, which is incorporated herein by reference. In certain embodiments, the LNPs are designed with ionizable lipid: mRNA weight ratios varying between 5 : 1 to 25 : 1. In certain embodiments, the ionizable lipid: mRNA weight ratio is 5: 1, 10: 1, 12.5: 1, 15: 1, 20: 1, or 25: 1.Other LNPs have been described and are useful herein. See, e.g., WO 2016 / 118724, US 10,413,618B2, US 10,723,692B2, and US8754062B2, each of which is incorporated herein by reference.In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation. Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- Icarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearioyl-2- oleoylphosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl- sn-glycero-3 phosphocholine (DSPC).In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8:1.In various embodiments, the LNPs further comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 5: 1 to 1:1.The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, Ndodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, Nglutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoylol eyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.In certain embodiments, the LNP comprises glycolipids (e.g., monosial oganglioside GM1).In certain embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term“pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monom ethoxy - polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like.In certain embodiments, the LNP comprises an additional, stabilizing-lipid which is a polyethylene gly col-lipid (pegylated lipid). Suitable polyethylene glycollipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG- modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene gly col-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene gly col-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene gly col-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monom ethoxy - polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradeca noxy)propyl)carba mate or 2,3-di(tetradecanoxy)propyl-N-(w- methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. U520120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.In certain embodiments, the LNP comprises a cargo. The term “cargo” or “drug” as used herein is meant any therapeutic, prophylactic, or diagnostic compound or reagent thatis contained within the flexible nanoparticles described herein. In one embodiment, the drug is a water-miscible compound. In another embodiment, the drug is an anti -rejection drug. Anti -rejection drugs include agents such as alemtuzumab, tacrolimus, and other drugs currently delivered systemically post-transplant to prevent rejection. In another embodiment, the drug is an anti-inflammatory agent. Anti-inflammatory agents include corticosteroids, methotrexate, mycophenolate mofetil, azathioprine and other agents intended to limit inflammation. In another embodiment, the drug is an anti -edema agent, e.g., albuterol. In still another embodiment the drug is a compound that prevent ischemiareperfusion injury, such as N-acetylcysteine, allopurinol, L-arginine, among known agents. In certain embodiments, the drug includes combinations of agents that act on multiple cell types, such as albuterol (acting on epithelial ENac to pump out alveolar fluid), dexamethasone (enhances endothelial barrier function and decreases neutrophil activity), and palifermin (enhances repair and regeneration of alveoli).In certain embodiments, the agent is a polynucleotide, e.g., RNA or DNA. The polynucleotide sequence molecule may express, for example, a therapeutic protein. The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.The phrase “nucleotide sequence encoding” refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.As used herein, the term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a polypeptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includestriple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequencespecific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and U in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.The skilled artisan will appreciate that the T bases in the codon maps disclosed herein are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding RNA sequences (comprising U) are considered codon-optimized nucleotide sequence of the present disclosure. A skilled artisan would also understand that equivalent codon-maps can be generated by replaced one or more bases with non-natural bases. Thus, e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn would correspond to a 'P'P codon (RNA map in which U has been replaced with pseudouridine).Also contemplated by the terms “modification” and “modified” as such terms related to the polynucleotide of the present invention are alterations which improve orenhance translation of nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence).In some embodiments, the polynucleotides described herein have undergone a chemical or biological modification to render them more stable. Exemplary modifications to a DNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring nucleic acids, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).In certain embodiments, the nanoparticle includes an agent that is an RNA molecule. In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridine (y) or 5-methylcytosine (m5C). Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one or ordinary skill in the art.In another embodiment the cargo is a nucleic acid. Illustrative genes which may be delivered via the LNP include, without limitation, glucose-6-phosphatase, associated with glycogen storage disease or deficiency type 1A (GSD1), phosphoenolpyruvatecarboxykinase (PEPCK), associated with PEPCK deficiency; cyclin-dependent kinase-like 5 (CDKL5), also known as serine / threonine kinase 9 (STK9) associated with seizures and severe neurodev el opmental impairment; galactose- 1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase (PAH), associated with phenylketonuria (PKU); gene products associated with Primary Hyperoxaluria Type 1 including Hydroxyacid Oxidase 1 (G0 / HA01) and AGXT, branched chain alpha-ketoacid dehydrogenase, including BCKDH, BCKDH-E2, BAKDH-Ela, and BAKDH-Elb, associated with Maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl -CoA mutase, associated with methylmalonic acidemia;medium chain acyl CoA dehydrogenase, associated with medium chain acetyl CoA deficiency; ornithine transcarbamylase (OTC), associated with ornithine transcarbamylase deficiency; argininosuccinic acid synthetase (ASS1), associated with citrullinemia; lecithin-cholesterol acyltransferase (LCAT) deficiency; amethylmalonic acidemia (MMA); NPC1 associated with Niemann-Pick disease, type Cl); propionic academia (PA); low density lipoprotein receptor (LDLR) protein, associated with familial hypercholesterolemia (FH), LDLR variant, such as those described in WO 2015 / 164778; ApoE and ApoC proteins, associated with dementia; lipoprotein lipase (LPL) (Lipoprotein Lipase Deficiency), UDP-glucouronosyltransf erase, associated with Crigler-Najjar disease; adenosine deaminase, associated with severe combined immunodeficiency disease; hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch-Nyan syndrome; biotimidase, associated with biotimidase deficiency; alphagalactosidase A (a-Gal A) associated with Fabry disease); beta-galactosidase (GLB1) associated with GM1 gangliosidosis; ATP7B associated with Wilson’s Disease; beta- glucocerebrosidase, associated with Gaucher disease type 2 and 3; peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; arylsulfatase A (ARSA) associated with metachromatic leukodystrophy, galactocerebrosidase (GALC) enzyme associated with Krabbe disease, alpha-glucosidase (GAA) associated with Pompe disease; sphingomyelinase (SMPD1) gene associated with Nieman Pick disease type A; camosinase (CN1); hypoxanthine-guanine phosphoribosyltransferase (HGPRT); erythropoietin (EPO); Carbamyl Phosphate Synthetase (CPS1), N-Acetylglutamate Synthetase (NAGS); Argininosuccinate Lyase (ASL) (Argininosuccinic Aciduria); and Arginase (AG); argininosuccsinate synthase associated with adult onset type II citrullinemia (CTLN2) (WO 2018 / 144709, which is incorporated herein by reference); carbamoyl -phosphate synthase 1 (CPS1) associated with urea cycle disorders; survival motor neuron (SMN) protein, associated with spinal muscular atrophy; ceramidase associated with Farber lipogranulomatosis; b-hexosaminidase associated with GM2 gangliosidosis and Tay-Sachs and Sandhoff diseases; aspartylglucosaminidase associated with aspartyl-glucosaminuria; a-fucosidase associated with fucosidosis; a-mannosidase associated with alpha-mannosidosis; porphobilinogen deaminase, associated with acute intermittent porphyria (AIP); alpha- 1 antitrypsin for treatment of alpha- 1 antitrypsin deficiency (emphysema); erythropoietin for treatment of anemia due to thalassemia or torenal failure; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; the beta adrenergic receptor, anti-sense to, or a mutant form of, phospholamban, the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for the treatment of congestive heart failure; a tumor suppressor gene such as p53 for the treatment of various cancers; a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; dystrophin or minidystrophin and utrophin or miniutrophin for the treatment of muscular dystrophies; and, insulin or GLP-1 for the treatment of diabetes.Examples of suitable transgenes for delivery include, e.g., those associated with familial hypercholesterolemia (e.g., VLDLr, LDLr, ApoE), muscular dystrophy, cystic fibrosis, and rare or orphan diseases. Examples of such rare disease may include spinal muscular atrophy (SMA), Huntingdon’s Disease, Rett Syndrome (e.g., methyl - CpG-binding protein 2 (MeCP2); UniProtKB - P51608), Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedrichs Ataxia (e.g., frataxin), progranulin (PRGN) (associated with non- Alzheimer’ s cerebral degenerations, including, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia), among others. Other useful gene products include, carbamoyl synthetase I, ornithine transcarbamylase (OTC), arginosuccinate synthetase, arginosuccinate lyase (ASL) for treatment of arginosuccinate lyase deficiency, arginase, fumarylacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, rhesus alpha- fetoprotein (AFP), rhesus chorionic gonadotrophin (CG), glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin gene product [e.g., a mini- or micro-dystrophin]. Still other useful gene products include enzymes such as may be useful in enzyme replacement therapy, which is useful in a variety of conditionsresulting from deficient activity of enzyme. For example, enzymes that contain mannose- 6-phosphate may be utilized in therapies for lysosomal storage diseases (e.g., a suitable gene includes that encoding p -glucuronidase (GUSB)).Other useful therapeutic products include those expressed in muscle, including heart muscle. Other useful therapeutic products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, glucagon-like peptide 1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor P superfamily, including TGF P, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT -4 / 5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, any one of the family of semaphorins / collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. Other transgenes useful herein include those for treating mucopolysaccharidosis type I- VII (IDUA, IDS, GNA, HGSNAT, NAGLU, SGSH, GALNS, GLB1, ARSB, GUSB).In still another embodiment, the agent is an imaging agent. An “imaging agent” as used herein is a compound that has one or more properties that permit its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds incorporating a labeled entity that permits detection. Among suitable imaging agents are molecules containing radionuclides that are amenable to SPECT or PET imaging (e.g., Indium-I l l for SPECT imaging); molecules containing moi eties that provide contrast for CT imaging (e.g., gold nanoparticles or iodinated contrast agents); molecules containing moieties that provide contrast for MRI imaging (e.g., gadolinium); nano- or micro-scale complexes that provide contrast forultrasound imaging (e.g., microbubbles filled with gas). A “detectable label” is a marker used for detection or imaging. Examples of such labels include: a radiolabel, a fluorophore, a chromophore, or an affinity tag. In one embodiment, the label is a radiolabel used for medical imaging, for example tc99m or iodine-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen- 15, oxygen- 17, gadolinium, manganese, iron, etc.In certain embodiments, an mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles.As described herein, in certain embodiments, the LNP is formulated with the galectin inhibitor. The galectin inhibitor may be formulated with the LNP as a prodrug. In certain embodiments, the galectin inhibitor is conjugated to a lipid component of the LNP. In other embodiments, the galectin inhibitor is conjugated to a negatively-charged species (e.g., sulfate, phosphate, peptides containing negatively charged amino acids, etc.) which will electrostatically condense with the positively charged ionizable lipid. Other methods of conjugation are known in the art. In other embodiments, the galectin inhibitor is loaded into the LNP as the parent molecule, i.e., without first conjugating the inhibitor to another molecule.In one embodiment, a nanoparticle includes mRNA encoding IL- 10 and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface. In certain embodiments, the nanoparticle comprises an outer surface layer comprising an ESCRT - recruiting cationic lipid. In another embodiment, a nanoparticle includes an outer surface layer comprising an ESCRT -recruiting cationic lipid, and mRNA encoding IL-10. In another embodiment, a nanoparticle includes an agent, an ESCRT -recruiting cationic lipid, and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.Pharmaceutical CompositionsIn another aspect, a pharmaceutical composition is provided that contains a nanoparticle and a gal ectin inhibitor. The pharmaceutical composition further contains a carrier, excipient, and / or preservative.In another aspect, a pharmaceutical composition is provided that contains a nanoparticle and an NFKB inhibitor. The pharmaceutical composition further contains a carrier, excipient, and / or preservative.In another aspect, a pharmaceutical composition is provided that contains a nanoparticle and an inhibitor of mitogen-activated protein kinase kinase (MEK). The pharmaceutical composition further contains a carrier, excipient, and / or preservative.In another aspect, a pharmaceutical composition is provided that contains a nanoparticle and a caspase inhibitor. The pharmaceutical composition further contains a carrier, excipient, and / or preservative.In another aspect, a pharmaceutical composition is provided that contains a nanoparticle and a cathepsin inhibitor. The pharmaceutical composition further contains a carrier, excipient, and / or preservative.In another aspect, a pharmaceutical composition is provided that contains a nanoparticle and a STAT3 inhibitor. The pharmaceutical composition further contains a carrier, excipient, and / or preservative.As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.In certain embodiments, the composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.Methods and agents well known in the art for making formulations are described, for example, in “Remington's Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa. Formulations may, for example, contain excipients, carriers, stabilizers, or diluents such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes, preservatives (such as octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol, and m-cresol), low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Pol oxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxy stearate), LABRASOL (Poly oxy capryllic glyceride), poly oxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter “P” (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.These above compositions may be administered in a variety of volumes of carrier, excipient or buffer formulations, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.Any suitable route of administration may be selected. Accordingly, pharmaceutical compositions may be formulated for any appropriate route of administration, for example, in the form of liquid solutions or suspensions (as, for example, for intravenous or intraarterial administration).MethodsThe compositions provided herein are useful for reducing or preventing inflammation resulting from administration of a nanoparticle composition.In certain embodiments, a method of delivering an agent to a target organ of a subject is provided, wherein the method comprises administering to the subject one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3 and a nanoparticle comprising the agent. In certain embodiments, the methods decrease inflammation in a target organ. In yet another embodiment, the methods decrease expression of one or more of IL-6, TNF-a, IL-la, or MCP-1.In certain embodiments, a method of treating ARDS in a subject in need thereof is provided, the method comprising administering to the subject a galectin inhibitor and a nanoparticle comprising mRNA encoding IL-10.In other embodiments, a method of treating ARDS in a subject in need thereof is provided, the method comprising administering to the subject a nanoparticle comprising an outer surface layer comprising an ESCRT -recruiting cationic lipid, the nanoparticle comprising mRNA encoding IL- 10.By “administering” or “route of administration” is meant delivery of composition described herein, with or without a pharmaceutical carrier, excipient, and / or preservative, to the subject. Routes of administration may be combined, if desired. In certain embodiments, the method includes intravenous delivery of a composition described herein. In certain embodiments, the method includes intraarterial delivery of a compositiondescribed herein. In certain embodiments, the method includes intrathecal delivery of a composition described herein. In certain embodiments, the method includes intrathecal delivery of a composition described herein.In certain embodiments, the nanoparticles are used to treat acute respiratory distress syndrome (ARDS). ARDS is an acute, diffuse, inflammatory lung injury with a variety of causes, most commonly pneumonia and sepsis. ARDS causes the lungs’ air sacs, called alveoli, to fill up with proteinaceous liquid, preventing the lungs from oxygenating the blood. The impact of ARDS is enormous, with 190,000 US cases per year, and a mortality rate of 35%. Decades of