Lipid nanoparticles formed by lipidoids for efficient delivery of nucleotide drugs and biologics

Abstract

The present technology provides a compound of Formula I, wherein PAO, R and n are defined herein. Also provided are lipid nanoparticles incorporating compounds of Formula I and methods of using same to deliver nucleotide, polynucleic acid or RNP to a cell.

Description

LIPID NANOPARTICLES FORMED BY LIPIDOIDS FOR EFFICIENT DELIVERY OF NUCLEOTIDE DRUGS AND BIOLOGICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Appl. No. 63 / 546,129, filed October 27, 2023, the contents of each of which are incorporated herein by reference in their entirety for any and all purposes.GOVERNMENT RIGHTS

[0002] This invention was made with government support under NS111688 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present technology relates to lipid nanoparticles formed by new lipidoid compounds of Formula I. The technology further relates to methods of delivering nucleotides (including nucleotide drugs) and biologies (e.g., polynucleic acids) to cells in vitro and in vivo.SUMMARY

[0004] In one aspect, the present technology provides a compound of Formula I,I whereinPAO is a linear or branched polyamine or poly(amidoamine) oligomer, each having 2 to 30 amine groups and 2 to 100 carbon atoms, wherein each ester sidechain is connected to the PAO by one of the amine groups;R at each occurrence is independently selected from a Ce-20 alkyl group, a Ce-20 alkenyl group, or TG;TG is a monosaccharide selected from the group consisting of glucose, O-protected glucose, galactose, O-protected galactose, fructose, O-protected fructose, O- protected N-acetylgalactosamine, and N-acetylgalactosamine; and n is 2 to 32.

[0005] In any embodiments of the compounds, it may be at least one occurrence of R is TG. In any embodiments, it may be the PAO is a polyethyleneimine oligomer (PEI). In any embodiments, it may be the PAO comprises a PAMAM dendrimer with an ethylenediamine core.[0006| In another aspect, the present technology provides a lipid nanoparticle (LNP) comprising a compound of Formula I as described in any embodiment disclosed herein and a PEG-lipid. The LNP may further comprise a nucleotide, polynucleic acid or ribonucleoprotein (RNP). In any embodiments, it may be the LNP comprises NAD+and / or NADH. LNPs of the present technology are stable without incorporating cholesterol or phospholipids.

[0007] In another aspect, the present technology provides a method of delivering a nucleotide, polynucleic acid or RNP to a cell comprising exposing the cell to an effective amount of an LNP. The method may comprise administering an effective amount of the LNP to a subject in need thereof. In any embodiments, it may be the subject is suffering from sepsis and / or inflammation, and the LNP comprises an effective amount of NAD+and / or NADH.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1E. FIGS. 1A and IB are schematics showing the synthesis of illustrative compounds of Formula I, wherein the R group is alkyl (FIG. 1 A) or alkyl and TG where TG is glucose (FIG. IB). Additional compounds of Formula 1 may be prepared by analogous methods using appropriate starting materials such as alkenyl acrylates and O-protectedgalactose, O-protected fructose, or O-protected N-acetylgalactosamine. FIG. 1C shows a representative 'H NMR (CDCh) spectrum of a C14P6 lipid prepared in accordance with one embodiment of the present technology. FIGS. ID and IE show the structure of C12N4 and C14N4 moieties, respectively.

[0009] FIGS. 2 A and 2B show in vitro optimization of GFP mRNA transfection in, respectively, NIH3T3 fibroblasts and in HEK293T cells using different lipidoid-formulated LNPs. GFP mRNA transfection efficiency was measured by flow cytometry quantifying GFP expression percentage. C8-C16 labels indicate the different carbon lengths of the alkyl group. P6 stands for PEI (Mn = 600 Da) and P12 stands for PEI (Mn = 1200 Da). PAMAM stands for polyamidoamine. PEG stands for DMG-mPEG2000. CPP stands for TAT47-57 peptide. UT stands for untreated. LP stands for Lipofectamine 2000.

[0010] FIGS. 3A and 3B show the in vitro cell viability of, respectively, human embryonic kidney (HEK293T) and murine fibroblasts (NIH 3T3) treated with C14P6-formulated LNP delivering mRNA at different doses (100 - 800 nanograms, ng). The cell viability was assayed by cell-counting kit-8 (CCK-8). UT stands for untreated. LP stands for Lipofectamine 2000. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test.

[0011] FIGS. 4 A and 4B show the in vivo Cre recombinase mRNA delivery (2 mg / kg) efficacy in the liver after intravenous injection of C14P6 LNP in Ail 4 mice. The total flux fold (FIG. 4B) was calculated based on IVIS images (FIG. 4A) with the tdTomato level of the PBS group set as the standard. AX1-4 show results for C14P6 LNP supplemented with PEG termini conjugated with (AX1) 10 mol% glucose, (AX2) 10 mol% galactose, (AX3) 20 mol% glucose, and (AX4) 20 mol% galactose.

[0012] FIGS. 5 A and 5B show the in vivo Cre recombinase mRNA delivery (2 mg / kg) efficacy in the lung after intravenous injection of C14P6 LNP in Ail 4 mice. The total flux fold (FIG. 5B) was calculated based on IVIS images (FIG. 5A) with the tdTomato level of the PBS group set at the standard. AX1-4 stands for C14P6 LNP supplemented with PEG terminal termini conjugated with (AX1) 10 mol% glucose, (AX2) 10 mol% galactose, (AX3) 20 mol% glucose, and (AX4) 20 mol% galactose.[0013J FIGS. 6 A and 6B show the in vivo Cre recombinase mRNA delivery (2 mg / kg) efficacy in the spleen after intravenous injection of C14P6 LNP in Ail 4 mice. The total flux fold (FIG. 6B) was calculated based on IVIS images (FIG. 6A) with the tdTomato level of the PBS group set at the standard. AX1-4 stands for C14P6 LNP supplemented with PEG termini conjugated with (AX1) 10 mol% glucose, (AX2) 10 mol% galactose, (AX3) 20 mol% glucose, and (AX4) 20 mol% galactose.

[0014] FIG. 7 shows immunofluorescence staining of liver sections from Ail4 mice receiving intravenous injection of C14P6 LNP encapsulating Cre mRNA. The ail 4 mouse genome employs a LoxP-flanked STOP cassette that consists of three SV40 polyA sequences to prevent transcription of the downstream tdTomato, a red fluorescent protein. Cre recombinase can remove the STOP cassette and then lead to the expression of tdTomato. The liver sections of mice to which C14P6 LNP encapsulating Cre mRNA were administered strongly fluoresced red compared to the PBS control. Scale bar: 100 pm.

[0015] FIG. 8 shows immunofluorescence staining of lung sections from Ail4 mice receiving intravenous injection of C14P6 LNP encapsulating Cre mRNA. The lung sections of mice to which C14P6 LNP encapsulating Cre mRNA were administered strongly fluoresced red compared to the PBS control. Scale bar: 100 pm.

[0016] FIG. 9 shows immunofluorescence staining of spleen sections from Ail4 mice receiving intravenous injection of C14P6 LNP encapsulating Cre mRNA. The spleen sections of mice to which C14P6 LNP encapsulating Cre mRNA were administered strongly fluoresced red compared to the PBS control. Scale bar: 100 pm.

