Phlip ®-LNP for targeted intracellular delivery of nucleic acid therapeutics

Abstract

Compositions comprising multiple pHLIP® peptides linked to a lipid nanoparticle encapsulating one or more nucleic acids (pHLIP®-LNP) and methods of preparing the pHLIP®-LNPs are described.

Description

PHLIP®-LNP FOR TARGETED INTRACELLULAR DELIVERY OF NUCLEIC ACID THERAPEUTICS CROSS REFERENCE TO A RELATED APPLICATION

[0001] This PCT application claims the benefit of U.S. Provisional Patent Application No.63 / 549,959, filed February 5, 2024. The entire teaching of the above-referenced application is incorporated herein by reference. FIELD

[0002] Compositions comprising multiple pHLIP®peptides linked to a lipid nanoparticle (“LNP”) encapsulating one or more nucleic acids (“pHLIP®-LNP”) and methods of preparing the pHLIP®-LNPs are disclosed herein. BACKGROUND

[0003] RNA- and gene-based therapeutics are rapidly emerging due to progress in the synthesis of stable nucleic acids. Also, the progress in lipid formulations (e.g., lipid nanoparticle (“LNP”)), in the use of viral envelopes, and in the application of other delivery vehicles has made it possible to encapsulate nucleic acid therapeutics for injection into biological systems. However, key challenges remain, including, but not limited to, i) targeted delivery of nucleic acid therapeutics to diseased tissues other than the liver, and ii) enhanced cytoplasmic delivery of nucleic acid payloads into specific cells.

[0004] Disclosed herein are pH Low Insertion Peptide (“pHLIP®”) linked LNPs encapsulating one or more nucleic acid payloads (“pHLIP®-LNP”) that solve the challenges discussed above. pHLIP®peptides on the surface of an LNP serve both as targeting moieties and as enhancers of intracellular delivery of pHLIP®-LNPs. Previously, pHLIP®-mediated targeted delivery of nanomaterials to acidic cell surfaces was reported, including liposomes and niosomes, but not for encapsulated nucleic acids. The pHLIP®- LNPs described herein comprise LNPs having a high content of positively charged / ionizable lipids, e.g. >10% molar ratio. Additionally, the pHLIP®-LNPsdescribed herein target delivery of the nucleic acid payloads to naturally occurring diseased, acidic tissues, and / or artificially created acidic loci. Therapeutic nucleic acid payloads in pHLIP®-LNP formulations could be used for prevention and / or treatment of a variety of diseases. BRIEF SUMMARY

[0005] Compositions comprising Formula (I): (pHLIP®peptides)n–LNP (I) wherein n is 1 to 10,000; wherein LNP is a lipid nanoparticle comprising (a) at least 10% molar ratio of cationic or ionizable lipids; and (b) at least 10% molar ratio of cholesterol and / or its naturally occurring analogs; and wherein each “–” is a covalent bond or a non-cleavable linker are described herein.

[0006] In some aspects, the composition of Formula (I) further encompasses one or more nucleic acid payloads (pHLIP®-LNPs). In some aspects, in the pHLIP®-LNPs, the one or more nucleic acid payloads are encapsulated within the LNP to which the pHLIP®peptide is covalently bonded or linked with a non-cleavable linker.

[0007] In some aspects, the one or more pHLIP®peptides comprise the following sequence: XnYm; YmXn; XnYmXj; YmXnYi; YmXnYiXj; XnYmXjYi; YmXnYiXjYl; XnYmXjYiXl; YmXnYiXjYlXh; XnYmXjYiXhYg; YmXnYiXjYlXhYg; XnYmXjYiXhYgXf; (XY)n; (YX)n; (XY)nYm; (YX)nYm; (XY)nXm; (YX)nXm; Ym(XY)n; Ym(YX)n; Xn(XY)m; Xn(YX)m; (XY)nYm(XY)i; (YX)nYm(YX)i; (XY)nXm(XY)i; (YX)nXm(YX)i; Ym(XY)n; Ym(YX)n; Xn(XY)m; or Xn(YX)m, wherein, (i) each Y is, individually, a non-polar amino acid with solvation energy, DGXcor> +0.50, or Gly; (ii) each X is, individually, a protonatable amino acid, (iii) n, m, i, j, l, h, g, f are each, individually, an integer from 1 to 8.