research have yielded myriad drug targets, but after the failure of more than a dozen large clinical trials, there are still no FDA approved drugs that improve survival in ARDS. From a pharmacology perspective, there are three reasons why many rationally chosen drugs have failed in ARDS. Firstly, ARDS patients are too fragile to tolerate drug side effects. These patients have multi-organ dysfunction, and thus cannot tolerate even mild side effects. Secondly, the inhalational route of delivery, useful for so many pulmonary problems, has limited benefit in ARDS, as the flooded alveoli (those filled with liquid) are covered by a column of fluid, which means that topical delivery to the alveoli is not possible via the inhaled route. Finally, ARDS is a very heterogeneous disease, so targeting a single pathway is unlikely to be sufficient.In another aspect, provided herein are methods of treating inflammation or treating an inflamed tissue. The method includes administering nanoparticles and one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3 to a subject in need thereof. In another embodiment, the method includes administering nanoparticles comprising an ESCRT -recruiting lipid and a galectin inhibitor to a subject in need thereof. The inflammation may be attributable, e.g., to an inflammatory disorder and / or infection in the subject. In certain embodiments, the inflamed tissue is lung.In certain embodiments, the method includes administration of a nanoparticle and one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3 intravenously. In certain embodiments, the method includes administration of a nanoparticle comprising ESCRT -recruiting lipid and a galectin inhibitor intravenously. Optionally, routes other than intravenous administrationmay be used, such as, e.g., oral, intranasal, intratracheal, intraarterial, intraocular, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.In certain embodiments, administration of the compositions described herein can be intravenous (iv) for delivery of the drug to the lungs. For example, where the disease is ARDS, pneumonia, interstitial lung disease, idiopathic pulmonary fibrosis, post- pulmonary embolism, pulmonary capilliaritis syndrome, emphysema, or a viral infection (such as SARS, influenza, or COVID), the composition may be administered intravenously. In certain embodiments, the methods employ injecting intravenously the compositions described herein carrying one or more of albuterol, dexamethasone, and palifermin for the treatment of ARDS. In certain embodiments, where the disease involves any other selected mammalian organ, the composition is administered in vivo intraarterially immediately upstream of the target organ for delivery of an agent. Additionally, for treatment of disease involving a selected mammalian organ designated for transplantation (other than the lung), the composition is administered ex vivo via feeding arterial opening into the organ prior to reperfusion and transplantation. In certain embodiments, the composition is administered via an arterial conduit of a selected organ using an arterial catheter. In any intra-arterial administration, the composition can be administered via an intra-arterial catheter. Such organs include, without limitation, the heart, brain, eyes, thyroid, kidney, liver, pancreas, spleen, intestines, or prostate.In certain embodiments, the nanoparticle composition and one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3 are delivered essentially simultaneously via the same route of administration. In certain embodiments, the nanoparticle composition is delivered subsequent to one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3. In other embodiments, the nanoparticle composition is delivered first.In certain embodiments, the method further includes administration of a therapeutic agent in addition to a nanoparticle composition and one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3. In certain embodiments, the therapeutic agent is an anticoagulant.As used herein, the term “administered in combination” or “combined administration” means that two or more compositions (e.g., a nanoparticle and one or more inhibitors of mitogen-activated protein kinase kinase (MEK), caspase, galectin, cathepsin, NF-KB and STAT3) are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each composition on the patient. In certain embodiments, they are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of one another. In certain embodiments, the administrations of the compositions are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved. In some embodiments, the administration in combination can be concurrent or consecutive.In still a further aspect, the compositions and methods can also be employed for imaging, such as to map capillary structure or pathology, wherein the drug is an imaging reagent.All scientific and technical terms used herein have their known and normal meaning to a person of skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, certain terms are defined as provided herein.Specific Embodiments: 1stembodiments1. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.2. The method of embodiment 1, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.3. The method of embodiment 1 or 2, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.4. The method of embodiment 3, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.5. The method of any of embodiments 1 to 3, wherein the galectin inhibitor is administered before the nanoparticle.6. The method of any of embodiments 1 to 3, wherein the galectin inhibitor is administered simultaneously with the nanoparticle.7. The method of any one of embodiments 1 to 6, wherein the nanoparticle comprises the galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.8. The method of any one of embodiments 1 to 7, wherein the galectin inhibitor is an inhibitor of galectin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and / or 15.9. The method of embodiment, wherein the galectin inhibitor is a pan-galectin inhibitor.10. The method of any one of embodiments 1 to 8, wherein the galectin inhibitor is thiodigalactoside.11. The method of any one of embodiments 1 to 8, wherein the galectin inhibitor is Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, P-D-lactosyl-steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)- modified peptide, DB16, DB21, OTX008, PTX013, anti-galectin antibodies, galectin- specific aptamers, siRNA and shRNA-coding vectors, LLS30, or LLS2.12. The method of any one of embodiments 1 to 11, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.14. The method of any one of embodiments 1 to 13, wherein the nanoparticle is administered intravenously, intrathecally, or intra-arterially.15. The method of any one of embodiments 1 to 14, wherein the nanoparticle and galectin inhibitor are administered to the subject via the same route.17. The method of any one of embodiments 1 to 16, wherein the agent is a therapeutic, diagnostic, or imaging agent.18. The method of any one of embodiments 1 to 17, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.19. The method of any one of embodiments 1 to 18, wherein administration of the galectin inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the galectin inhibitor.21. The method of embodiment 20, wherein the target organ is the lung.22. The method of any one of embodiments 1 to 21, wherein the nanoparticle comprises RNA and administration of the galectin inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the galectin inhibitor.23. A nanoparticle comprising an agent and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.24. The nanoparticle of embodiment 23, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.25. The nanoparticle of embodiment 23 or 24, wherein the galectin inhibitor is a pan- galectin inhibitor.26. The nanoparticle of any one of embodiments 23 to 25, wherein the galectin inhibitor is thiodigalactoside, Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, 0- D-lactosyl-steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)-modified peptide, DB16, DB21, OTX008, PTX013, anti-galectin antibodies, galectin-specific aptamers, siRNA and shRNA-coding vectors, LLS30, or LLS2.27. The nanoparticle of any one of embodiments 23 to 26, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.28. The nanoparticle of embodiment 27, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.29. The nanoparticle of any one of embodiments 23 to 28, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV.30. The nanoparticle of any one of embodiments 23 to 29, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.31. The nanoparticle of any one of embodiments 23 to 30, wherein the agent is a therapeutic, diagnostic, or imaging agent.32. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and the nanoparticle of any one of embodiment 23 to 31, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.33. The method of embodiment 32, further comprising administering to the subject a galectin inhibitor.34. The method of any one of embodiments 32 or 33, wherein the nanoparticle is administered intravenously or intra-arterially.35. The method of any one of embodiments 32 to 34, wherein the nanoparticle and galectin inhibitor are administered to the subject via the same route.36. The method of any one of embodiments 32 to 35, wherein the nanoparticle and galectin inhibitor are administered sequentially.37. The method of any one of embodiments 32 to 36, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.38. The method of any one of embodiments 32 to 37, wherein administration of the galectin inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the galectin inhibitor.39. The method of embodiment 38, wherein the target organ is the lung.40. The method of any one of embodiments 32 to 39, wherein the nanoparticle comprises RNA and administration of the galectin inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the galectin inhibitor.41. A pharmaceutical composition comprising the nanoparticle of any one of embodiments 23 to 31 and a pharmaceutically acceptable carrier, excipient and / or preservative.42. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.43. The method of embodiment 44, wherein the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.44. The method of embodiment 42 or 43, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.45. The method of any one of embodiments 42 to 44, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.46. The method of embodiment 45, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.47. The method of any one of embodiments 42 to 46, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV.48. The method of any one of embodiments 42 to 47, wherein the agent is a therapeutic, diagnostic, or imaging agent.2ndEmbodiments1. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an NFKB inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.2. The method of embodiment 1, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.3. The method of embodiment 1 or 2, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.4. The method of embodiment 3, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.5. The method of any of embodiments 1 to 3, wherein the NFKB inhibitor is administered before the nanoparticle.6. The method of any of embodiments 1 to 3, wherein the NFKB inhibitor is administered simultaneously with the nanoparticle.7. The method of any one of embodiments 1 to 6, wherein the nanoparticle comprises the NFKB inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.8. The method of any one of embodiments 1 to 7, wherein the NFKB inhibitor is celastrol or carfilzomib.9. The method of any one of embodiments 1 to 8, wherein the NFKB inhibitor is celastrol and is formulated with the nanoparticle.10. The method of any one of embodiments 1 to 7, wherein the NFKB inhibitor is N50 (VQRKRQKLMP).11. The method of any one of embodiments 1 to 10, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.12. The method of any one of embodiments 1 to 11, wherein the nanoparticle is administered intravenously, intrathecally, or intra-arterially.13. The method of any one of embodiments 1 to 12, wherein the nanoparticle and NFKB inhibitor are administered to the subject via the same route.14. The method of any one of embodiments 1 to 13, wherein the agent is a therapeutic, diagnostic, or imaging agent.15. The method of any one of embodiments 1 to 14, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.16. The method of any one of embodiments 1 to 15, wherein administration of the NFKB inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the NFKB inhibitor.17. The method of embodiment 16, wherein the target organ is the lung.18. The method of any one of embodiments 1 to 17, wherein the nanoparticle comprises RNA and administration of the NFKB inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the NFKB inhibitor.19. A nanoparticle comprising an agent and an NFKB inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.20. A nanoparticle comprising an agent and an NFKB inhibitor encapsulated therein.21. A nanoparticle formulated with an NFKB inhibitor and encapsulating an agent.22. The nanoparticle of any one of embodiments 19 to 21, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.23. The nanoparticle of any one of embodiments 19 to 22, wherein the NFKB inhibitor is celastrol.24. The nanoparticle of any one of embodiments 19 to 22, wherein the NFKB inhibitor is N50.25. The nanoparticle of any one of embodiments 19 to 24, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.26. The nanoparticle of any one of embodiments 19 to 25, wherein the agent is a therapeutic, diagnostic, or imaging agent.27. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an NFKB inhibitor and the nanoparticle of any one of embodiments 19 to 26, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.28. The method of embodiment 27, further comprising administering to the subject a galectin inhibitor.29. The method of any one of embodiments 27 or 28, wherein the nanoparticle is administered intravenously or intra-arterially.30. The method of any one of embodiments 27 to 29, wherein the nanoparticle and NFKB inhibitor are administered to the subject via the same route.31. The method of any one of embodiments 27 to 30, wherein the nanoparticle and NFKB inhibitor are administered sequentially.32. The method of any one of embodiments 27 to 31, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.33. The method of any one of embodiments 27 to 32, wherein administration of the NFKB inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the galectin inhibitor.34. The method of embodiment 33, wherein the target organ is the lung.35. The method of any one of embodiments 27 to 34, wherein the nanoparticle comprises RNA and administration of the NFKB inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the NFKB inhibitor.36. A pharmaceutical composition comprising the nanoparticle of any one of embodiments 19 to 26 and a pharmaceutically acceptable carrier, excipient and / or preservative.37. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an NFKB inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.38. The method of embodiment 37, wherein the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.39. The method of embodiment 37 or 38, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.40. The method of any one of embodiments 37 to 39, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.41. The method of embodiment 40, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.42. The method of any one of embodiments 37 to 41, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV.43. The method of any one of embodiments 37 to 42, wherein the agent is a therapeutic, diagnostic, or imaging agent.3rdEmbodiments1. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3, and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.2. The method of embodiment 1, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.3. The method of embodiment 1 or 2, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.4. The method of embodiment 3, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.5. The method of any of embodiments 1 to 3, wherein the inhibitor is administered before the nanoparticle.6. The method of any of embodiments 1 to 3, wherein the inhibitor is administered simultaneously with the nanoparticle.7. The method of any one of embodiments 1 to 6, wherein the nanoparticle comprises the inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.8. The method of any one of embodiments 1 to 6, wherein the inhibitor is encapsulated by the nanoparticle.9. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3, and a nanoparticle comprising the agent and an siRNA against NFkB, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.10. The method of embodiment 9, wherein the inhibitor is administered before the nanoparticle.11. The method of embodiment 9, wherein the inhibitor is administered simultaneously with the nanoparticle.12. The method of any one of embodiments 9 to 11, wherein the nanoparticle comprises the inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.13. The method of any one of embodiments 9 to 11, wherein the inhibitor is encapsulated by the nanoparticle.14. A nanoparticle comprising an agent and an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3 conjugated to a lipid component of the nanoparticle’s outer surface.15. A nanoparticle comprising an agent and an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3 encapsulated therein.16. A nanoparticle formulated with an inhibitor of MEK, caspase, galectin, cathepsin, NFkB or STAT3and encapsulating an agent.17. The nanoparticle of any one of embodiments 14 to 16, further comprising an NFkB siRNA encapsulated therein.18. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject an NFKB inhibitor and the nanoparticle of any one of embodiments 14 to 17, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.19. A pharmaceutical composition comprising the nanoparticle of any one of embodiments 14 to 17 and a pharmaceutically acceptable carrier, excipient and / or preservative.EXAMPLESThe following examples disclose specific embodiments of preparation of compositions of this invention, their characteristics, and methods of use thereof. These examples should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.Example 1 :LNP FormulationLNPs were formulated using the microfluidic mixing method. An organic phase containing a mixture of lipids dissolved in ethanol at a designated molar ratio was mixed with an aqueous phase (50 mM citrate buffer, pH 4) containing Luciferase mRNA that was either purchased by TriLink (most experiments) or made in-house via in vitro transcription (IVT)