[0017] FIG. 10 shows IVIS images of major organs from the Ail4 mice receiving intravenous injection of C14P6 LNPs delivering Cre mRNA (2 mg / kg). The fluorescence levels in livers were quantified. Details for each LNP sample are: (C) PBS, (1) C14P6 LNP with 75 wt% C14P6 lipidoid + 25 wt% glucose-conjugated C14P6 lipidoid, (2) C14P6 LNP with 50 wt% C14P6 lipidoid + 50 wt% glucose-conjugated C14P6 lipidoid, (3) C14P6 LNP with 25 wt% C14P6 lipidoid + 75 wt% glucose-conjugated C14P6 lipidoid, (4) C14P6 LNP with 75 wt% C14P6 lipidoid + 25 wt% glucose-conjugated C14P6 lipidoid + 25 mol% PEG termini conjugated with CPP, (5) C14P6 LNP with 50 wt% C14P6 lipidoid + 50 wt% glucose-conjugated C14P6 lipidoid + 50 mol% PEG termini conjugated with CPP.

[0018] FIGS. 11 A and 1 IB show results of in vitro Cas9 RNP and mRNA delivery efficiency using C14P6 LNP. GFP-expressing HEK293T cells were treated with C14P6 LNP encapsulating the indicated amounts of Cas9 RNP (FIG. 11 A) or Cas9 mRNA (FIG.1 IB) per Example 13. The payload amount for lipofectamine was 100 ng (LP) or 200 ng (LP2X). Gene editing efficiency was measured by flow cytometry quantifying GFP expression percentage 96 h after treatment.

[0019] FIGS. 12A and 12B show dynamic light scattering (DLS) data of hydrodynamic diameters of NAD+-loaded LNPs. FIG. 11 A: NAD+-loaded C14P6 LNP. FIG. 1 IB: NAD+- loaded C l 4P AM AM LNP.

[0020] FIG. 13 shows effective NAD+delivery by NAD oaded C14P6 LNP in HEK293T cells verified by a bioluminescent assay. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test.

[0021] FIG. 14 shows intracellular NAD(H) levels in HEK293T cells incubated with free NAD+ and C14P6-, C8PAMAM-, C12PAMAM-, C14PAMAM-, and C16PAMAM- LNPs verified by a bioluminescent assay.

[0022] FIG. 15 shows intracellular NAD(H) levels in HEK293T cells incubated with free NAD+ and various concentrations of C12PAMAM- LNP verified by a bioluminescent assay.

[0023] FIG. 16 shows intracellular NAD(H) levels in HEK293T cells incubated at various temperatures and time in University of Wisconsin (UW) solution with fresh NAD+ and C12PAMAM- LNP verified by a bioluminescent assay.

[0024] FIG. 17A shows intracellular NAD(H) levels in murine macrophages and FIG. 17B shows intracellular NAD(H) levels in HEK293T cells, both incubated with free NAD+ and C12PM-, C12N4-, and C14N4- LNPs verified by a bioluminescent assay.

[0025] FIG. 18A shows cell viability of murine macrophages and FIG. 17B shows cell viability of HEK293T cells, both incubated with free NAD+ and C12PM-, C12N4-, and C14N4- LNPs verified by a bioluminescent assay.

[0026] FIG. 19 shows the therapeutic outcome of NAD+-loaded C14P6 LNP (20 mg / kg of NAD+), in combination with free rifampin (10 mg / kg) in sepsis mice of E. coli blood infection, as demonstrated by the improvement in mouse 14-day survival rates versus PBS control and rifampin alone. Statistical significance was calculated via a log-rank test.

[0027] FIGS. 20A, 20B, and 20C show in vivo biocompatibility study in healthy mice IV injected with PBS (control) or NAD+-loaded C14P6 LNP (60 mg / kg of NAD+, 3-fold of the therapeutic dose used) on Day 0 and monitored for 7 days. FIG. 18 A: No significant body weight loss was found in mice treated with NAD+-loaded C14P6 LNP. FIG. 18B: Blood tests indicated no significant variation in mice treated with NAD+-loaded C14P6 LNP. Statistical significance was calculated via Student’s t-test. ns, not significant, P > 0.05. Abbreviations: RBC, red blood cell; HGB, hemoglobin; WBC, white blood cell; PLT, platelet; ALT, alanine transaminase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CRE, creatinine. FIG. 18C: H&E staining of major organs of mice injected with PBS or NAD+-loaded C14P6 LNP. Scale bars: 200 pm.DETAILED DESCRIPTION

[0028] The following terms are used throughout as defined below. All other terms and phrases used herein have their ordinary meanings as one of skill in the art would understand.

[0029] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0030] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt%” would be understood to mean “9 wt% to 11 wt% ” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” - for example, “about10 wt%” discloses “9 wt% to 11 wt%” as well as disclosing “10 wt% ”

[0031] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and / or C” would mean “A or B or C; A and B; A and C; B and C; or the combination of A, B, and C.”

[0032] Alkyl groups include straight chain and branched chain alkyl groups having (unless indicated otherwise) from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups.

[0033] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carbon-carbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, and -CH=CH(CH3), among others.

[0034] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups.

[0035] As used herein, the terms “effective amount” or “therapeutically effective amount,” or “pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the full or partial amelioration of disease or disorders or symptoms associated with sepsis in a subject (e.g., a mammal) in need thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. A person of ordinary skill in the art will be able to determine appropriate dosagesdepending on these and other factors. The compositions can also be administered in combination with one or more additional compounds. Multiple doses may be administered. Additionally or alternatively, multiple therapeutic compositions or compounds may administered. In the methods described herein, the compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder described herein.

[0036] As used herein, with respect to polymers, the term “poly dispersity” (or “PDI”) means the ratio Mw, / Mn, where Mw is weight average molecular weight and Mnis number average molecular weight. Polymer poly dispersity cannot be less than 1 (monodisperse).

[0037] As used herein, with respect to nanoparticles, the term “poly dispersity” (or PDI) is a number calculated from a two-parameter fit to correlation data per the ISO 22412:2017 standard. Numbers closer to 0 indicate a monodisperse population of nanoparticles.

[0038] As used herein, the term "protecting group" refers to a chemical group that exhibits the following characteristics: 1) reacts selectively with the desired functionality in good yield to give a protected substrate that is stable to the projected reactions for which protection is desired; 2) is selectively removable from the protected substrate to yield the desired functionality; and 3) is removable in good yield by reagents compatible with the other functional group(s) present or generated in such projected reactions. Examples of suitable protecting groups can be found in Greene et al. (1991) Protective Groups in Organic Synthesis, 3rd Ed. (John Wiley & Sons, Inc., New York). Thus, an O-protected group is a hydroxyl that has been masked with a protecting group. Such hydroxyl protecting groups include ethers (including alkyl, aralkyl, and silyl ethers), esters, acetals, ketals, and carbonates, among others. Hydroxyl protecting groups include but are not limited to: methoxymethyl ethers (MOM), methoxy ethoxymethyl ethers (MEM), benzyloxymethyl ethers (BOM), tetrahydropyranyl ethers (THP), benzyl ethers (Bn), p- methoxybenzyl ethers, trimethyl silyl ethers (TMS), triethyl silyl ethers (TES), triisopropyl silyl ethers (TIPS), Z-butyldimethylsilyl ethers (TBDMS), Z-butyldiphenylsilyl ethers (TBDPS), o-nitrobenzyl ethers, -nitrobenzyl ethers, trityl ethers, acetate, chloroacetate, dichloroacetate, tri chloroacetate, trifluoroacetate, benzoate (Bz), acetonide (acetone ketal of, e.g., vicinal diols), methyl carbonate, allyl carbonate (alloc), dimethylthiocarbamate (DMTC), benzyl carbonate (Cbz), / -butyl carbonate (Boc), and 9- (fluorenylmethyl) carbonate (Fmoc).