[0042] at a flow rate ratio of 1 :3 and at a total lipid / mRNA weight ratio of 40: 1 in a microfluidic mixing device (NanoAssemblr Ignite, Precision Nanosystems).LNPs were dialysed against 1 * PBS in a 10 kDa molecular weight cut-off cassette for 2 h, sterilized through a 0.22 pm filter and stored at 4 °C.Cell CultureRAW 264.7 macrophages were cultured at 37°C in DMEM media containing 10% FBS and 1% Penicillin Streptomycin.Luciferase Delivery for measurement of mRNA Transfection EfficiencyLuciferase mRNA LNPs fabricated as described above were injected into mice intravenously or instilled intratracheally for a circulation time of 6 hours. Select organs were then flash-frozen until the day of analysis or homogenized immediately. Samples were suspended in 900 uL of homogenization buffer (5mM EDTA, lOmM EDTA, 1 : 100 diluted stock protease inhibitor (Sigma), and lx (PBS), samples were then loaded with a steel bead (Qiagen), then placed in a tissue homogenizer (Powerlyzer 24, Qiagen) using the following settings:Speed (S) 2000 rpm, 2 Cycles (C), T time 45 sec, and pause for 30 sec). After this, 100 uL of lysis buffer (10% Triton-X 100 and PBS) was added into each tube and then allowed to incubate for 1 hr at 4C. After this, they were immediately transferred into fresh tubes, and sonicated, using a point sonicator to remove in excess DNA, using an amplitude of 30%, 5 cycles of 3 secs on / off After this, samples were then centrifuged at 160,000 x g for 10 minutes. The resultant lysate is either frozen or prepared for luminometry analysis. For luciferase expression 20 uL of undiluted sample was loaded onto a black 96 well-plate then lOOuL luciferin solution (Promega) added immediately before reading on a luminometer (Wallac). Last, a Lowry assay (Bio-Rad) is performed according to manufacturer specification using diluted samples, specifically a 1 :40 dilution forlung and spleen tissues and a 1 :80 dilution for liver tissues. Final luminescence readings were then normalized based on total protein concentration obtained from Lowry Assay.Cytokine MeasurementsCytokine measurements were carried out with a LegendPlex 13-plex Mouse Inflammation Panel (Biolegend) according to the manufacturer’s instructions.Example 2: Drug Loading:Small Molecule Drug LoadingOrganic Loading: Small molecule loading initial tests were all loaded in the organic (lipid) phase during LNP synthesis. They were loaded in a drug to lipid mole ratio of 0.1. The LNPs then were put through a 7kDa cutoff zeba column to remove any unencapsulated drug. LNP samples before and after zeba were run on UPLC to assess encapsulation efficiency.Active Loading: Liposomes were formed with an internal buffer high in ions (300mM ammonium sulfate, sucrose, and hexametaphosphate). The drug of interest was then introduced into the solution at a high concentration driving the drug into the liposomes where the internal buffer would result in the drug crashing out and therefore being successfully encapsulated. JSH was selected for these loading experiments as its primary amine made it a good candidate for this process. However, in the end JSH was still unable to be encapsulated. FIG. 3 ACelastrol was initially loaded in the organic phase. However, after our lab discovered cholesterol leaves LNPs almost immediately after introduction to the blood and given celastrol’ s homology to cholesterol it raised concerns that celastrol loaded in the lipid phase would leave the LNPs not making it to the same cell the LNP does. Therefore, celastrol was also loaded in the aqueous phase to mitigate this issue and the two loading techniques were compared. Celastrol in the lipid phase replaced a portion of the cholesterol normally added resulting in a five lipid component LNP rather than our typical four component LNP (Ionizable lipid, Cholesterol, DOPE, and DMG-PEG). Cholesterol loaded in the mRNA phase was done in a drug to lipid molar ratio equivalent to the amounts added in the lipid phase. FIGs. 3B -3D.Peptide LoadingHydrophobic peptides were loaded in the lipid phase the same way as small molecule loading while positively charged peptides were loaded in the aqueous phase. The positively charged peptides were complexed with negatively charged nucleic acids prior to LNP synthesis; however, this complexation led to a significant reduction in protein expression of mRNA therefore requiring further optimization of this loading method. FIGs. 4 A and 4B.Example 3: Materials and MethodsDOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, DMG-PEG 2000 (l,2dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), 18: 1 PE TopFluor AF 594 (l,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(TopFluor® AF594) (ammonium salt)), and DSPE-PEG2000-azide (l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N[azido(polyethylene glycol)-2000] (ammonium salt)) were purchased from Avanti Polar Lipids. Ionizable lipids 4A3-SC8, cKK-E12, SM-102, ALC- 0315, and C12-200 were purchased from Echelon Biosciences. All other ionizable lipids were purchased from Broadpharm. L-leucyl-Lleucine, methyl ester (LLOMe) was purchased from Cayman Chemical. Thiodigalactoside was purchased from MedChemExpress.AnimalsAll experiments involving animals were conducted following the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Approval for all animal protocols was obtained from the University of Pennsylvania Institutional Animal Care and Use Committee. Male C57BL / 6 mice aged 6-8 weeks (23-25 g) sourced from The Jackson Laboratory, Bar Harbor, ME, were utilized for the experiments. The mice were housed in a controlled environment at temperatures between 22-26°C, adhering to a 12 / 12h dark / light cycle, and had unrestricted access to food and water.LNP FormulationLNPs were formulated using the microfluidic mixing method. An organic phase containing a mixture of lipids dissolved in ethanol at a designated molar ratio (50% ionizable lipid, 38.5% cholesterol, 10% DOPE, 1.5% DMG-PEG [Avanti]) was mixed with an aqueous phase (50 mM citrate buffer, pH 4) containing 5moU modified LuciferasemRNA (unless stated otherwise) or mCherry (for the NFKB reporter line) that was purchased from TriLink Biotechnologies, at a flow rate ratio of 1 :3 and at a total lipid to mRNA weight: weight ratio of 40: 1 in a microfluidic mixing device (NanoAssemblr Ignite, Precision Nanosystems). LNPs were dialyzed against 1 * PBS in a 10 kDa molecular weight cut-off cassette for 2 h, sterilized through a 0.22 pm filter, and stored at 4 °C.LNP CharacterizationMeasurements of hydrodynamic nanoparticle size, distribution, poly dispersity index, and zeta potential were conducted through dynamic light scattering using a Zetasizer Pro ZS from Malvern Panalytical. The encapsulation efficiencies and concentrations of LNP RNA were determined using a Quant-iT RiboGreen RNA assay (Invitrogen).Cell Culture:Raw264.7 mouse macrophages were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin (PS). For 96 well-plate experiments, RAW 264.7 macrophages and Luciferase reporter lines were seeded at 100k density using 100 uL volume and treated with 100 uL of LNP volume (diluted in DMEM).NFKB reporter cell line of Raw 264.7 mouse macrophages was purchased from BPS Bioscience Inc and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin / streptomycin (PS), 1% GlutaMAX, and 700ug / mL geneticin. Experiments were performed in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin / streptomycin (PS), and 1% GlutaMAX. For 96 well-plate experiments, RAW 264.7 macrophages and Luciferase reporter lines were seeded at 100k density using 100 uL volume and treated with 100 uL of LNP volume (diluted in DMEM)In Vitro Luciferase Delivery for measurements of mRNA Transfection Efficiency Luciferase mRNA LNPs fabricated as described above were incubated with cells for 6 hours. Supernatants were collected (spun down at 10,000 x g for 15 mins) for cytokine analysis using LegendPlex 13-plex Mouse Inflammation Panel (Biolegend). Adhered cells were lysed with 20 uL of lx Promega Luciferase Assay System Cell Culture Lysis Reagent for 15 mins. Luminescence was read on a luminometer (Promega) after 100 pL of luciferin solution (Promega) was added well by well by an autosampler.Cell viability assay:Cells were seeded in 96-well plates and incubated overnight (Raw264.7 cells: 1E5 cells / well; MLE-12 cells: 8E3 cells / well). Following different LNP treatments, 10% cell counting kit-8 (CCK8, ALX850039KI01, Enzo Life Sciences) reagent in complete medium was added to cells for 2 h incubation. Then, CCK-8 absorbance was recorded by microplate reader at 450 nm (660 nm absorbance as reference). Cell viability rates were normalized to control cells.Cytokine measurements:Cytokine measurements were carried out on plasma, BAL, or cell culture supernatant with a LegendPlex 13-plex Mouse Inflammation Panel (Biolegend) according to the manufacturer’s instructions.Intracerebral Hemorrhage:Experimental ICH was induced using bacterial collagenase as described see Lei B, Sheng H, Wang H, Lascola CD, Warner DS, Laskowitz DT, et al. Intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage. J Vis Exp 2014. 10.3791 / 51439 and Krafft PR, Rolland WB, Duris K, Lekic T, Campbell A, Tang J, et al. Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase. J Vis Exp 2012:e4289. 10.3791 / 4289 and Rosenberg GA, Mun- Bryce S, Wesley M, Kornfeld M. Collagenase-induced intracerebral hemorrhage in rats. Stroke 1990;21 :801-7. 10.1161 / 01. str.21.5.801 Briefly, mice were anesthetized using 2% isoflurane in 2 L / min 100% oxygen. They were placed in a stereotaxic frame and the skull was exposed. A 1 mm hole was drilled at 2.5 mm lateral, 0.5 mm anterior. A 35G needle (WPI) was slowly lowered 3.5 mm ventrally through skull opening. After 5 mins, 0.1 units of collagenase IV (Millipore) in 600 nL sterile saline were infused at a rate of 100 nL / min using a syringe pump. After injection, the needle was left in place for an additional 10 mins before slow withdrawal and wound closure. Sham animals underwent the same procedure with injection of sterile saline without collagenase.Grid walkAnalysis of motor deficit in mice after acute ICH was performed using grid walk (or foot-fault) testing as described Schaar KL, Brenneman MM, Savitz SI. Functional assessments in the rodent stroke model. Exp Transl Stroke Med 2010;2: 13. 10.1186 / 2040- 7378-2-13 and Ruan J, Yao Y. Behavioral tests in rodent models of stroke. BrainHemorrhages 2020; 1 : 171-84. 10.1016 / j.hest.2020.09.001. Briefly, animals were placed on an elevated grid with 1.69cm2 square openings and allowed to freely move across the grid. Their movements were recorded for 3 mins via a digital camera for subsequent analysis. Percent ratios of foot-fault over total number of steps for the ipsilateral versus contralateral hind paws were calculated. Statistical analysis was performed using a two-way ANOVA with a post-hoc Sidak's multiple comparisons test.Hematoma quantificationFor all animals used in this study, hematomas were quantified via ImageJ as described. See Wasserman JK, Yang H, Schlichter LC. Glial responses, neuron death and lesion resolution after intracerebral hemorrhage in young vs. aged rats. Eur J Neurosci 2008;28: 1316-28. 10.1111 / j .1460-9568.2008.06442.x. and iu X-C, Wu C-Z, Hu X-F, Wang T-L, Jin X-P, Ke S-F, et al. Gastrodin Attenuates Neuronal Apoptosis and Neurological Deficits after Experimental Intracerebral Hemorrhage. J Stroke Cerebrovasc Dis 2020,29: 104483. 10.1016 / j.jstrokecerebrovasdis.2019.104483. and Yu M, Tian T, Zhang J, Hu T. miR-141-3p protects against blood-brain barrier disruption and brain injury after intracerebral hemorrhage by targeting ZEB2. J Clin Neurosci 2022;99:253-60. 10.1016 / j.jocn.2022.03.010. Briefly, after perfusion brains were removed from the skull and fixed in 10% formalin. 1 mm coronal sections were prepared and layered onto a slide. Images were then captured using a digital camera and computerized analysis of hematoma size in each section was done using FIJI (NIH), from which volume was calculated by the sum of all slice volumes.Example 3 : Cotreatment with small molecule inhibitorsCotreatment of the small molecule inhibitors with 98N12 LNPs showed celastrol, Bot64, and JSH to be the three top hits. FIGs. 5A-5D, 6A and 6B. However, of these three top hits only celastrol was able to load and show efficacy. FIGs. 5E and 5F. Interestingly carfilzomib, which was originally not expected to show efficacy given the free drug treatments, was able to load into LNPs and showed efficacy. Given carfilzomib does not meet lipinsky’s rule of five, it makes sense it was unable to enter the cells without the LNP as a vehicle.Dose responses of these two top candidates showed carfilzomib’ s efficacy is very dependent on the D / L ratio used. It also showed celastrol is capable of resolving LNP-associated inflammation in vitro at low doses. This is especially promising as efficacy at lower doses will help reduce off target side effects including immunosuppression. FIGs. 6C-6H. Therefore, moving forward we chose celastrol as our top candidate for small molecule based NFKB inhibition.Recently, our lab has shown that cholesterol leaves LNPs very quickly once they enter the bloodstream. This has been supported by other groups having the same or very similar efficacy of LNPs with no cholesterol to ones that have it. Given celastrol’s homology to cholesterol, it is very likely that celastrol would leave the LNP quickly after reaching the bloodstream. This would result in very quick clearance of the drug and an inability to resolve LAI. However, celastrol could be protected from this leak by being loaded into the aqueous phase alongside mRNA. FIGs. 7A-7C.The NFKB Macrophage reporter line expresses firefly luciferase driven by four copies of the NFKB response element. FIG. 8A. Luciferase assay can be performed to measure the activation of NFKB. Using the NFKB Macrophage reporter line, we were able to show that in vitro aqueous and organic loaded celastrol are nearly identical in NFKB inactivation. Celastrol was capable of reducing NFKB activation to below the baseline shown with untreated cells from 0.1925 down to 0.012 D / L. Fig. 8BExample 5: siRNA InhibitionAlthough we’ve found inhibitors capable of resolving LNP associated inflammation (LAI), there are many therapeutic uses that would benefit from local and long-term inhibition of inflammation. Therefore, we started by investigating potential siRNAs for this purpose given the ease of loading into LNPs and that they’ve been thoroughly studied. p50 and p65 are the two most important NFKB subunits in the canonical NFKB pathway so we started by investigating the pool provided by Horizon Discovery as well as one from Santa Cruz Biotechnology.We treated the NFKB reporter cell line with the siRNAs loaded in sml02 LNPs for either 24 or 48 hours before treating with inflammatory 98N12 mCherry LNPs. FIG. 9A. The results showed that p65_7 or p65_SCB were the two most promising in reducing NFKB activation in comparison to mRNA treated cells. FIGs. 9B-9E.Example 6: Treatment of StrokeOne such therapeutic use for long-term NFKB inhibition is in stroke. There is an initial injury from the stroke injury itself, but there is also a secondary injury due to an inflammatory response in the brain. NFKB has been characterized in hemorrhagic strokes in patients showing that 13-48 hours post injury shows the highest levels of p65 activation. See Zhang, Z., Liu, Y., Huang, Q. et al. NF-KB activation and cell death after intracerebral hemorrhage in patients. Neurol Sci 35, 1097-1102 (2014). It has also been shown that the pan inhibition of NFKB in early intervention betters therapeutic outcome in rats while pan inhibition in late intervention worsens therapeutic outcome. This is due to cRel, another NFKB subunits, self-regulation ability creating a ceiling for NFKB activation. See de Jesus TJ, Ramakrishnan P. NF-KB c-Rel Dictates the Inflammatory Threshold by Acting as a Transcriptional Repressor. iScience. 2020 Mar 27;23(3): 100876. Both of these findings make siRNA for specific NFKB subunits very promising for therapeutic treatment of stroke.FIG. 10A provides a treatment scheme for ICH injury and VCAM targeted 4A3 siRNA LNPs. Better behavioral response for siRNA treated animals versus PBS treated is extremely promising for therapeutic use of NFKB inhibition in diseases where inflammation plays a major role. FIG. 10B.Example 7: LNPs induce potent inflammation in vitro and in vivo across speciesWe first probed the inflammation induced in various systems by mRNA-LNPs fabricated with one of the best-studied ionizable lipids, cKK-E12, which is among the topreported ionizable lipids for driving strong expression from mRNAs (Fig. 43). Into healthy, wildtype mice, we intratracheally instilled LNPs containing an mRNA dose ranging from 2.5 pg - lOpg per mouse, which are therapeutically relevant doses for intratracheal LNP treatment, and animals were sacrificed 24h after LNP treatment. Gross anatomical inspection of the lungs of mice instilled with 7.5 pg of mRNA in LNPs reveals severe “hepatization,” which is the classical pathology term for lung so inflamed that it looks like liver in some regions (Fig. 11 A). Furthermore, we assessed lung-specific inflammation by analyzing the gold standard for lung inflammation, which is the protein and leukocyte content of the bronchoalveolar lavage (BAL) fluid, which assess capillary leak and leukocyte infiltration into the alveoli (air sacs). We observed a large, dose-dependent increase in BAL protein (Fig. 1 IB) and BAL leukocyte (Fig. 11C) levels from 2.5 pg - 10 g of mRNA. We also observed dose-dependent changes in other markers associated with inflammation (Fig. 20). To put this inflammation severity into context, we gave the mice nebulized LPS, which is the most common animal model of the human disease acute respiratory distress syndrome (ARDS), which is the acute alveolar inflammation caused by severe COVID-19, sepsis, and other insults. Mice given nebulized LPS had lower BAL inflammatory markers than intermediate doses of mRNA-LNPs (Fig. 40).To generalize the inflammation stimulated by LNPs to other species, we instilled cKK-E12 LNPs or saline into ex vivo pig lungs for 3 hours at a dose of 0.8mg of mRNA in LNPs and subsequently extracted the BAL fluid. Similarly to mice, we observed evidence of hepatization after LNP administration (Fig. 1 ID). We observed a > 3 -fold increase in BAL protein levels (Fig. 1 IE) and a > 1.8-fold increase in BAL leukocyte count (Fig. 1 IF) relative to saline-treated control samples. Furthermore, the BAL concentrations of the pro-inflammatory cytokines IL-6 (Fig. 11G) and TNF-a (Fig 11H) increased by ~2.5-fold and ~4-fold respectively.In mice, we investigated more deeply the cytokine profile post-mRNA-LNP treatment in vivo and in vitro. We intratracheally instilled LNPs in mice at a dose of 7.5pg of mRNA in LNPs, waited two hours, and harvested BAL fluid. We observed a significant increase in the BAL concentrations of pro-inflammatory cytokines (particularly IL- la, IL- 6, TNF-a, IFN-P) and chemokines (MCP-1) (Fig 1 II). Although the magnitudes differed, we observed an upregulation of the same cytokines and chemokines in plasma 2 hours after intravenous LNP injection in mice at the same dose (Fig. 11 J), showing a common cytokine response profile across different tissues and delivery routes that localize LNPs to different cell types. We also observed that 24h after intravenous injection, LNPs induce leukocytosis, lymphopenia, and neutrophilia as well as histological evidence of inflammation in the liver and spleen (Fig. 22). Furthermore, intradermally administered LNPs also lead to significant cytokine upregulation in skin tissues Fig. 22).Next, we wanted to determine the cell types that underlie LNP-associated inflammation. We had previously shown that when LNPs were delivered to mice with preexisting inflammation, the LNPs worsened the inflammation, and this effect was abrogated by the removal of phagocytes1. Therefore, we tested here if the LNP -induced cytokineprofile noted above was recapitulated by delivering LNPs to a macrophage-derived cell line (RAW 264.7). We exposed these cells to cKK-E12 mRNA-LNPs at a dose of 400ng / ml for 6h. To isolate the inflammatory effects of the lipid component from that of the mRNA cargo, we also administered the same lipid dose of empty LNPs with no cargo, and LNPs loaded with a negatively charged polymer, polystyrene sulfonate (PSS), as a model cargo that mimics the negatively charged property of mRNA20. We found that mRNA, PSS, and empty LNPs all upregulate the same pro-inflammatory cytokines and chemokines as in the in vivo studies, namely IL-la, IL-6, TNF-a, IFN-P and MCP-1 (Fig.1 IK). However, empty LNPs led to the highest cytokine concentrations, inducing a -20- fold and a 500-fold higher IL-6 concentration than mRNA and PSS LNPs respectively. These results indicate that the inflammation stimulated by LNPs is primarily due to the lipid component, is made worse by removing the negative charges complexed with ionizable lipids and is recapitulated in vitro by macrophages.To determine how cell types other than macrophages respond to LNPs, we compared the effects of LNP treatment on the viability of RAW 264.7 macrophages vs an epithelial cell line (MLE-12) (Fig. 1 IL). In these studies, cells were treated with varying doses of LNPs for 24 hours and the fraction of viable cells was quantified. While there is a significant decrease in cell viability with increasing LNP dose in RAW macrophages, there is a much larger reduction in cell viability in MLE-12 epithelial cells as revealed by the IC-50 values (i.e. LNP doses at which cell viability is 50%) (IC50 = 1789ng / ml for macrophages and 290ng / ml for epithelial cells). Indeed, the epithelial cells died so easily that we could not detect a cytokine response from them (Fig. 2 IB).We further probed the mechanisms of cell death in macrophages, by analyzing the fraction of cells that were apoptotic, necrotic, or pyroptotic 24h after LNP treatment at a dose of 400ng / ml of mRNA in LNPs. Apoptosis is a type of programmed cell death which is typically non-inflammatory. On the other hand, necrosis and pyroptosis are inflammatory cell death mechanisms that are typically triggered by membrane rupture and inflammasome assembly, respectively. While most cells were healthy under control conditions (Fig. 1 IM), LNP treatment increased the fraction of apoptotic, necrotic, and pyroptotic cells by 3-fold, 13-fold, and 6-fold respectively (Fig. UN). Therefore, LNPs have variable effects on different cell types, with LNPs inducing not only strong cytokine responses in macrophages, but also inflammatory forms of cell death.Example 8: LNP-induced inflammation strongly depends on the particular ionizable lipid and has a positive correlation with mRNA expressionTo understand the mechanisms of LNP-induced inflammation, we began by varying the identity of LNPs’ ionizable lipids, which are the key component that enables transfection. Ionizable lipids facilitate the process of endosomal escape of RNA cargo21. Once protonated in the acidic endosome, ionizable lipids form ion pairs with the anionic lipids of the endosomal membrane, which leads to the formation of an inverse hexagonal phase that disrupts the endosomal membrane and releases the RNA cargo into the cytosol22-24. It has been shown that different ionizable lipids have different endosomal disruption capabilities based on those lipid structural properties which influence their ability to form the inverse hexagonal phase25,26. These structural characteristics correspond to differences in RNA payload release and transfection efficiency. We therefore hypothesized that these differences also correlate with inflammatory responses elicited by LNPs. As shown in the schematic of Fig. 12A, we propose that LNPs formulated with less potent ionizable lipids not only induce less endosomal escape of RNA, but also lead to lower RNA expression, and less inflammation. Conversely, more potent ionizable lipids induce higher RNA expression but more severe inflammatory responses. This is part of our central hypothesis that some aspect of endosomal escape itself induces inflammation, though it is not the presence of escaped RNA itself (Fig 1 IK).To test this part of the hypothesis, we screened LNPs formulated with varying ionizable lipids and compared their ability to translate cargo RNA (a measure of endosomal escape) to their induction of inflammation. Out of the hundreds of published ionizable lipids, our primary screen consisted of 15 of the most studied and potent ionizable lipids27,28, aiming for representation across 4 key classes: unsaturated (e.g. Dlin-MC3-DMA), multi-tail (e.g C12-200), biodegradable (e.g. SM-102) and branched tail (98N12-529) (Fig. 43)30. We first confirmed that the uptake of LNPs in RAW macrophages and A549 cells was similar regardless of ionizable lipid (Fig. 22). We then compared the mRNA expression capacities and found that the luciferase expression of these LNPs had a wide range, with almost a 700-fold difference between the lowest expressing (93-O17S, Lipid C24, Dlin-MC3-DMA) and the highest expressing (cKK-E12, 4A3-SC8) ionizable lipids (Fig 12B). 