[0039] In one aspect, the present technology provides a compound of Formula I,I whereinPAO is a linear or branched polyamine or poly(amidoamine) oligomer, each having 2 to 32 amine groups and 2 to 100 carbon atoms, wherein each ester sidechain is connected to the PAO by one of the amine groups;R at each occurrence is independently selected from a Ce-20 alkyl group, a Ce-20 alkenyl group, or TG;TG is a monosaccharide selected from the group consisting of glucose, O-protected glucose, galactose, O-protected galactose, fructose, O-protected fructose, O- protected N-acetylgalactosamine, and N-acetylgalactosamine; and n is 2 to 32.[0040| The PAO of Formula I may be a linear or branched polyamine or poly (amidoamine) oligomer having 2 to 32 amine groups, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 32 amine groups or a range between any two of the foregoing values. For example, the PAO may have 4 to 30, 5 to 28, 6 to 26, or 8 to 26 amine groups. The PAO may have 2 to 120 carbon atoms, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or a range between any two of the foregoing values. For example, the PAO may have 4 to 120 carbon atoms, 6 to 120 carbon atoms, 8 to 110 carbon atoms, 4 to 100 carbon atoms, 6 to 100 carbon atoms, or 10 to 100 carbon atoms. In any embodiments the PAO may be linear. In any embodiments, the PAO may be branched. In any embodiments, the PAO may have a carbon chain of 2-4 carbons connecting a pair of amine groups. The carbon chain of 2-4 carbons may be a linear alkylene group. In any embodiments, each pair of amine groups may be connected by a carbon chain of 2-4carbons. In any embodiments, the PAO may have 6 to 24 or 8 to 26 amine groups. In any embodiments, the PAO may have 10 to 100 carbon atoms. In any embodiments, the PAO may have 10 to 16 amine groups and 18 to 60 carbon atoms. In any embodiments, the PAO may be branched.

[0041] In the compound of Formula I, n is 2 to 32, i.e., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 or a range between and / or including any two of the foregoing values. For example, n may be 4 to 8.

[0042] In any embodiments, the PAO may be a linear polyamine oligomer. In any embodiments, the PAO may be a branched polyamine oligomer, e.g., a polyethyleneimine (PEI) oligomer. In any embodiments, the PAO (including but not limited to the PEI oligomer) may have a number average molecular weight of about 200 Da to about 1800 Da, e.g., any of about 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 or a range between and including any two of the foregoing values. Thus, in any embodiments, the PAO may have a number average molecular weight of about 400 Da to about 800 Da.

[0043] In any embodiments, the PAO may comprise a poly(amidoamine) oligomer (PAMAM), e.g., a PAMAM dendrimer with an ethylenediamine core. In any embodiments, the PAMAM dendrimer may be a generation 0 or 1. In any embodiments, the PAMAM dendrimer may be capped with a C2-4 alkylenediamine, e.g., ethylenediamine. Thus, in any embodiments, the PAO may have the structure of Formula II:lt will be understood by those skilled in the art that the squiggly lines indicate the attachment points to the ester side chains of Formula I.

[0044] As a small polymer, the PAO may or may not be polydisperse. Typically, the PAO may have a poly dispersity of about 1 to about 2. For example, the poly dispersity may be any of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or may be within a range between and / or including any two of the foregoing values, such as about 1 to about 1.5.

[0045] In any embodiments of the compound of Formula I, it may be at least one occurrence of R is a Ce-20 alkylene group. In any embodiments, it may be at least one occurrence of R is a Cio-is alkylene group. In any embodiments, it may be at least one occurrence of R is an Ce-20 alkenyl group. In some such embodiments, it may be the Ce-20 alkenyl group may have 1, 2 or 3 carbon-carbon double bonds. In any embodiments, it may be at least one occurrence of R is a Cio-is alkenyl group having 1 or 2 carbon-carbon double bonds. In any embodiments, it may be at least one occurrence of R is TG. In any embodiments, TG may be glucose, galactose, fructose, or N-acetylgalactosamine. In any embodiments, it may be TG is glucose. In any embodiments, TG may be O-protected galactose, fructose, O-protected fructose, or O-protected N-acetylgalactosamine. In some such embodiments, it may be the O-protected moi eties are protected as ketals, e.g., acetonides.

[0046] The compound of Formula I may include various amounts of different R groups. For example, about 10% to about 90% or about 25% to about 75% of R groups may be Ce-20 alkyl groups (e.g., Cio-is alkyl groups or any of those disclosed herein), and the remaining R groups may be TG. In any embodiments, about 25% to about 50% of R groups may be a Ce-20 alkyl group (e.g., Cio-is alkyl groups or any of those disclosed herein) and the remaining R groups may be TG. In any embodiments, about 50% of R groups may be Ce-20 alkyl groups, e.g., Cio-is alkyl groups or any of those disclosed herein.

[0047] In another aspect, the present technology provides lipid nanoparticles (LNP) that include a compound of Formula I of any embodiment disclosed herein and a PEG-lipid. In any embodiments, it may be the LNP includes about 75 wt% to about 95 wt% of the compound of Formula I and about 5 wt% to about 25 wt% of the PEG-lipid. In any-l iembodiments, it may be the LNP includes about 85 wt% to about 90 wt% of the compound and about 10 wt% to about 15 wt% of the PEG-lipid.

[0048] LNPs of the present technology may include a variety of PEG-lipids. For example, the LNP may include DMG-PEG, DSPE-PEG and / or ceramide-PEG. In any embodiments, the PEG-lipid may include DMG-mPEG. The PEG-lipid of any embodiment may further include CPP -DMG-mPEG and / or glucose-DMG-mPEG.

[0049] LNPs of the present technology may further include a diagnostic agent, or a prophylactic or therapeutic for the treatment or prevention of a disease, disorder or condition. For example, the present LNPS may further include a nucleotide, polynucleic acid or a ribonucleoprotein complex (RNP). In any embodiments, it may be the LNP further includes a nucleotide. The nucleotide may be, e.g., NAD+, NADH, NADP+, NADPH, ADP, ATP, cGAMP, remdesivir (a nucleotide drug), or combinations of two or more thereof. In any embodiments, it may be the LNP further includes a polynucleic acid or protein-nucleic acid complex, which, e.g., may be selected from the group consisting of plasmid DNA (pDNA), single-stranded donor oligonucleotide (ssODN), complementary (cDNA), messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), transfer RNA (tRNA), ribozymes, Cas9 RNP, Cas9 RNP+ssODN or a base editor, and combinations of two or more thereof.

[0050] LNPs of the present technology exhibit average hydrodynamic diameters and polydispersities favorable for the delivery of therapeutics, nucleotides, polynucleic acids and RNPs. In any embodiments, the LNPs may exhibit an average hydrodynamic diameter of about 10 to less than 1000 nm, including any of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or less than 1000 nm or a range between and including any two of the foregoing values. Thus, in any embodiments, the LNPs may exhibit an average hydrodynamic diameter of about 30 to about 250 nm. In any embodiments, the LNPs may exhibit a poly dispersity of 0.4 or below. In any embodiments the poly dispersity of the LNPs may range from 0.05 to 0.4, e.g., 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4 or a range between and including any two of the foregoing values. In any embodiments, the poly dispersity of the LNPs may be about 0.2.

[0051] LNPs of the present technology may have a surface potential (i.e., zeta potential) near zero, e.g., -10 mV to +10 mV. LNP surface potential may be measured by DLS in an applied electric field at any suitable voltage (e.g., 40 V; the measured surface potential will be independent of the exact voltage used) at 0.1 mg / mL, pH 7.4, 25°C. Examples of the surface potential of the present LNPs include -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, +5, +6, +7., +8, +9, +10 mV or a range between and including any two of the foregoing values.

[0052] Nanoparticle surface potential may be measured by DLS in an applied electric field at any suitable voltage (e.g., 40 V; the measured surface potential will be independent of the exact voltage used) at 0.1 mg / mL, pH 7.4, 25°C. Examples of the surface potential of the present SNPs include -45, -30, -25, -20, -15, -10, -5, +5, +15, +20, +25, +30, or +45 mV, or a range between and including any two of the foregoing values.