4A3-SC8 emerged as the highest-expressingionizable lipid in vitro31,32. To determine if this could be recapitulated in vivo, we tested a subset of these LNPs intravenously and intratracheally in mice. Similarly to the expression profile in vitro, the luciferase expression in the liver and spleen after intravenous injection follows the trend Dlin-MC3-DMA LNPs< SM-102 LNPs « cKK- E12 LNPs < 4A3-SC8 LNPs (Fig 12C, D). Additionally, 4A3-SC8 LNPs have a >3-fold higher luciferase expression in the lung than cKK-E12 LNPs after intratracheal administration (Fig. 12E).Having established the different ionizable lipid expression profiles, we next measured their provocation of inflammation, with the goal of correlating expression vs inflammatory profiles. We first measured the effect of treatment with different LNP formulations on cell viability in RAW macrophages (Fig. 23) and A549 cells (Fig. 24). We then treated RAW macrophages with LNPs formulated with the 15 ionizable lipids from Fig 2B and measured the concentrations of the cytokines IL-6 (Fig. 12F) and TNFa (Fig. 12G) in the cell culture supernatant 6h post-treatment. We found that the highest expressing ionizable lipids (C12-200, 98N12-5, cKK-E12) also generated the most cytokines, except for the highest expressing ionizable lipid 4A3-SC8, which did not increase cytokine levels above control. This is shown in the scatter plots of IL-6 (Fig. 12H, y-axis) or TNFa (Fig. 121, y-axis) concentrations vs luciferase expression (x-axis) for the 15 ionizable lipid LNP formulations. If we exclude one outlier (4A3-SC8), there is a positive correlation between luciferase expression and inflammatory cytokines (black trendlines, with r2 = 0.8662 & 0.7703 for Fig 12H & 121, respectively). The inclusion of this outlier brings the correlations down significantly (red trendlines, r2 = 0.3314 & 0.2933 for Fig 12H & 121, respectively). Importantly, these trends are very similar in vivo after intravenous injection of LNPs into mice: 2 hours post-LNP injection, the plasma concentrations of IL-6 (Fig. 12J) and TNFa (Fig. 12K) follow the trend Dlin-MC3-DMA LNPs< SM-102 LNPs < cKK-E12 as does the trend for the luciferase expression. As seen in vitro in RAW macrophages, 4A3-SC8 LNPs do not significantly increase in vivo plasma cytokine levels despite having the highest luciferase expression.To generalize this correlation of ionizable lipid identity vs inflammation, we next measured a much broader array of cytokines when cells were exposed to LNPs with different ionizable lipids (Fig. 25). We also compared cKK-E12 and 4A3-SC8 LNPs in human macrophages and observed that cKK-E12 LNPs lead to higher translocation of NF-KB from the cytoplasm to the nucleus (Fig. 26). Furthermore, to obtain more detailed structure-function relationships, we analyzed a library of lipidoids with the same amine head group with one-carbon differences in their tail structures33 and we found that there was also a positive correlation between expression and inflammation (Fig. 17). In RAW macrophages in vitro, we found that Dlin-MC3-DMA LNPs, which have low mRNA expression, do not significantly increase the concentrations of other pro-inflammatory cytokines beyond IL-6 and TNF-a (IL-la, IL-27, IFN-P, MCP-1) (Fig. 12L). Conversely, cKK-E12 LNPs, which have high mRNA expression, significantly upregulate the entire array of pro-inflammatory cytokines we described earlier (Fig. 12M). By contrast, the one outlier, 4A3-SC8 is a high-expressing ionizable lipid and is not inflammatory across the entire cytokine profile (Fig. 12N). Thus, in general, our sub-hypothesis in Figure 2A appears to be correct, in that more strongly expressing ionizable lipids also drive greater endosomal escape. However, we found an outlier, 4A3-SC8, whose unusual properties (high expresser but non-inflammatory) might allow us to further probe the mechanisms of LNP -induced inflammation.Example 9: Endosomal escape induces endosomal damage, with the size of endosomal holes predicting inflammationSince we found a general correlation between endosomal escape and inflammation, we next sought to investigate endosomal escape through the lens of “endosomal damage.” We hypothesized that in order to achieve endosomal escape of cargo RNA, the endosome must sustain some level of damage, with endosomal damage including “endosomal holes.” Such holes in the endosomal membrane would help permit mRNA to escape but would also expose the cytosol to toxic endogenous endosomal contents, which set off inflammation34.To study LNP -induced endosomal damage, we began by confirming that the RNA- LNPs were indeed localizing primarily to endosomes. We visualized the co-localization of endosomes and LNPs in RAW macrophages (Fig. 13A & Fig, 30B) and A549 cells (Fig. 30A) 30 minutes, Ih, and 6h after cKK-E12 LNP treatment. We fluorescently labeled the endosomes and lysosomes with the dye Lysotracker and added a fluorescent lipid into the LNPs (18: 1 PE-TopFluor AF59). At all time points tested, there was a strong correlation between the LNP signal and the endosome / lysosome signal, as shown by thecorresponding Pearson’s coefficient values (Fig. 12B). This shows that at early and late time points post-LNP administration, the lipid components of the LNPs are co-localized with the endolysosomal system (hereafter called “endosomes” for simplicity).We then investigated if LNPs were inducing enough endosomal damage / holes to cause endosomes to lose their pH gradient with respect to the cytosol. To measure this, we employed the pH-sensitive dye Acridine Orange (AO). In acidic vesicles such as the endosome, AO becomes protonated - which inhibits its ability to cross the vesicle membrane - and exhibits a red fluorescence. However, under non-acidic conditions, such as in the cytoplasm or nucleus, AO exhibits a green fluorescence. Therefore, we expect that damage to the endosome will cause a loss of endosomal proton gradient, leading to leakage of AO into the cytosol, thus increasing the green fluorescence and decreasing the red fluorescence of AO. We expect that because AO is a small molecule, there will be a loss of the endosomal proton gradient from both small endosomal holes that do not allow mRNA through, and from large endosomal holes that permit mRNA escape (Fig. 3C). We AO-stained control or cKK-E12 LNP -treated RAW macrophages (D) and A549 epithelial cells (E) and visualized the green and red fluorescence signals via microscopy. In both cell types, we observed a visible increase in the green mean fluorescence intensity (MFI) and a decrease in the red MFI, indicating significant endosomal damage. We quantified the extent of endosomal damage in RAW macrophages using flow cytometry. As a positive control, we treated cells with L-leucyl-L-leucine methyl ester (LLOMe) which is a known lysosomotropic agent that severely damages the endosomal membrane. We then quantified the fraction of ruptured endosomes in each sample using the ratio of the red MFI to the green MFI as a fraction of that of control samples (Fig. 31). We found that while <1% of endosomes are ruptured in control cells, LLOME-treated cells have >90% of their endosomes ruptured (Fig. 13F). In cells treated with cKK-E12 LNPs, >50% of endosomes are ruptured badly enough to have lost their pH gradient. This is a surprisingly high fraction of damaged endosomes, given that it is usually estimated that only ~1% of cargo mRNA escape to the cytosol35-38, and further suggesting that endosomal escape of RNA comes with a large amount of collateral endosomal damage.We next endeavored to determine if the fraction of endosomes with their pH gradient lost correlates with expression. We performed the same quantification as above on cells treated with LNPs formulated with some of the ionizable lipids screened in Fig.12, 6h after LNP treatment (Fig. 3G, Fig. 31). We also performed this quantification for select ionizable lipids 30 minutes, Ih, 2h, and 4h after LNP treatment (Fig. 22). While there was a range, over 80% of the ionizable lipids caused endosomal rupture severe enough to detect by the AO assay above control. This even includes ionizable lipids such as Dlin-MC3-DMA and ALC-0315 which were not significantly inflammatory and did not lead to the highest mRNA expression levels. Importantly, we observed a weak correlation when we compared mRNA expression levels and inflammatory responses to the fraction of endosomal rupture for each formulation. As shown in Fig. 13C, AO is a small molecule (MW = 265 g / mol) compared to the mRNA used (MW >652,000 g / mol). Therefore, AO would be able to leak from the endosome to the cytosol through smaller holes that would be impermeable to mRNA. However, when we observed the endosomal escape of an intermediately sized split GFP peptide, (MW = 6314g / mol), the escape efficiency follows the trend MC3 LNPs < C12-200 LNPs < SM-102 LNPs < cKK-E12 LNPs < 4A3-SC8 LNPs which more closely follows the trend for mRNA expression (Fig. 33). This indicated to us that the amount of RNA expression and inflammation induced by LNPs is not simply a function of the quantity of small endosomal holes (but just big enough to allow small molecules through). We therefore investigated if the size of endosomal holes plays an additional role.To answer this question, we first looked to how cells sense different types of endosomal damage induced by pathogens, particularly by viruses, which also have to achieve endosomal escape to deliver their nucleic acid cargo39. Microbiology studies showed that large endosomal ruptures (> 100 nm) recruit the sugar-binding proteins galectins, which recognize the glycans present on the intra-luminal leaflet of the endosomal membrane which become exposed upon rupture. Upon detecting such large endosomal holes, at least those induced by pathogens, galectins modulate inflammatory cascades and facilitate the process of lysophagy - the process through which cells degrade damaged endosomes by fusing them with lysosomesl4,15,40. Conversely, small endosomal ruptures (<100 nm) trigger the leakage of calcium ions into the cytosol, which mediate the recruitment of the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT machinery consists of a group of proteins that form filaments that promote budding, leading to the repair of the endosomal membrane 16-19. While ESCRT repair has been studied for different pathogens and particles, the response of the ESCRTmachinery to LNP treatment is not known. These pathways are illustrated in the schematic in Fig. 13H. We sought to then analyze the recruitment of galectins and ESCRT proteins to the endosomes induced by multiple LNP formulations measured by the formation of clusters (puncta).First, to assay galectin recruitment to large endosomal holes, we treated RAW macrophages with Dlin-MC3-DMA, 4A3-SC8, or cKK-E12 LNPs, using LLOME as a positive control and stained for galectins. While there are 15 galectins that we could have examined, previously nanomedicine researchers had shown that galectins 1, 3, 8, and 9 can be used as tracers to measure the extent of endosomal escape, which was previously viewed as helpful in screening for high expressing LNP formulations41-43. Therefore, we tested Gal-1, 3, 8, and 9, but show below the results for Gal-9, which was most predictive (Fig. 34). We stained for Gal-9 and quantified the puncta formed 6 hours post- LNP treatment. The images (Fig 131) and subsequent quantifications (Fig. 13K) reveal that low RNA-expressing and less inflammatory Dlin-DMA-MC3 LNPs led to the lowest levels of Gal-9 recruitment, while high RNA-expressing and highly inflammatory cKK- E12 LNPs led to the highest levels of Gal-9 recruitment. This suggests that in general, higher RNA-expressing ionizable lipids such as cKK-E12 lead to more severe endosomal damage and generate a higher fraction of large endosomal ruptures. While this facilitates the release of more RNA, it also leads to a more severe inflammatory response. The reverse is the case for lower RNA-expressing ionizable lipids such as Dlin-MC3-DMA which does not appear to generate a significant number of endosomal ruptures.We then quantified the amount of small, reparable endosomal ruptures induced by the LNPs 6 hours post-LNP treatment by analyzing the number of puncta of ALG-2- interacting protein X (ALIX), which is a protein that is part of the ESCRT machinery44. The images (Fig. 13 J) and quantifications (Fig. 13L) show that there is no significant ALIX recruitment with Dlin-MC3-DMA LNPs, cKK-E12 LNPs, or LLOME. However, there is a significant number of ALIX puncta formed with LNPs of the outlier ionizable lipid from Figure 12, 4A3-SC8 (Fig. 13J, L & Fig. 35). This indicates that while 4A3-SC8 LNPs cause some large endosomal ruptures (Fig 13K), they also cause a great amount of intermediate-sized, reparable endosomal ruptures. This provides great insight into why this outlier ionizable lipid induces high RNA expression without significant inflammation. Repair of endosomes by ESCRTs has been shown to actively limit inflammation, bypreventing exposure of endosomal contents to the cytosol and by preventing the inflammasome-mediated, highly inflammatory process of pyroptosis that we showed LNPs can cause in Figure 145,46. We hypothesize that because 4A3-SC8 is predicted to be rapidly biodegradable (numerous esters and adjacent thioethers), it is quickly degraded upon entry into the endosome, which limits the size and severity of the resulting endosomal holes, providing holes big enough for endosomal escape of RNA, but still in the reparable size range. We did test other biodegradable lipids (all with esters instead of amides, the same as 4A3-SC8), but they did not achieve as good expression as 4A3-SC8, probably because they do not have the same multiplicity of alkyl tails, which has previously been shown to correlate with high expression. Collectively, this data set shows that large endosomal holes (galectin-marked) associate with inflammatory LNPs, while a great number of intermediate-sized / reparable holes (ESCRT-marked) associate with inflammation-free expression.Example 10: Inhibition of endosomal escape detection by galectins ameliorates LNP- induced inflammationGalectins are one of the earliest and primary sensors of large endosomal holes induced by LNPs, and we have shown that more inflammatory ionizable lipids recruit more galectins. Therefore, we hypothesized that inhibiting galectins upstream would prevent or delay the downstream initiation of inflammatory pathways moderated by galectins. In addition to facilitating the removal of damaged endosomes through lysophagy, galectins have been shown to promote inflammation by facilitating inflammasome assembly, activating and recruiting innate immune cells, and promoting cytokine functionl5,47. Therefore, we hypothesized that inhibiting galectins would have a net anti-inflammatory effect. We inhibited galectins using the small molecule drug thiodigalactoside (TG), which is a pan-galectin inhibitor that functions by competitively binding to the carbohydrate-binding domains of galectins. In all experiments, we tested the efficacy of TG against inflammation induced by cKK-E12 LNPs, which were the most inflammatory LNPs revealed in our screening. We first verified that TG pretreatment reduces the formation of galectin puncta. We treated RAW macrophages in vitro with TG (2.5mg / ml) for 1 hour and then administered cKK-E12 LNPs (400ng / ml of mRNA in LNPs) for a period of 6 hours. The images and subsequent quantifications revealed that thenumber of Gal-9 puncta formed is significantly reduced with TG pre-treatment, indicating that TG inhibits galectin recruitment following LNP treatment (Fig 14A). We then measured the effects of galectin inhibition with TG pre-treatment on the expression of pro- inflammatory cytokines. In RAW macrophages, TG pre-treatment largely attenuates the LNP -induced production of IL-6, TNFa, IL- la, and MCP-1 (Fig. 14B) and other pro- inflammatory cytokines (Fig. 36). We then sought to determine if galectin inhibition is efficacious in vivo through different routes of administration. We first injected TG (lOOpg per mouse) intravenously and 1 hour later, injected cKK-E12 LNPs (7.5pg of mRNA in LNPs) for a circulation time of 2 hours. As with RAW macrophages, intravenous TG pretreatment significantly decreased the LNP-induced increases in plasma concentrations of IL-6, TNFa, IL- la, and MCP-1 (Fig. 14C) and other pro-inflammatory cytokines (Fig.36). Furthermore, pre-treatment with TG prevented LNP-induced leukocytosis (increased circulating white blood cell count) and neutrophilia (increased circulating neutrophils) (Fig. 14D). Finally, we tested the ability of TG to prevent LNP-induced inflammation via a different LNP delivery route, instilling the TG intratracheally before intratracheally administering cKK-E12 LNPs 1 hour later and extracting the BAL fluid from the lungs 2 hours after LNP treatment. In the BAL fluid, TG significantly reduced the concentrations of IL-6, TNFa, and IL- la (Fig. 14E) and other pro-inflammatory cytokines (Fig. 36). Furthermore, TG reduces the leukocyte count in the BAL which indicates a decrease in leukocyte infiltration into the alveolar space (Fig. 14F). This reduction in BAL leukocyte count persists even 24 hours after LNP instillation (Fig. 36). Thus, galectin inhibition prevents LNP-induced inflammation in vitro and across multiple routes of in vivo LNP delivery.Example 11 : Inhibition of endosomal escape detection positively impacts mRNA expressionAfter showing that galectin inhibition ameliorates LNP-induced inflammation, we sought to determine its effects on RNA expression. We first treated RAW macrophages with TG (2.5mg / ml) for 1 hour. We then administered cKK-E12 LNPs formulated with luciferase mRNA and measured the luciferase expression 6 hours later. TG pre-treatment increases mRNA expression by >2.5-fold compared to cKK-E12 LNPs alone (Fig. 15A). We then administered TG intratracheally into mice (lOOpg per mouse) and then treatedthem with cKK-E12 LNPs for 6 hours. We found that TG pre-treatment does not attenuate the mRNA expression in the lung (Fig. 15B). Finally, we injected TG into mice intravenously (lOOpg per mouse) and then injected them with cKK-E12 LNPs for a circulation time of 6 hours. TG pre-treatment leads to a ~2.7-fold and a ~2.4-fold increase in mRNA expression in the liver and spleen respectively (Fig. 15C). These results indicate that in addition to ameliorating inflammation induced by LNPs, inhibition of large endosomal rupture detection also generally improves RNA expression, presumably by limiting the degradation of RNA-containing endosomes.Having demonstrated that LNP -induced inflammation can be ameliorated while preserving high mRNA expression by utilizing an ESCRT -recruiting ionizable lipid or inhibiting galectins, we sought to test the therapeutic efficacy of our non-inflammatory formulations in settings with pre-existing inflammation. We and others have shown that in addition to the inflammation induced by LNPs under naive conditions, LNPs exacerbate pre-existing inflammation by orders of magnitude, whether in the diseased organ or an organ with a comorbid inflammationl. This phenomenon makes the use of inflammatory LNPs prohibitive in patients with conditions such as ARDS, stroke, and heart attack which form a large fraction of potential LNP indications. We therefore tested our noninflammatory LNP formulations in a model of endotoxemia-induced, ARDS-like acute lung injury induced by administering nebulized-LPS. Despite the therapeutic potential of ARDS and the high patient mortality rate (>45%), particularly in cases associated with COVID-1948, there are currently no mRNA LNPs being tested for this disease. This is in part because, as we have demonstrated, LNPs severely increase inflammation in the nebulized-LPS ARDS model by orders of magnitude (Fig. 37). We have previously attempted to ameliorate this exacerbated inflammation induced by LNPs in this inflammatory model using small molecule drugs (dexamethasone, MCC950), proteins (IL- IRa), and antibodies (anti -IL-6) but the levels of inflammation were so severe that none of these seemingly obvious therapeutic approaches were efficacious (Fig. 38). We also attempted to understand the signaling pathways underlying LNP -induced inflammation exacerbation using knockout mice (ASC, CCR2, MyD88, caspase-3) but were unable to isolate a clear pathway (Fig. 39). Having subsequently elucidated and ameliorated LNP- induced inflammation, we sought to revisit the use of LNPs in inflammatory models by testing our non-inflammatory LNP formulations namely: 1) 4A3-SC8 LNPs and 2) cKK-E12 LNPs with TG pre-treatment, loaded with mRNA encoding for the potent antiinflammatory cytokine IL-10 as a therapeutic for ARDS. We targeted LNPs to the lung by conjugating the lung-targeting moiety PECAM (Platelet Endothelial Cell Adhesion Molecule) to the surface of LNPs49. Mice were either intravenously injected with TG (lOOpg per mouse) followed by cKK-E12 LNPs 1 hour later (7.5pg of mRNA in LNPs) or intravenously injected with 4A3-SC8 LNPs (7.5pg of mRNA in LNPs). Nebulized LPS was administered immediately following LNP injection and mice were sacrificed 4 hours later (Fig 15D). Both 4A3-SC8 and TG + cKK-E12 LNPs induced potent upregulation of IL- 10 by ~40-fold and ~60-fold respectively in the BAL fluid (Fig. 5E). Both formulations also reduced the BAL concentrations of pro-inflammatory cytokines such as IL-6, TNF-a, IL- la, and IL-ip compared to nebulized LPS mice levels (Fig. 40). Furthermore, IL- 10 4A3-SC8 LNPs and TG + IL-10 cKK-E12 LNPs completely abrogated the hallmark physiological characteristics of ARDS including leukocyte infiltration (Fig. 15F) and capillary leak (Fig. 15G) into the alveolar space, even at longer timepoints (Fig. 41). Notably, TG pre-treatment alone did not affect ARDS phenotypes (Fig. 42).We have demonstrated that we can ameliorate LNP-induced inflammation by 1) preventing sensing of severe, irreparable endosomal damage by inhibiting galectins thus preventing downstream galectin-modulated inflammatory responses or 2) formulating LNPs with branched, multi -tail, biodegradable ionizable lipids such as 4A3-SC8 which induce less severe, reparable endosomal damage that can be repaired by ESCRT proteins before inflammation is triggered (Fig. 15H).Example 12:We first tested our non-optimized LNP formulations in a model of endotoxemia- induced, ARDS -like acute lung injury induced by administering nebulized-LPS. In these experiments, we formulated LNPs encapsulating a model luciferase mRNA cargo with the ionizable lipid cKK-E12 and conjugated the lung-targeting moiety PECAM (Platelet Endothelial Cell Adhesion Molecule) which has been shown to localize LNPs to the lung3. cKK-E12 PECAM LNPs severely increase the bronchoalveolar (BAL) protein levels, which is a measure of capillary leak into the alveolar space, in a dose-dependent manner. cKK-E12 LNPs also decrease the peripheral lymphocyte count, increase the neutrophil count, and decrease the platelet count in the blood (FIG. 16A-C). These LNPsalso increase the levels of pro-inflammatory cytokines in the plasma compared to nebulized-LPS control levels (FIG. 16D). We have previously attempted to ameliorate this exacerbated inflammation induced by LNPs in inflammatory models of ARDS using small molecule drugs, proteins, and antibodies but the levels of inflammation were so severe that none of these seemingly obvious therapeutic approaches were efficacious. Amongst these, we tested dexamethasone palmitate LNPs, MCC950 (an NLRP3 inflammasome inhibitor), IL-lra protein, and anti-IL-6 antibodies (FIG. 17).We therefore tested our optimized, non-inflammatory LNP formulations namely: 1) 4A3-SC8 LNPs and 2) cKK-E12 LNPs (with TG pre-treatment), loaded with mRNA encoding for the potent anti-inflammatory cytokine IL-10 as a therapeutic for ARDS. We targeted LNPs to the lung by conjugating PEC AM to the surface of LNPs. Mice were either intravenously injected with TG (lOOpg per mouse) followed by cKK-E12 LNPs 1 hour later (7.5pg of mRNA in LNPs) or intravenously injected with 4A3-SC8 LNPs (7.5pg of mRNA in LNPs). Nebulized LPS was administered immediately following LNP injection and mice were sacrificed 4 hours later (FIG. 18A). Both IL-10 4A3-SC8 and IL- 10 TG + cKK-E12 LNPs induced potent upregulation of IL-10 by ~40-fold and ~60-fold respectively in the BAL fluid (FIG. 18B). Furthermore, IL-10 4A3-SC8 LNPs and IL-10 TG + cKK-E12 LNPs completely abrogated the hallmark physiological characteristics of ARDS including leukocyte infiltration (FIG. 18C) and capillary leak (FIG. 18D) into the alveolar space. Both formulations also reduced the BAL concentrations of pro- inflammatory cytokines such as IL-6, TNF-a, IL- la, and IL-ip compared to nebulized LPS mice levels (FIG. 18E, F). Notably, TG alone did not affect ARDS phenotypes, indicating that it ameliorates only the LNP -induced inflammation (FIG. 19).Example 13: Materials and MethodsMaterialsDOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, DMG-PEG 2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), 18:1 PE TopFluor AF 594 (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(TopFluor® AF594) (ammonium salt)), and DSPE-PEG2000-azide (l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (ammonium salt)) were purchased from Avanti Polar Lipids. Ionizable lipids 4A3-SC8, cKK-E12, SM-102, ALC-0315, and C12-200 were purchased from Echelon Biosciences. All other ionizable lipids were purchased from Broadpharm. L-leucyl-L-leucine, methyl ester (LLOMe) was purchased from Cayman Chemical. Thiodigalactoside was purchased from MedChemExpress.AnimalsAll experiments involving animals were conducted following the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Approval for all animal protocols was obtained from the University of Pennsylvania Institutional Animal Care and Use Committee. Male C57BL / 6 mice aged 6-8 weeks (20-25 g) sourced from The Jackson Laboratory, Bar Harbor, ME, were utilized for the experiments. The mice were housed in a controlled environment at temperatures between 22-26°C, adhering to a 12 / 12h dark / light cycle, and had unrestricted access to food and water.LNP FormulationLNPs were formulated using the microfluidic mixing method. An organic phase containing a mixture of lipids dissolved in ethanol at a designated molar ratio was mixed with an aqueous phase (50 mM citrate buffer, pH 4) containing 5moU modified Luciferase mRNA (unless otherwise stated) that was purchased from TriLink Biotechnologies, at a flow rate ratio of 1 :3 and at a total lipid to mRNA weight: weight ratio of 40: 1 in a microfluidic mixing device (NanoAssemblr Ignite, Precision Nanosystems). LNPs were dialyzed against 1 * PBS in a 10 kDa molecular weight cut-off cassette for 2 h, sterilized through a 0.22 pm filter, and stored at 4 °C. IL-10 mRNA was synthesized via in vitro transcription as described previously 1.LNP CharacterizationMeasurements of hydrodynamic nanoparticle size, distribution, poly dispersity index, and zeta potential were conducted through dynamic light scattering using a Zetasizer Pro ZS from Malvern Panalytical. The encapsulation efficiencies and concentrations of LNP RNA were determined using a Quant-iT RiboGreen RNA assay (Invitrogen).Cell CultureRaw264.7 mouse macrophages were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovineserum (FBS) and 1% penicillin / streptomycin (PS). A549GFP1-10 cells were generously gifted by Prof. Andrew Tsourkas and cultured in DMEM with 10% FBS, 1% PS and 2 ug / mL puromycin. MLE-12 lung epithelial cells were kindly gifted by Prof. Jeremy Katzen and cultured in DMEM-F12K containing 2% FBS, 1% PS, 10 nM P-estradiol, 10 nM hydrocortisone, 2 mM L-glutamine and 1 : 100 insulin-transferrin-selenium- ethanolamine supplement. All cells were incubated with 5% CO2 at 37 °C.For 24 well-plate experiments, RAW 264.7 macrophages were seeded at 400k density using 500 uL volume and treated with 300 uL of LNP volume (diluted in DMEM). A549GFP1-10 and MLE-12 cells were seeded at a density of 60k.In Vitro Cytokine and