[0053] In any embodiment disclosed herein, it may be the LNPs do not include cholesterol and / or do not include a phospholipid.

[0054] In another aspect, the present technology provides methods of delivering a diagnostic, prophylactic or therapeutic agent to a cell. The methods include exposing the cell to an effective amount of LNP comprising such agents as described herein. The methods may be carried out in vitro or in vivo. More specifically, the LNPs may include a nucleotide, polynucleic acid or a ribonucleoprotein complex (RNP) as described herein. Thus, the nucleotide, polynucleic acid or protein-nucleic acid complex to be delivered to the cell may be selected from the group consisting of NAD+, NADH, NADP+, NADPH, ADP, ATP, cGAMP, remdesivir, plasmid DNA (pDNA), single-stranded donor oligonucleotide (ssODN), complementary (cDNA), messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), transfer RNA (tRNA), ribozymes, Cas9 RNP, Cas9 RNP+ssODN, a base editor, or a combination of any two or more thereof.

[0055] In any embodiments, the methods may comprise administering an effective amount of the LNP to a subject in need thereof. The subject may be an animal, such as, but not limited to, a dog, cat, rodent, bird, primate, cattle, pig, horse, or sheep. In any embodiments, the subject may be a human. In any embodiments of the methods, the subjectmay be suffering from sepsis and / or inflammation, and the LNP comprises an effective amount of NAD+or NADH. In any embodiments, the sepsis and / or inflammation may result from an infection and the methods further include administering an effective amount of an antimicrobial to the subject. In any embodiments, the sepsis and / or inflammation may be caused by a bacterial infection and the antimicrobial is an antibacterial. In any embodiments, the sepsis and / or inflammation is caused by a viral infection and the antimicrobial is an antiviral. In any embodiments, the sepsis and / or inflammation may be caused by a fungal infection and the antimicrobial is an antifungal.

[0056] In yet another aspect, the present technology provides a method of decreasing the level of TNF-a or IL-6 in a cell, comprising administering an effective amount of any of the LNPs comprising NAD+ / NADH of any embodiment described herein to the cell. The cell may be in vitro or in vivo.

[0057] In certain embodiments, the LNP comprises an aqueous carrier and is administered to the subject intravenously or intraperitoneally (e.g., directly to the cancer site in the animal body). The aqueous carriers can include saline or an aqueous carbohydrate solution, for example, 0.9 % NaCl solution or a 5% aqueous saccharide solution. The aqueous saccharide solution can be, for example, dextrose or glucose. The aqueous carrier can also be any sterile aqueous solutions of water-soluble salts, for example, NaCl. The aqueous solutions can also be isotonic. The aqueous solutions may be suitably buffered. Aqueous solutions, described herein, can be suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, and intratumoral injection. Appropriate sterile aqueous media can be purchased or can be prepared by standard techniques well known to those skilled in the art.EXAMPLESMaterials and General Procedures

[0058] Materials. P-Nicotinamide adenine dinucleotide (NAD+) sodium salt, PAMAM dendrimer (generation 0), 1 -decanol, 1 -undecanol, 1 -tridecanol, 1 -pentadecanol, triethylamine, acryloyl chloride, dichloromethane, and ethanol were purchased from Sigma- Aldrich. Branched oligoethylenimine (PELoo, average Mn-600 by GPC), hexane, ethyl acetate, and methanol were purchased from Thermo Fisher Scientific. Dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, and stearyl acrylate were purchased from TCIAmerica. l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- mPEG2ooo), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2ooo), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2ooo-maleimide) were purchased from Avanti Polar Lipids. 3,5-O-Benzylidene-l,2-O-isopropylidene-a-D-glucofuranose was purchased from Synthose. Nuclease water was purchased from Integrated DNA Technologies. All the chemicals were used as received without further purification. Cellpenetrating peptide Cys-TAT (47-57) (CPP, sequence: CYGRKKRRQRRR-NH2) was obtained from GenScript, Inc.

[0059] Cell Culture. Human embryonic kidney (HEK293T) cells, green fluorescent protein (GFP)-expressing HEK293T cells, and NIH 3T3 fibroblasts were cultured at 37 °C in a 5% CO2 atmosphere with the Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% (v / v) fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (v / v) Penicillin-Streptomycin (Pen-Strep, Thermo Fisher Scientific).

[0060] Animals. All animal studies strictly conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin- Madison (Protocol ID: M006286, M006549). B6.CgA;i ld)SA)26SorlmNd'4G-GT',m(!l',dGe / J mice (Ail4 mice, 4-8 weeks old, male and female, JAX stock #007914) were randomly divided into groups and used to assay the in vivo Cre recombinase-mediated gene recombination and Cas9-mediated gene editing efficiency of Cre-mRNA encapsulating LNPs and Cas9 encapsulating LNPs. CD1 mice (Crl:CDl(ICR), 4-8 weeks old, male and female, Charles River Laboratories) were randomly divided into groups and used to assay the in vivo therapeutic efficacy of NAD+delivery for sepsis treatments. All mice were maintained under a tightly controlled temperature (22 °C), humidity (40-50%), and light / dark (12 / 12 h) cycle conditions, with water and food ad libitum. Before injections, the mice were anesthetized with 3% isoflurane for 8-10 min followed by 1% isoflurane during the injection.

[0061] Statistical Analysis. Data for each experiment were analyzed without further preprocessing. Results are presented as mean ± standard deviation (s.d.). Assignments of the mice to treatments and selections of fields of microscopic inspection were made at random.The small sample sizes, combined with no apparent data evidence of deviations from ANOVA assumptions (not shown), supported analysis using one-way ANOVA followed by Tukey’s post hoc comparison test. The survival benefit was determined using a log-rank test. P > 0.05 was considered not statistically significant in all analyses (95% confidence level). Statistical analyses were performed using GraphPad Prism 9.0 software.Example 1 - Synthesis of acrylate fatty chains

[0062] To a solution of hydroxyl-terminal fatty chains (14.7 mmol, 1 equiv.) in dichloromethane (DCM, 30 ml), triethylamine (44.1 mmol, 3 equiv.) and 4-methoxyphenol (1,000 ppm) were added. This solution was cooled to 0 °C in an ice bath, and acryloyl chloride (18.4 mmol, 1.5 equiv.) in 20 ml dichloromethane was added dropwise. The reaction mixture was allowed to stir for 24 hours at room temperature. The organic solvent was removed by rotary evaporator. The crude product was purified by column chromatography using silica gel as the stationary phase and a mixture of ethyl acetate / hexane (1 / 5, v / v) as eluent to yield acrylate fatty chains as a colorless liquid.Example 2 - Synthesis of PEIeoo-based lipidoids

[0063] PEIeoo-based lipidoids were synthesized via Michael addition reaction followed by deprotection as generally shown in FIG. 1 A. Branched PEIeoo (0.2 mmol, 1 equiv.) was mixed with various acrylate fatty chains (2.8 mmol, 14 equiv.). The reaction continued at 90 °C for 72 h to yield PEIeoo-based lipidoids, which were finally purified by column chromatography using silica gel as the stationary phase and a mixture of DCM / methanol (10 / 1, v / v) as eluent to yield PEIeoo-based lipidoids as a yellow liquid. TheJH NMR (CDCh) spectrum of C14P6 is representative and is shown in FIG. 1C.Example 3- Synthesis of PAMAM-based lipidoids

[0064] PAMAM generation 0 (0.2 mmol, 1 equiv.) in methanol solution was mixed with various acrylate fatty chains (1.6 mmol, 8 equiv.). The reaction continued at 90 °C for 72 h to yield PAMAM-based lipidoids, which were finally purified by column chromatography using silica gel as the stationary phase and a mixture of DCM / methanol (10 / 1, v / v) as eluent to yield PAMAM-based lipidoids as a yellow liquid.Example 4- Synthesis ofN4-based lipidoids

[0065] Triethylenetetramine (2 mmol, 1 equiv.) was mixed with various acrylate fatty chains (24 mmol, 12 equiv.). The reaction continued at 90 °C for 72 h to yield N4-based lipidoids, which were finally purified by column chromatography using silica gel as the stationary phase and a mixture of DCM / methanol (10 / 1, v / v) as eluent to yield N4-based lipidoids as a yellow liquid. The structures of C12N4 and C14N4 moi eties are shown in FIGS. ID and IE, respectively.Example 5 - Synthesis of acrylate glucose precursors

[0066] 3,5-O-Benzylidene-l,2-O-isopropylidene-a-D-glucofuranose (1.6 mmol, 1 eq) and triethylamine (4 mmol, 2.5 eq) were dissolved in dichloromethane (20 ml). Under magnetic stirring and in an ice bath, acryloyl chloride (1 mmol, 1.25 eq) in dichloromethane (40 ml) was added to the reaction mixture dropwise. 4-methoxyphenol (1,000 ppm) was added as an inhibitor to avoid polymerization. The reaction continued overnight in the dark. The organic solvent was then removed by a rotary evaporator. The product was then purified by column chromatography with silica gel and eluents with hexane / ethyl acetate (2 / 1, v / v). The final product was dried under vacuum and a white powder was yielded.Example 6 - Synthesis of glucose-conjugated PEIeoo-based lipidoids

[0067] Glucose-conjugated PEIeoo-based lipidoids were synthesized via Michael addition reaction followed by deprotection as generally shown in FIG. IB. Branched PEIeoo (0.2 mmol, 1 equiv.) was mixed with tetradecyl acrylate (2.4 mmol, 12 equiv.) and acrylate glucose precursor (0.4 mmol. 2 eq) dissolved in a minimal amount of anhydrous methanol. The reaction continued at 90 ° C for 72 h to yield protected glucose-conjugated PEIeoo-based lipidoids. Deprotection was done by dissolving the crude product in TFA / H2O (8 / 2, vol / vol) in an ice bath. Thereafter, the reaction was stirred at 20 ° C for 1 hour and the solvent was removed by high vacuum. The glucose-conjugated PEIeoo-based lipidoid was finally purified by column chromatography using silica gel as the stationary phase and a mixture of DCM / methanol (10 / 1, v / v) as eluent to yield glucose-conjugated PEIeoo-based lipidoid as a yellow liquid.Example 7- Fabrication of LNP

[0068] All LNPs were fabricated through the same procedure. Lipids (i.e., lipidoids with DMG-mPEG2ooo or DMG-mPEG2ooo and DSPE-mPEG2ooo-Mal, where Mai = maleimide) were dissolved in anhydrous ethanol and mixed proportionally according to the specific formulation. mRNA, Cas9 RNP, and NAD+were diluted with 10 mM citrate buffer (pH = 3.0), 10 mM Tris-HCl buffer (pH = 7.0), and 10 mM citrate buffer (pH = 5.0), respectively. The mixing can be done by either vortexing or a microfluidic device. For mixing by vortexing, 30 pl of payload aqueous solution was transferred to a 1.5-ml Eppendorf tube. While the solution was being vortexed, 10 pl of lipid ethanol solution was quickly added into the same tube and vortexed for another 15 seconds. For mixing using a microfluidic device, the lipid ethanol solution and the payload aqueous solution were loaded into two 1- ml syringes, respectively, and loaded on two syringe pumps. The flow rates of the ethanol solution and the aqueous solution were set at 600 pl / min and 200 pl / min, respectively, and the mixing was done in a staggered herringbone microfluidic mixer. Payloads ranged from 100 ng to 400 ng as indicated in the Figure descriptions. After the mixing process by vortexing or microfluidic device, the resulting solution was dialyzed against isotonic PBS IX buffer using a dialysis unit with a molecular weight cut-off at 20 kDa for 2 hours. For CPP-conjugated LNPs, DSPE-mPEG2000-maleimide was used to fabricate the LNP. The LNP was prepared as mentioned above and dialyzed for 2h against PBS. The dialyzed LNP suspension was then mixed with CPP solution and reacted at 20°C for 30 min. The mixture was then dialyzed again in PBS for 2h to yield CPP-conjugated LNP. Table 1 below shows six example formulations prepared with a wt / wt ratio of 10 / 1 (lipids / mRNA) in which the R group has 14 carbons and PAO was PEI having a number average weight of 600 Da. Table 2 shows NAD+LNP formulations prepared with a wt / wt ratio of 1 / 6 (lipids / NAD+). Table 3 shows PAMAM-based formulations prepared with differing R group lengths (8-16 carbons) at a wt / wt ratio of 10 / 1 (Iipids / Cas9 RNP or Cas9 lipids / mRNA+sgRNA).Example 8 - Characterization of Lipidoids

[0069] The chemical structures of synthesized molecules and lipidoids were characterized by proton nuclear magnetic resonance (*HNMR, 400 MHz, Bruker) and matrix-assisted laser desorption / ionization-time of flight (MALDLTOF) mass spectrometry (MS, Bruker microflex LRF). The hydrodynamic diameters and zeta potentials of LNPs were measured by dynamic light scattering (DLS, ZetaSizer Nano ZS90, Malvern Panalytical Ltd) andanalyzed using Zetasizer software v7.01 (Malvern Panalytical Ltd). The loading efficiencies and loading content of NAD+in LNPs were characterized by high-performance liquid chromatography (HPLC, Hitachi) with a UV detector at 260 nm. Results are shown in Tables 1-6 and in FIGS. 12A and 12B.Table 1*wt%* Formulation: Lipidoid (C14P6 or C14PAMAM) / DMG-mPEG2000 = 7 / 1, wt / wt.Table 3Table 4Table 5Table 6Example 9 - In vitro mRNA transfection assay

[0070] HEK293T and NIH 3T3 cells were seeded in 96-well plates (10,000 cells per well). After 24 h, LNP formulations encapsulating GFP-mRNA were added to the cells with a dose of mRNA at 100 ng per well. A Lipofectamine 2000 (Thermo Fisher Scientific) formulation complexed with the same doses of GFP-mRNA was prepared according to the manufacturer’s manual. After 48 h, cells were detached by Trypsin-EDTA (0.25%, Thermo Fisher Scientific) and collected for flow cytometry analysis (Attune NxT, Thermo Fisher Scientific). The percentage of GFP -positive cells and the mean fluorescence intensity (MFI) of GFP were both used to quantify GFP mRNA transfection efficiency.

[0071] A series of LNPs prepared from lipidoids with C12 to C15 alkyl esters and PEI ranging from 600-1200 Da were mixed with various percentages of DMG-mPEG2ooo and tested in NIH 3T3 cells. A second series of LNPs prepared from lipidoids with C8 to C14 alkyl esters and PAMAM were mixed with DMG-mPEG2ooo and tested in HEK293T cells. Results are shown in FIGS. 2 A and 2B.Example 10 - In vitro cytotoxicity assay

[0072] HEK293T cells were seeded in 96-well plates (10,000 cells per well). After 24 h, cells were treated with various LNP formulations. After 24 h of incubation, a standard cell counting kit-8 assay (CCK-8, Dojindo Molecular Technologies) was performed according to the manufacturer's instructions. Each well was added with 10 pl CCK-8 solution and incubated at 37 ° C for 4 h. Thereafter, the absorbance at 450 nm and 650 nm (as a reference) of each well was measured using a microplate reader (Promega Corporation). The cell viability relative to the untreated cells was finally calculated.