Luciferase Delivery for measurements of mRNA Luciferase mRNA LNPs fabricated as described above were incubated with cells for 6 hours. Supernatants were collected (spun down at 10,000 x g for 15 mins) for cytokine analysis using LegendPlex 13-plex Mouse Inflammation Panel (Biolegend). Adhered cells were washed with lx PBS and collected using 0.25% trypsin. Cell counts were determined using Cell Countess after staining with trypan blue to determine cell viability. Then, cells were spun down at 300 x g for 5 mins to pellet, washed with lx PBS, and lysed with 120 uL of lx Promega Luciferase Assay System Cell Culture Lysis Reagent. For luciferase expression 20 uL of lysed sample was loaded onto a white 96 well-plate then lOOuL luciferin solution (Promega) added immediately before reading on a luminometer (Promega). Final luminescence readings were then normalized based on cell count.In Vivo Luciferase Delivery for measurement of mRNA Transfection Efficiency Luciferase mRNA LNPs fabricated as described above were injected into mice intravenously or instilled intratracheally for a circulation time of 6 hours. Select organs were then flash-frozen until the day of analysis or homogenized immediately. Samples were suspended in 900 uL of homogenization buffer (5mM EDTA, lOmM EDTA, 1 : 100 diluted stock protease inhibitor (Sigma), and lx (PBS), samples were then loaded with a steel bead (Qiagen), then placed in a tissue homogenizer (Powerlyzer 24, Qiagen) using the following settings: Speed (S) 2000 rpm, 2 Cycles (C), T time 45 sec, and pause for 30 sec). After this, 100 uL of lysis buffer (10% Triton-X 100 and PBS) was added into each tube and then allowed to incubate for 1 hr at 4C. After this, they were immediately transferred into fresh tubes, and sonicated, using a point sonicator to remove in excessDNA, using an amplitude of 30%, 5 cycles of 3 secs on / ofif After this, samples were then centrifuged at 16,000 x g for 10 minutes. The resultant lysate was either frozen or prepared for luminometry analysis.For luciferase expression 20 uL of undiluted sample was loaded onto a black 96 well-plate then lOOuL luciferin solution (Promega) added immediately before reading on a luminometer (Wallac). Last, a Lowry assay (Bio-Rad) is performed according to manufacturer specifications using diluted samples, specifically a 1 :40 dilution for lung and spleen tissues and a 1 :80 dilution for liver tissues. Final luminescence readings were then normalized based on total protein concentration obtained from Lowry Assay.Bronchoalveolar lavage (BAL) Protein and Leukocyte measurements mRNA LNPs were administered intratracheally at various doses for various timepoints. After blood collection, bronchoalveolar lavage was performed by inserting a catheter intratracheally, dispensing 800uL of ice cold lx PBS (0.5uM EDTA) using ImL BD syringe and performing a total of three washes prior to collection. Total leukocyte count was measured by diluting BAL fluid with trypan blue (1 : 1) and using cell countess for quantification. BAL fluid was spun down at 300g for 5 mins to pellet cells. The supernatant was collected and used to perform DC Protein Assay (Bio-Rad) using manufacture instructions for total BAL protein quantification. Total BAL protein and leukocyte count was normalized using total BAL volume collected.Cell viability assayCells were seeded in 96-well plates and incubated overnight (Raw264.7 cells: le5 cells / well; MLE-12 cells: 8e3 cells / well). Following different LNP treatments, 10% cell counting kit-8 (CCK-8, ALX850039KI01, Enzo Life Sciences) reagent in complete medium was added to cells for 2 h incubation. Then, CCK-8 absorbance was recorded by microplate reader at 450 nm (660 nm absorbance as reference). Cell viability rates were normalized to control cells.Apoptosi s / Necrosi s / Pyroptosi s AssayApoptotic cells were detected with fluorescent phosphatidylserine. Necrotic cells were detected with fluorescent 7-AAD. Pyroptotic cells were detected with fluorescent caspase-1. Healthy cells were detected with fluorescent cytocalcein. Markers were then quantified with flow cytometry. All markers were purchased from Abeam and used according to the manufacturer’s instructions.Complete Blood Count (CBC)Blood was collected from mice drawing from the inferior vena cava (terminal procedure). VetScan was used to obtain cell counts from whole blood according to the manufacturer’s instructions.In Vitro and In Vivo Cytokine MeasurementsCytokine measurements were carried out with a LegendPlex 13-plex Mouse Inflammation Panel (Biolegend) according to the manufacturer’s instructions.Pig Lung Cytokine MeasurementsLNPs were instilled into ex vivo pig lungs at a dose of 0.8mg of mRNA in LNPs for a duration of 3 hours at 37°C after which the BAL was harvested. TNF-a and IL-6 pig ELIS As were purchased from Abeam and used according to the manufacturer’s instructions. hMDM Cell Culture and isolationHuman peripheral blood mononuclear cells (PBMC) were separated from blood obtained from de-identified healthy donors (New York Blood Center, Long Island City, New York) by Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) gradient centrifugation. Following isolation, the percentage of monocytes in the PBMC was quantified using a monocyte isolation kit (MACS, Miltenyi Biotechnology) and PBMC were plated at a density of approximately 1 x 105 monocytes / cm2 to obtain a pure culture of human monocyte-derived macrophages (hMDM) via adherence isolation. PBMC were cultured in RPML1640 supplemented with 10% FBS, 5% human AB serum, 10 mM HEPES, 1% penicillin / streptomycin, and M-CSF (10 ng / mL) for 3 days then washed with fresh media to remove non-adherent cells. Adherent cells were cultured another 3 days in fresh media containing M-CSF. After 6 days in culture, cells are considered matured to hMDM. Experiments were performed at day 6 or 7. hMDM Immunocytochemical Staining and AnalysisHuman monocyte-derived macrophages (hMDM) were cultured in Nunc™ MicroWell™ 96-well optical-bottom plates (Thermo Fisher Scientific, Waltham, MA) and left untreated or treated with vehicle (sterile IX PBS), LPS (Ing / mL) or LNPs for 90 minutes. Treatment with LPS (1 ng / mL) for 90min was used as a positive control. Cells were then fixed using 4% paraformaldehyde for 10 mins and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were blocked for 30 min in 1% BSA and 300 mMglycine in 0.1% Tween-20 in PBS. The primary antibody rabbit monoclonal NF-KB (CST8242, 1 :400) diluted in blocking solution and incubated at 4 C overnight. Alexa Fluor 488 goat anti-rabbit secondary antibody (Thermo Fisher, 1 : 1000) was used for detection, nuclei were stained with DAPI (0.2 pg / mL) and plasma membranes were stained with cell mask deep red (CMDR, Thermo Fisher Scientific, 250 ng / mL). Images were acquired on a Cell Insight CX7 High Content screening platform, an automated confocal scanning microscope, acquiring 20 fields per well using a 20x objective. Four wells were imaged for each condition, acquiring approximately 1,000 - 1,500 cells per well (4,000 - 6,000 cells per condition). Images were acquired with a fixed exposure time of 0.07 s, 1.68 s, 0.035 s (DAPI, NF-KB, CMDR, respectively) and intra-well autofocusing at every field. Images were analyzed using HCS studio software and the Cellomics Colocalization bioapplication (Cellomics, ThermoFisher, Pittsburgh, PA). This analysis creates a binary mask for the cytoplasmic (cell mask deep red) and nuclear (DAPI) regions of interest (ROI), and then quantifies the intensity of NF-KB staining in each ROI for every cell. The NF-KB intensity in the nuclear ROI is divided by the NF-KB intensity in the cytoplasmic ROI in each individual cell to generate a nuclear colocalization ratio for each individual macrophage. This quantifies the relative amount of NF-KB in the nucleus, while also controlling for cell size and differences in the total amount of NF-KB in different cells. The nuclear colocalization ratio is averaged across all cells and wells from a particular condition, and then the average ratios from all conditions across all donors are compared using an ANOVA.Acridine orange assay by confocal imaging and flow cytometer Lysosomal pH changes were tested by acridine orange staining. To analyze acridine orange by flow cytometry, Raw264.7 cells were seeded at 4e5 cells / well and then incubated with 400 ng / mL LNP for 6 h. For positive control, 6.8 mM LLOMe was cocultured with cells for 1 h. Then, cell pellets were harvested and stained by 1 ug / mL acridine orange for 10 min. At the end of staining, add 1 mL / tube PBS immediately. Then cells were washed by centrifugation at 4°C, 300 g, 5 min and finally analyzed by Guava easyCyte flow cytometer (Luminex). To record acridine orange images, cells were seeded to 8-well p-Slide chambers (Raw264.7 cells: 1.5E5 cells / well; A549GFP1-10 cells: 1.5E4 cells / well) and then treated with LNP for 6 h. As for the positive control group, Raw264.7 cells were incubated with 6.8 mM LLOMe for 1 h and A549GFP1-10 cells were treatedwith 68 pM LLOMe for 2 h. Next, cells were labeled by 10 pg / mL acridine orange for 10 min and then imaged by LSM980 confocal microscope (Zeiss).Colocalization imaging of LNPs and endo-lysosomesCells were seeded to 8-well p-Slide chambers (Raw264.7 cells: 1.5e5 cells / well; A549GFP1-10 cells: 1.8e4 cells / well) and then treated with cKK-E12 LNPs formulated with 0.3 mol % of 18: 1 TopFluor PE-Alexa Fluor 594 for 0.5, 1 and 6 h. Endo-lysosomes were labeled by 200 nM LysoTracker DeepRed (L12492, Invitrogen) for 30 min and nuclei were stained by Hoechst33342 (R37650, Invitrogen). Fluorescent images were acquired by LSM980 microscopy (Zeiss) and colocalization of LNPs and endo-lysosomes was analyzed by Pearson’s coefficient in ImageJ.Split GFP assayA549GFP1-10 cells were seeded at 5.5e4 cells / well in 24-well plates. As reported previously58, 2 pL of Lipofectamine (13778030, Thermo Fisher) was firstly mixed with 8 uL Opti-MEM, and then 2.87 pL Img / mL SI 1 protein was added by gender pipetting. The final mixture was incubated at room temperature for 15 min. Next, the Lipofectamine: SI 1 mixture was added to cells as the total SI 1 concentration of 500 nM, followed by a 6-h incubation. For the LLOMe-treated group, cells were pretreated with 3.4 mM LLOMe for 30 min and then cocultured with SI 1 protein or Lipofectamine: SI 1 mixture for 6 h. For LNP delivery, SI 1 -loaded LNPs were diluted with complete medium and then added to cells for 6 h. Split GFP expression was measured by Guava easyCyte flow cytometer (Luminex). Si l peptides were synthesized (Lifetein) as reported previously58.Galectin and Alix puncta imaging and quantificationAfter LNP and LLOMe treatments, cells were fixed in 4% fresh paraformaldehyde for 15 min. Permeability was then performed with 0.05% saponin buffer (J63209.AK, Invitrogen) for 10 min. Then, samples were incubated with 10% normal goat serum (50062Z, Life Technologies Corp.) for 1 h. After PBS washing, cells were cocultured with 1 : 150 primary antibodies (anti Gal-1, sc-166618, Santa Cruz Biotechnology Inc.; anti-Gal- 3, sc-32790, Santa Cruz Biotechnology Inc; anti-Gal-8, sc-377133, Santa Cruz Biotechnology Inc; anti-Gal-9, ab275877, Abeam; anti-Alix, ab275377, Abeam) at 4°C overnight. Unbound antibodies were washed with PBS. Next, 1 :750 secondary antibodies were added to cells (Alexa Fluor 488-conjugated goat anti -rabbit antibody, Al 1008; Alexa Fluor 647-conjugated goat anti-rabbit antibody, A21244; Alexa Fluor 647-conjugated goatanti-mouse antibody, Al 1029; Alexa Fluor 488-conjugated goat anti-mouse antibody, Al 1008; all from Invitrogen) and incubated at 37 °C for 1.5 h. Cell nuclei were labeled by DAPI. Images were acquired by LSM980 microscopy (Zeiss).For intracellular puncta quantification, images were analyzed with Gaussian blur function by ImageJ. Simply, images were duplicated and processed with Gaussian blur to obscure dispersed background signals. Then, image subtraction was performed to remove the background noises. For Gal-9 puncta, the radius sigma was set as 1 for the duplicated image. Intracellular Gal-9 puncta were selected by size (>5). For Alix puncta, the raw image was set blurring with the radius sigma of 1 while the duplicated one was performed by the radius sigma of 2. Alix puncta were selected by size (>10). Total puncta numbers were measured and normalized to cell number in each image.Nebulized LPS ModelMice were exposed to nebulized LPS in a “whole-body” exposure chamber, with separate compartments for each mouse (MPC-3 AERO; Braintree Scientific, Inc.; Braintree MA). To maintain adequate hydration, mice were injected with 1 mL of sterile saline, 37°C, intraperitoneally, immediately before exposure to LPS. LPS (L2630-100 mg, Sigma Aldrich) was reconstituted in PBS to 10 mg mL-1 and stored at -80 °C until use. Immediately before nebulization, LPS was thawed and diluted to 5 mg mL-1 with PBS. LPS was aerosolized via a mesh nebulizer (Aerogen, Kent Scientific) connected to the exposure chamber (NEB-MED H, Braintree Scientific, Inc.). 5 mL of 5 mg mL-1 LPS was used to induce the injury. Nebulization was performed until all liquid was nebulized (~20 min).StatisticsAll results are expressed as mean ± SEM unless specified otherwise. 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Claims