[0073] The cytotoxicity of C14P-LNP was tested as above. Results are shown in FIGS. 3 A and 3B. C14P6-LNP did not induce any significant toxicity at the tested doses in both cell types.Example 11 - In vivo Cre-mRNA transfection study

[0074] Ail 4 mice (n = 3 per group, 4-8 weeks old) were i.v. injected with 100 pl of PBS (negative control) and LNP solutions (with an mRNA dose of 2.5 mg / kg) via retro-orbital injections. After 7 days, the mouse was perfused with PBS IX, and their major organs including the brain, heart, liver, spleen, lung, and kidneys were collected. Ex vivo imaging was performed by IVIS to evaluate TdTomato expression levels in organs. The organs were then stored in 4% neutral -buffered formalin at 4 ° C or flash-frozen by liquid nitrogen and stored at -80 ° C.

[0075] This study was carried out using the LNPs prepared from the lipidoids indicated in Table 7. Results are shown in FIGS. 4A-4B (liver), 5A-5B (lung), and 6A-6B (spleen). Tdtomato expression was observed in each of these organs.Table 7Example 12 - Immunofluorescence staining

[0076] TdTomato expression was further investigated via immunofluorescence staining. The collected tissues and organs were embedded in optimal cutting temperature (OCT) compound blocks (Thermo Fisher Scientific), and the blocks were sectioned into 8-pm slices. Organ sections were stained with anti-RFP primary antibody (Abeam, abl52123, 1 : 1,000) for TdTomato. Sections were further stained by secondary antibodies (Abeam, abl50080, 1 : 1,000) and DAPI (Abeam, ab228549, 1 : 1,000) for visualization. After mounting on glass slides, the sections were imaged using a Nikon ARI confocal microscope. Images were exported using NIS Viewer v5.21 (Nikon).

[0077] Results are shown for liver (FIG. 7), lung (FIG. 8), and spleen (FIG. 9) with the LNPs of Table 4 and in FIG. 10 with the LNPs of Table 3.Example 13 - In vitro Cas9 genome editing assay

[0078] GFP-expressing HEK293T cells were seeded in 96-well plates (5,000 cells per well). After 24 h, LNP formulations encapsulating Cas9 RNP were added to the cells with a dose of Cas9 RNP at 300 ng per well, and LNP formulations encapsulating Cas9 mRNA + sgRNA were added to the cells with a dose of mRNA and sgRNA at 200 ng and 50 ng, respectively, per well. Lipofectamine 2000 (Thermo Fisher Scientific) formulations complexed with the same doses of Cas9 RNP or Cas9 mRNA + sgRNA were prepared according to the manufacturer’s manual. After 96 h, cells were detached by Trypsin-EDTA (0.25%, Thermo Fisher Scientific) and collected for flow cytometry analysis (Attune NxT, Thermo Fisher Scientific). The percentage of GFP-negative cells was used to quantify GFP gene disruption efficiency.

[0079] Results are shown in FIGS. 11 A (Cas9 RNP) and 1 IB (Cas9 mRNA + gRNA).Example 14 - In vitro NAD+and NAD (H) functional delivery assay

[0080] HEK293T cells were seeded into a 96-well plate (10,000 cells per well) and cultured overnight. Thereafter, the cells were treated with 10 pM free NAD+or NAD+-loaded LNPs with an equivalent amount of NAD+(10 pM) and cultured for 12 h. Subsequently, the cell culture media were removed and 50pl PBS and 50pl of NAD / NADH-Glo™ DetectionReagent (Promega) were added to each well. The cell plate was gently shaken to mix and lyse the cells and then incubated for 30 - 60 min at 200C.

[0081] Results for the in vitro NAD+delivery assay are shown in FIG. 13 (C14P6-NAD- LNP).

[0082] The cellular NAD(H) level was quantified by bioluminescence intensity using a microplate reader. As above, the cell culture media were removed and 50pl PBS and 50pl of NAD / NADH-Glo™ Detection Reagent (Promega) were added to each well. The cell plate was gently and briefly shaken to mix and lyse the cells and then incubated for 30-60 min at room temperature. The luminescence was recorded using a luminometer.

[0083] Results are shown in FIG. 14 for C14P6-, C8PAMAM-, C12PAMAM-, C14PAMAM-, and C16PAMAM-, NAD-LNPs. All CxPAMAM (x = 8-16) formulations are formed with CxPAMAM : C14PEG2000 = 7 / 1, wt / wt. The cellular NAD(H) levels in HEKs incubated with free NAD+ and LNPs for varying concentrations C12PAMAM were tested as above. Results are shown in FIG. 15 for 87.5 %, 80 %, 70 %, 60 % and 50 % C12PAMAM. FIG. 16 shows results for intracellular NAD(H) levels in HEK cells incubated NAD-C12PAMAM LNPs incubated at various temperatures and time in University of Wisconsin (UW) solution.

[0084] UW solution (e.g., Belzer UW® Cold Storage Solution) includes, in a 1000 ml solution, Pentafraction (HES) 50 g (50 g / 1); Lactobionic Acid (as Lactone) 35.83 g (105 mmol / 1); Potassium Phosphate monobasic 3.40 g (25 mmol / 1); Magnesium Sulfate heptahydrate 1.23 g (5 mmol / 1); Raffinose pentahydrate 17.83 g (30 mmol / 1); Adenosine 1.34 g (5 mmol / 1); Allopurinol 0.136 g (1 mmol / 1); Total Glutathione 0.922 g (3 mmol / 1); Potassium Hydroxide 5.61 g (100 mmol / 1); Sodium Hydroxide / Hydrochloric Acid qs pH: 7.4; Water for Injection (qs 1000 ml).

[0085] FIGS. 17A and 17B show results for intracellular NAD(H) levels in murine macrophages and HEK293T cells incubated with free NAD+ and LNPs. FIGS. 18A and 18B show cell viability results for murine macrophages and HEK293T cells incubated with free NAD+ and LNPs.Example 15 - In vivo NAD+delivery study

[0086] CD-I Mice were infected with E. coli (CFT073, 1 * 108CFU per mouse in 0.1 mL PBS) by i.v. injection and the infected mice were randomly separated into groups (n = 8). Six hours after infection, the mice were treated with PBS, free rifampin (10 mg / kg), or free rifampin (10 mg / kg) with NAD+loaded LNPs (with NAD+dose at 20 mg / kg) via i.v. injection, respectively. Their survival and body weights were monitored every 12 hours or 24 hours for 14 days.

[0087] Results are shown in FIG. 19, showing a significant survival benefit for mice administered NAD+loaded LNP and Rifampin, compared to Rifampin alone or PBS control.Example 16 - In vivo safety study for C14P6-NAD-LNP

[0088] CD-I mice (n = 3) were treated with a single i.v. injection of PBS or NAD+loaded LNPs (with NAD+dose at 60 mg / kg) on Day 0. On Day 7, the mice were sacrificed, and their blood and major organs including heart, liver, spleen, lung, and kidneys were collected. The collected blood was centrifuged at 3,000 rpm for 10 min at 4°C to obtain plasma. The organs were embedded in paraffin and sectioned into 5-pm sections.Haematoxylin and eosin (H&E) staining was carried out for histological examinations. The blood biochemical profile was assessed using VetScan Preventive Care Profile Plus rotors (Abaxis) on a VetScan VS2 blood chemistry analyzer (Abaxis), following the manufacturer's instructions. The blood routine test was assessed using a VetScan HM5 hematology analyzer (Abaxis), following the manufacturer's instructions.

[0089] Results are shown in FIGS. 20A-20C. No significant toxicity was observed in vivo.Example 17 - Preparation of Red blood cell membrane (RBCM) coated, NAD+ loaded C12N4LNP

[0090] To prepare NAD-loaded N4PEG 2.5 nanoparticles, 60 pL of 40 mg / mL NAD in lx PBS buffer (pH 5) was mixed with 20 pL of 10 mg / mL lipid solution (C12N4 / C14-PEG = 97.5 / 2.5, wt / wt). The mixture was vortexed thoroughly to form N4PEG2.5_NAD nanoparticles. The resulting nanoparticle suspension was dialyzed in 10 mM PBS (pH 7.4) for 2 hours to remove unencapsulated materials. The hydrodynamic diameter and zeta potential of the N4PEG2.5 NAD nanoparticles were measured using dynamic lightscattering (DLS). The NAD loading efficiency was quantified by high-performance liquid chromatography (HPLC).

[0091] RBCM-coated N4PEG2.5 NAD nanoparticles were prepared by mixing 1 mg / mL RBCM solution with N4PEG2.5_NAD nanoparticles in different weight ratios (1 : 1, 1.5: 1, and 2:1). Specifically, 200 pL of 1 mg / mL RBCM solution was combined with 200 pL of N4PEG2.5_NAD nanoparticle solution (1, 0.667, or 0.5 mg / mL). The mixture was subjected to extrusion through polycarbonate membranes with pore sizes of 1 pm and 400 nm (five passes each) using an Avanti mini-extruder. The RBCM-coated nanoparticles were concentrated by centrifugal filtration (14,000 * g for 15 minutes). The hydrodynamic diameter and zeta potential of the RBCM-coated nanoparticles were measured by DLS. Additionally, the NAD loading content was determined by HPLC. Results are shown in Table 8.Table 8Example 18 - Preparation of platelet membrane coated, NAD+ loaded C12N4 LNP

[0092] To prepare Cy5-labeled platelet membrane-coated N4PEG2.5 NAD nanoparticles, 44 pg of Cy5-NHS dye was added to 1 mL of a 1.5 mg / mL platelet membrane solution. Then, 500 pL of the labeled platelet membrane solution was mixed with 500 pL of a 0.75 mg / mL N4PEG2.5_NAD solution under vortexing for 1 minute, followed by 3 minutes of sonication. The resulting mixture was extruded through polycarbonate membranes with pore sizes of 1 pm, followed by 400 nm, each for five passes using an Avanti mini -extruder. This procedure was repeated to prepare a total of 4 mL of Cy5-labeled platelet membrane-coatedN4PEG2.5 NAD nanoparticle solution. The nanoparticle solution was then concentrated to 700 pL via centrifugal filtration at 5000 RPM for 15 minutes, and the NAD loading content was analyzed using high-performance liquid chromatography (HPLC). Results are shown in Table 9.Table 9EQUIVALENTS

[0093] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compositions of the present technology as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed regarding any or all of the other aspects and embodiments.

[0094] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, or compositions, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[0095] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0096] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the technology. This includes the generic description of the technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0097] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0098] All publications, patent applications, issued patents, and other documents (for example, journals, articles and / or textbooks) referred to in this specification are hereinincorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0099] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A compound of Formula I,I whereinPAO is a linear or branched polyamine or poly(amidoamine) oligomer, each having 2 to 30 amine groups and 2 to 100 carbon atoms, wherein each ester side-chain is connected to the PAO by one of the amine groups;R at each occurrence is independently selected from a Ce-20 alkyl group, a Ce- 20 alkenyl group, or TG, provided that at least one occurrence of R is TG;TG is a monosaccharide selected from the group consisting of glucose, O- protected glucose, galactose, O-protected galactose, fructose, O- protected fructose, O-protected N-acetylgalactosamine, and N- acetylgalactosamine; and n is 2 to 32.B. The compound of Paragraph A, wherein the PAO is a branched poly amine oligomer.C. The compound of Paragraph A, wherein the PAO is a branched poly(amidoamine) oligomer.D. The compound of any one of Paragraphs A-C, wherein the PAO has a carbon chain of 2-4 carbons connecting a pair of amine groups.E. The compound of Paragraph D, wherein the carbon chain of 2-4 carbons connecting each pair of amine groups in the PAO is a linear alkylene group.F. The compound of any one of Paragraphs A-E, wherein the PAO has 10 to 16 amine groups and 18 to 60 carbon atoms.G. The compound of any one of Paragraphs A-F, wherein the PAO oligomer has a number average molecular weight of about 200 Da to about 1800 Da.H. The compound of Paragraph G, wherein the PAO oligomer has a number average molecular weight of about 400 Da to about 800 Da.I. The compound of any one of Paragraphs A-B or D-H, wherein the PAO is a polyethyleneimine oligomer (PEI).J. The compound of any one of Paragraphs A or C-H, wherein the PAO comprises aPAMAM dendrimer with an ethylenediamine core.K. The compound of Paragraph J, wherein the PAMAM dendrimer is generation 0 or 1.L. The compound of Paragraph J or Paragraph K, wherein the PAO has the structure ofFormula II:M. The compound of any one of Paragraphs A-L, wherein the PAO has a poly dispersity of1 to about 2.N. The compound of any one of Paragraphs A-M, wherein at least one occurrence of R is aCe-20 alkylene group.O. The compound of any one of Paragraphs A-N, wherein at least one occurrence of R is aCio-18 alkylene group.P. The compound of any one of Paragraphs A-M, wherein at least one occurrence of R is aCe-20 alkenylene group.Q. The compound of Paragraph P, wherein at least one occurrence of R is a Ce-20 alkenylene group having 1, 2 or 3 carbon-carbon double bonds.R. The compound of Paragraph P or Paragraph Q, wherein at least one occurrence of R is aCio-18 alkenylene group having 1 or 2 carbon-carbon double bonds.S. The compound of any one of Paragraphs A-R, wherein TG is glucose, galactose, fructose, or N-acetylgalactosamine.T. The compound of any one of Paragraphs A-S, wherein TG is glucose.U. The compound of any one of Paragraphs A-T, wherein about 25% to about 75% of R groups are a C6-20 alkylene group, and the remaining R groups are TG.V. The compound of Paragraph U, wherein about 50% of R groups are a C6-20 alkylene group.W. A lipid nanoparticle (LNP) comprising a compound of any one of Paragraphs A-V and a PEG-lipid.X. The LNP of Paragraph W comprising 75 wt% to 95 wt% of the compound and 5 wt% to25 wt% of the PEG-lipid.Y. The LNP of Paragraph W or Paragraph X comprising 85 wt% to 90 wt% of the compound and 10 wt% to 15 wt% of the PEG-lipid.Z. The LNP of any one of Paragraphs W-Y, wherein the PEG-lipid comprises one or more selected from the group consisting of DMG-PEG, DSPE-PEG and ceramide-PEG.AA. The LNP of any one of Paragraphs W-Z, wherein the PEG of the PEG-lipid has a terminal moiety selected from the group consisting of OH, O-C1-4 alkyl, NH2, NHR’, COOH, COOR’, wherein R’ at each occurrence is an alkyl or alkenyl group, a dye, a targeting ligand, and a metal chelating ligand.AB. The LNP of any one of Paragraphs W-AA, wherein the PEG of the PEG-lipid has a number average molecular weight ranging from 100 to 10,000 Da.AC. The LNP of any one of Paragraphs W-AB, wherein the PEG-lipid comprises one or more of DMG-mPEG, CPP-DMG-mPEG or glucose-DMG-mPEG.AD. The LNP of any one of Paragraphs W-AC, further comprising a nucleotide, polynucleic acid or a ribonucleoprotein complex (RNP).AE. The LNP of Paragraph AD, wherein the nucleotide, polynucleic acid or protein-nucleic acid complex is selected from the group consisting of NAD+, NADH, NADP+, NADPH, ADP, ATP, cGAMP, remdesivir, plasmid DNA (pDNA), single-stranded donor oligonucleotide (ssODN), complementary (cDNA), messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA(shRNA), single guide RNA (sgRNA), transfer RNA (tRNA), ribozymes, Cas9 RNP, Cas9 RNP+ssODN or a base editor, and combinations of two or more thereof.AF. The LNP of any one of Paragraphs W-AE, wherein the LNP comprises 1 wt% to 90 wt% of the nucleotide, poly nucleic acid or RNP.AG. The LNP of any one of Paragraphs W-AF, having an average hydrodynamic diameter of about 20 to less than 1000 nm.AH. The LNP of any one of Paragraphs W-AG, having zeta potential of from -10 to + 10 mV.Al. The LNP of any one of Paragraphs W-AH, wherein the LNP does not comprise cholesterol and / or does not comprise a phospholipid.AJ. A method of delivering a nucleotide, polynucleic acid or RNP to a cell comprising exposing the cell to an effective amount of an LNP of any one of Paragraphs W-AI.AK. The method of Paragraph AJ, wherein the method is carried out in vitro.AL. The method of Paragraph AJ, wherein the method comprises administering an effective amount of the LNP to a subject in need thereof.AM. The method of Paragraph AL wherein the subject is suffering from sepsis and / or inflammation, and the LNP comprises an effective amount of NAD+or NADH.AN. The method of Paragraph AM wherein the sepsis and / or inflammation result from an infection and the methods further include administering an effective amount of an antimicrobial to the subject.AO. The method of Paragraph AN, wherein the sepsis and / or inflammation is caused by a bacterial infection and the antimicrobial is an antibacterial.AP. The method of Paragraph AN, wherein the sepsis and / or inflammation is caused by a viral infection and the antimicrobial is an antiviral.AQ. The method of Paragraph AN, wherein the sepsis and / or inflammation is caused by a fungal infection and the antimicrobial is an antifungal.