CLAIMS:

1. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.

2. The method of claim 1, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.

3. The method of claim 1 or 2, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.

4. The method of claim 3, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.

5. The method of any of claims 1 to 3, wherein the galectin inhibitor is administered before the nanoparticle.

6. The method of any of claims 1 to 3, wherein the galectin inhibitor is administered simultaneously with the nanoparticle.

7. The method of any one of claims 1 to 6, wherein the nanoparticle comprises the galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.

8. The method of any one of claims 1 to 7, wherein the galectin inhibitor is an inhibitor of galectin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and / or 15.

9. The method of claim, wherein the galectin inhibitor is a pan-galectin inhibitor.

10. The method of any one of claims 1 to 8, wherein the galectin inhibitor is thiodigalactoside.

11. The method of any one of claims 1 to 8, wherein the galectin inhibitor is Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, P-D-lactosyl-steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers:galactose- or lactose-conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)- modified peptide, DB16, DB21, OTX008, PTX013, anti-galectin antibodies, galectin- specific aptamers, siRNA and shRNA-coding vectors, LLS30, or LLS2.

12. The method of any one of claims 1 to 11, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.

14. The method of any one of claims 1 to 13, wherein the nanoparticle is administered intravenously, intrathecally, or intra-arterially.

15. The method of any one of claims 1 to 14, wherein the nanoparticle and galectin inhibitor are administered to the subject via the same route.

17. The method of any one of claims 1 to 16, wherein the agent is a therapeutic, diagnostic, or imaging agent.

18. The method of any one of claims 1 to 17, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.

19. The method of any one of claims 1 to 18, wherein administration of the galectin inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the galectin inhibitor.

21. The method of claim 20, wherein the target organ is the lung.

22. The method of any one of claims 1 to 21, wherein the nanoparticle comprises RNA and administration of the galectin inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the galectin inhibitor.

23. A nanoparticle comprising an agent and a galectin inhibitor conjugated to a lipid component of the nanoparticle’s outer surface.

24. The nanoparticle of claim 23, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.

25. The nanoparticle of claim 23 or 24, wherein the galectin inhibitor is a pan-galectin inhibitor.

26. The nanoparticle of any one of claims 23 to 25, wherein the galectin inhibitor is thiodigalactoside, Belapectin (GR-MD-02), modified citrus pectin (MCP), OTX008 (Calixarene 0118), Selvigaltin (GB1211), Davanat® (GM-CT-01), GB1107, P-D-lactosyl- steroid, thiodigalactose (TDG), lactulose-L-leucine, dendrimers: galactose- or lactose- conjugated porphyrin derivatives, pectasol-C, GCS100, anginex peptide, 6BDF7 dibenzofuran (DBF)-modified peptide, DB16, DB21, OTX008, PTX013, anti-galectinantibodies, galectin-specific aptamers, siRNA and shRNA-coding vectors, LLS30, or LLS2.

27. The nanoparticle of any one of claims 23 to 26, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.

28. The nanoparticle of claim 27, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.

29. The nanoparticle of any one of claims 23 to 28, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV.

30. The nanoparticle of any one of claims 23 to 29, where the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.

31. The nanoparticle of any one of claims 23 to 30, wherein the agent is a therapeutic, diagnostic, or imaging agent.

32. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and the nanoparticle of any one of claim 23 to 31, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.

33. The method of claim 32, further comprising administering to the subject a galectin inhibitor.

34. The method of any one of claims 32 or 33, wherein the nanoparticle is administered intravenously or intra-arterially.

35. The method of any one of claims 32 to 34, wherein the nanoparticle and galectin inhibitor are administered to the subject via the same route.

36. The method of any one of claims 32 to 35, wherein the nanoparticle and galectin inhibitor are administered sequentially.

37. The method of any one of claims 32 to 36, wherein the nanoparticle is administered intravenously and inflammation in the lung is reduced or prevented.

38. The method of any one of claims 32 to 37, wherein administration of the galectin inhibitor does not alter localization of the nanocarrier when compared to delivery of the nanoparticle without administering the galectin inhibitor.

39. The method of claim 38, wherein the target organ is the lung.

40. The method of any one of claims 32 to 39, wherein the nanoparticle comprises RNA and administration of the galectin inhibitor does not alter expression of the RNA when compared to delivery of the nanoparticle without administering the galectin inhibitor.

41. A pharmaceutical composition comprising the nanoparticle of any one of claims 23 to 31 and a pharmaceutically acceptable carrier, excipient and / or preservative.

42. A method of delivering an agent to a target organ of a subject, the method comprising administering to the subject a galectin inhibitor and a nanoparticle comprising the agent, wherein inflammation resulting from administration of the nanoparticle is reduced or prevented.

43. The method of claim 44, wherein the nanoparticle is about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 5 nm to about 120 nm, about 5 nm to about 130 nm, about 5 nm to about 140 nm, about 5 nm to about 150 nm across its largest dimension.

44. The method of claim 42 or 43, wherein the nanoparticle is a lipid nanoparticle (LNP) or liposome.

45. The method of any one of claims 42 to 44, wherein the nanoparticle comprises an outer surface layer comprising a cationic lipid.

46. The method of claim 45, wherein the cationic lipid is l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DOTMA), DLin-KC2-DMA, or D-Lin-MC3-DMA.

47. The method of any one of claims 42 to 46, wherein the nanoparticle has a zeta potential greater than 0 mV, at least about 5 mV, at least about 10 mV, or at least about 15 mV.

48. The method of any one of claims 42 to 47, wherein the agent is a therapeutic, diagnostic, or imaging agent.

Images