[0100] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula I,whereinPAO is a linear or branched polyamine or poly(amidoamine) oligomer, each having 2 to 30 amine groups and 2 to 100 carbon atoms, wherein each ester side-chain is connected to the PAO by one of the amine groups;R at each occurrence is independently selected from a Ce-20 alkyl group, a Ce- 20 alkenyl group, or TG, provided that at least one occurrence of R is TG;TG is a monosaccharide selected from the group consisting of glucose, O- protected glucose, galactose, O-protected galactose, fructose, O- protected fructose, O-protected N-acetylgalactosamine, and N- acetylgalactosamine; and n is 2 to 32.

2. The compound of claim 1, wherein the PAO is a branched polyamine oligomer.

3. The compound of claim 1, wherein the PAO is a branched poly(amidoamine) oligomer.

4. The compound of claim 1, wherein the PAO has a carbon chain of 2-4 carbons connecting a pair of amine groups.

5. The compound of claim 4, wherein the carbon chain of 2-4 carbons connecting each pair of amine groups in the PAO is a linear alkylene group.

6. The compound of claim 1, wherein the PAO has 10 to 16 amine groups and 18 to 60 carbon atoms.

7. The compound of claim 1, wherein the PAO oligomer has a number average molecular weight of about 200 Da to about 1800 Da.

8. The compound of claim 7, wherein the PAO oligomer has a number average molecular weight of about 400 Da to about 800 Da.

9. The compound of claim 1, wherein the PAO is a polyethyleneimine oligomer (PEI).

10. The compound of claim 1, wherein the PAO comprises a PAMAM dendrimer with an ethylenediamine core.

11. The compound of claim 10, wherein the PAMAM dendrimer is generation 0 or 1.

12. The compound of claim 10, wherein the PAO has the structure of Formula II:

13. The compound of claim 1, wherein the PAO has a poly dispersity of 1 to about 2.

14. The compound of claim 1, wherein at least one occurrence of R is a Ce-20 alkylene group.

15. The compound of claim 1, wherein at least one occurrence of R is a Cio-is alkylene group.

16. The compound of claim 1, wherein at least one occurrence of R is a Ce-20 alkenylene group.

17. The compound of claim 16, wherein at least one occurrence of R is a C6-20 alkenylene group having 1, 2 or 3 carbon-carbon double bonds.

18. The compound of claim 16, wherein at least one occurrence of R is a Cio-18 alkenylene group having 1 or 2 carbon-carbon double bonds.

19. The compound of claim 1, wherein TG is glucose, galactose, fructose, or N- acetylgalactosamine.

20. The compound of claim 1, wherein TG is glucose.

21. The compound of claim 1, wherein about 25% to about 75% of R groups are a C6-20 alkylene group, and the remaining R groups are TG.

22. The compound of claim 21, wherein about 50% of R groups are a C6-20 alkylene group.

23. A lipid nanoparticle (LNP) comprising a compound of any one of claims 1-22 and aPEG-lipid.

24. The LNP of claim 23 comprising 75 wt% to 95 wt% of the compound and 5 wt% to 25 wt% of the PEG-lipid.

25. The LNP of claim 23 comprising 85 wt% to 90 wt% of the compound and 10 wt% to15 wt% of the PEG-lipid.

26. The LNP of claim 23, wherein the PEG-lipid comprises one or more selected from the group consisting of DMG-PEG, DSPE-PEG and ceramide-PEG.

27. The LNP of claim 23, wherein the PEG of the PEG-lipid has a terminal moiety selected from the group consisting of OH, O-C1-4 alkyl, NH2, NHR’, COOH, COOR’, wherein R’ at each occurrence is an alkyl or alkenyl group, a dye, a targeting ligand, and a metal chelating ligand.

28. The LNP of claim 23, wherein the PEG of the PEG-lipid has a number average molecular weight ranging from 100 to 10,000 Da.

29. The LNP of claim 23, wherein the PEG-lipid comprises one or more of DMG-mPEG,CPP-DMG-mPEG or glucose-DMG-mPEG.

30. The LNP of claim 23, further comprising a nucleotide, polynucleic acid or a ribonucleoprotein complex (RNP).

31. The LNP of claim 30, wherein the nucleotide, polynucleic acid or protein-nucleic acid complex is selected from the group consisting of NAD+, NADH, NADP+, NADPH, ADP, ATP, cGAMP, remdesivir, plasmid DNA (pDNA), single-stranded donor oligonucleotide (ssODN), complementary (cDNA), messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), transfer RNA (tRNA), ribozymes, Cas9 RNP, Cas9 RNP+ssODN or a base editor, and combinations of two or more thereof.

32. The LNP of claim 23, wherein the LNP comprises 1 wt% to 90 wt% of the nucleotide, polynucleic acid or RNP.

33. The LNP of claim 23, having an average hydrodynamic diameter of about 20 to less than 1000 nm.

34. The LNP of claim 23, having zeta potential of from -10 to + 10 mV.

35. A method of delivering a nucleotide, polynucleic acid or RNP to a cell comprising exposing the cell to an effective amount of an LNP of any one of claims 25-34.

36. The method of claim 35, wherein the method is carried out in vitro.

37. The method of claim 35, wherein the method comprises administering an effective amount of the LNP to a subject in need thereof.

38. The method of claim 37, wherein the subject is suffering from sepsis and / or inflammation, and the LNP comprises an effective amount of NAD+or NADH.

39. The method of claim 38, wherein the sepsis and / or inflammation result from an infection and the methods further include administering an effective amount of an antimicrobial to the subject.

40. The method of claim 39, wherein the sepsis and / or inflammation is caused by a bacterial infection and the antimicrobial is an antibacterial.

41. The method of claim 39, wherein the sepsis and / or inflammation is caused by a viral infection and the antimicrobial is an antiviral.

42. The method of claim 39, wherein the sepsis and / or inflammation is caused by a fungal infection and the antimicrobial is an antifungal.

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