[0008] In some aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®- LNPs described herein comprises a sequence comprising the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys),Azido-containing amino acid or other modified amino acids. In other aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence consisting of the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids. In some aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence comprising the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227). In other aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence consisting of the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).

[0009] In one aspect, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence selected from the group consisting of any one of SEQ ID NOs.1-225. In some aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein consists of a sequence selected from the group consisting of any one of SEQ ID NOs.1-225.

[0010] In some aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®- LNPs described herein comprises a polar flanking sequence at the membrane non- inserting end of the pHLIP®peptide, which is linked to a lipid, e.g., PEGylated (polyethylene glycol, e.g., PEG2000) lipid or pSar (polysarcosine) lipid. In some aspects, the pHLIP®polar flanking sequence of the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises 2 to 10 negatively-charged and / or polar residues. In some aspects, the pHLIP®polar flanking sequence of the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein consists of one or more of the following residues: Asp, Glu, Asn, Gln, Tyr, Ser, and Thr. In an additional aspect, the pHLIP®polar flanking sequence of the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence consisting of 2 to 10 of the sequence repeats Asp-Asn.

[0011] In some aspects, all amino acids in the pHLIP®peptide of Formula (I) or pHLIP®- LNPs described herein are D-amino acids.

[0012] In some aspects, the LNP of Formula (I) or pHLIP®-LNPs described herein comprises from about 10% to about 60% of one or more cationic or ionizable lipids. In some aspects, the ionizable lipids have a pK of about 5.5 to about 9.5. In some aspects,the ionizable lipids are propargylamine-based lipids. In an additional aspect, the cationic / ionizable lipids are selected from the following lipids: 11-10-8 (((2-(pyrrolidin-1- yl)ethyl)azanediyl)bis(decane-1,2-diyl) dioctanoate); 119-23 (9Z-octadecenoic acid, 6- [[3-(dimethylamino)propyl][(9Z)-1-oxo-9-octadecen-1-yl]amino]-7-oxo-7- (tricyclo[3.3.1.13,7]dec-1-ylamino)heptyl ester); 244cis (4-[2-[bis[9-[(2Z)-2-nonen-1- yloxy]-9-oxononyl]amino]ethyl]-1-piperazinenonanoic acid, (2Z)-2-nonen-1-yl ester); 304-O13 (tri-N-tridecyl 3-(ethyl(methyl)amino)propanoate or trimidin); 306-N16B (3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)) tetrakis(N-(2- (dodecyldisulfaneyl) ethyl)propanamide) or disulpax); 306-O12B (tetrakis(2- (octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl)) tetrapropionate or triscormin); 306Oi9-cis2 (tetra((Z)-non-2-en-1-yl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate); 80- O18 (N-[3-(dimethylamino)propyl]-N-[3-(octadecyloxy)-3-oxopropyl]-β-alanine, octadecyl ester); 98N12-5 (^^1,^^16-didodecyl-4,7,13-tris[3-(dodecylamino)-3- oxopropyl]-4,7,10,13-tetraazahexadecanediamide); 9A1P9 (2-(dioctylamino)ethyl nonyl hydrogen phosphate or lidophos); A18-ISO5-2DC18 (1H -Imidazole-2-carboxylic acid, 1-[3-(2-ethyl-1-piperidinyl)propyl]-5,5-di-(8Z )-8-heptadecen-1-yl-2,5-dihydro-, ethyl ester (ACI) or pimidol); AA3-Dlin (piperazine-1,4-diylbis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); ALC-0315 (2-hexyl-decanoic acid, 1,1'- [[(4-hydroxybutyl)imino]di-6,1-hexanediyl]); AMAP-O16B (bis(2- (dodecyldisulfaneyl)ethyl) 3,3'-((4-methyl-11-oxo-12-oxa-15,16-dithia-4,8- diazaoctacosyl)azanediyl)dipropionate) and analogues; ATX-100 (4,4′-[[[[3- (dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1′-bis(1-heptyloctyl) ester); BAmP-O16B (bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-(4-(3-((3-(2- (dodecyldisulfaneyl)ethoxy)-3-oxopropyl)amino)propyl)piperazin-1- yl)propyl)azanediyl)dipropionate); C12-200 (1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl) amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2- dodecanol); C13-112-tetra-tail (14,23-bis(2-hydroxytridecyl)-17,20-dioxa-14,23- diazahexatriacontane-12,25-diol); C14-A1 (1,1'-((5-(4-(2-((5-(bis(2-hydroxytetradecyl) amino)pentyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1- yl)pentyl)azanediyl)bis(tetradecan-2-ol)); C3-K2-E14 (3,3'-(propylazanediyl)bis(N-(2- (bis(2-hydroxytetradecyl)amino) ethyl)propanamide); Cationic Lipid A or Lipid III-45 (2-butyl-octanoic acid, 1,1′-[[(3-hydroxypropyl)imino]di-9,1-nonanediyl] ester); Cho-Arg (cholest-5-en-3-ol (3β)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl)amino]-1- oxopentyl]amino]hexanoate], 2,2,2-trifluoroacetate (1:2)); cKK-E12 (3,6-bis[4-[bis(2- hydroxydodecyl)amino]butyl]-2,5-piperazinedione); cKK-E15 (3,6-bis[4-[bis(2- hydroxypentadecyl)amino]butyl]-2,5-piperazinedione); CL15F6 (1-methyl-4- piperidinecarboxylic acid, 11-[(2-hexyl-1-oxodecyl)oxy]-5-[6-[(2-hexyl-1- oxodecyl)oxy]hexyl]-5-hydroxyundecyl ester); CL4F8-6 (7-(4-(dipropylamino)butyl)-7- hydroxytridecane-1,13-diyl bis(2-hexyloctanoate)); CP-LC-0743 (3-[[4-[[2- (dimethylamino)ethyl]amino]-3-[(2-hexyl-1-oxodecyl)amino]-4-oxobutyl]thio]-propanoic acid, 9Z-octadecen-1-yl ester); CP-LC-0867 (3-[[4-[[2-(dimethylamino)ethyl]amino]-3- [(2-octyl-1-oxododecyl)amino]-4-oxobutyl]thio]-propanoic acid, 2-ethylhexyl ester); CP- LC-1254 (2-[[[2-(dimethylamino)ethyl]amino]carbonyl]-4-[[3-[(2-hexyldecyl)oxy]-3- oxopropyl]thio]-butanoic acid, 2-hexyldecyl ester); CP-LC-1428 (6-methylheptyl 7-(2- ((3-((2-butyloctyl)oxy)-3-oxopropyl)thio)ethyl)-2-methyl-14-((3-((6-methylheptyl)oxy)- 3-oxopropyl)thio)-6,9-dioxo-17-thia-2,5,8-triazaicosan-20-oate); DC-1-16 (N-hexadecyl- N,N-dimethyl-1-hexadecanaminium, monobromide); DC-Cholesterol ((3^^)-3-[N-[2- (dimethylamino)ethyl]carbamate cholest-5-en-3-ol hydrochloride); DDAB (dimethyldioctadecylammonium (Bromide Salt)); DGTS (diacylglyceryl-N,N,N- trimethylhomoserine); Dios-Arg ((3β,25R)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl) amino]-1-oxopentyl]amino]hexanoate] spirost-5-en-3-ol, bis(2,2,2-trifluoroacetate)); DLin-DMA (N,N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-1- propanamine); DLin-KC2-DMA (N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl- 1,3-dioxolane-4-ethanamine); DLin-KC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DLin- MC3 DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien- 1-yl-10,13-nonadecadien-1-yl ester); Dlin-MC4-DMA (5-(dimethylamino)-pentanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DMT7 (1-(2-(dimethylamino)ethyl)-3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl)thiourea); DMTAP (N,N,N-trimethyl-2,3-bis[(1-oxotetradecyl)oxy]-1- propanaminium, monochloride); DOBAQ (N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium); DODAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9- octadecen-1-aminium, monochloride); DODAP (1,2-dioleoyl-3-dimethylammoniumpropane); DODMA (N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanamine); DODMAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9-octadecen-1- aminium, monochloride); DOEPC (4-ethoxy-N,N,N-trimethyl-10-oxo-7R-[[(9Z)-1-oxo- 9-octadecenyl] oxy]-3,5,9-trioxa-4-phosphaheptacos-18Z-en-1-aminium 4-oxide, monochloride); DOG-IM4 (N-[(25Z)-14-[(9Z)-9-octadecen-1-yloxy]-3,6,9,12,16- pentaoxatetratriacont-25-en-1-yl]-1H-imidazole-5-carboxamide); DOSPA (N-[2-[[2,5- bis[(3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(9Z)-9- octadecen-1-yloxy]-1-propanaminium, chloride, hydrochloride (1:1:4)); DOTAP (N,N,N- trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanaminium, monochloride) and analogues; DOTMA (N,N,N-trimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanaminium, monochloride); DPDAP (hexadecanoic acid, 1,1′-[1- [(dimethylamino)methyl]-1,2-ethanediyl] ester); E12CA1A3 (2-hexyl-decanoic acid, 2- [4-(dimethylamino)-1-oxobutoxy]dodecyl ester); FTT5 (9,9′,9′′,9′′′,9′′′′,9′′′′′-[1,3,5- benzenetriyltris(carbonylimino-3,1-propanediylnitrilo)]hexakis-nonanoic acid, 1,1′,1′′,1′′′,1′′′′,1′′′′′-hexakis(1-ethylhexyl) ester); GL67 (3β-[(3-aminopropyl)[4-[(3- aminopropyl)amino]butyl]carbamate] cholest-5-en-3-ol); H1L1A1B3 (7- (cyclohexylamino)-6-(N-(3-(dimethylamino)propyl)-4-methylnonanamido)-7-oxoheptyl dodecanoate); Hexa Lipid 114 (ditetradecyl 4,8,13,17-tetrakis(3-oxo-3- (tetradecyloxy)propyl)-4,8,13,17-tetraazaicosanedioate); IZ-Cholesterol ([3-(1H- imidazol-1-yl)propyl]carbamate, cholest-5-en-3β-ol); L319 (9-[4-(dimethylamino)-1- oxobutoxy]-heptadecanedioic acid, 1,17-di-(2Z)-2-nonen-1-yl ester); Lipid 14 (((2-((4- (dimethylamino)butanoyl)oxy)ethyl)azanediyl) bis(octane-8,1-diyl) bis(2- hexyldecanoate)); Lipid 16 (7-(((4-(dimethylamino)butanoyl)oxy) ((9Z,12Z)-octadeca- 9,12-dien-1-yl)amino)heptyl decanoate); Lipid 2-10 (10-[[3-(dimethylamino)propyl](1- oxooctyl)amino]-nonadecanedioic acid, 1,19-bis(1-hexylheptyl) ester); Lipid 29 (8-[[8- [(1-ethylnonyl)oxy]-8-oxooctyl][3-[[2-(methylamino)-3,4-dioxo-1-cyclobuten-1- yl]amino]propyl]amino]-octanoic acid, 1-octylnonyl ester); Lipid 369 (9-[4- (dimethylamino)-1-oxobutoxy]-heptadecanedioic acid, 1,17-bis(3-pentyloctyl) ester); Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1- octylnonyl ester); Lipid 50 (2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy) decanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)); Lipid A (2-hexyl-decanoic acid, 1,1′-[[[1-(hydroxymethyl)propyl]imino]di-6,1-hexanediyl] ester); Lipid A4 (1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxydodecyl) amino)ethoxy)ethyl)(2-hydroxydodecyl)amino) ethyl) piperazin-1-yl)ethoxy)ethyl) azanediyl)bis(dodecan-2-ol)); Lipid A9 (bis(2-butyloctyl) 10-(N-(3-(dimethylamino) propyl)nonanamido)nonadecanedioate); Lipid C2 (N1,N4-bis[2-[bis(2- hydroxytetradecyl)amino]ethyl]-1,4-piperazinedipropanamide); Lipid DIM1 (1,1',1'',1'''- (((((3R,3aS,6S,6aS)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(propane-3,1- diyl))bis(azanetriyl))tetrakis(dodecan-2-ol)); Lipid OA2 (di((Z)-octadec-9-en-1-yl) L- lysyl-L-glutamate, dihydrochloride); Lipid R6 (2-((3-(diethylamino)propyl)amino)-N- undecylheptadecanamide); Lipid U105 (di(heptadecan-9-yl) 6,6'-((3-(bis(2- hydroxyethyl)amino)propyl)azanediyl)dihexanoate); LP-01 (9,12-Octadecadienoic acid (9Z,12Z)-, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy] carbonyl]oxy]methyl]propyl ester); MVL5 (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4- [di(3-amino-propyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); ND98 (N1,N16-didodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13- tetraazahexadecanediamide); O12-D3-I3 (didodecyl 3,3'-((3-(2-amino-3-(1H-imidazol-4- yl)propanamido)propyl)azanediyl) dipropionate); OC2-K3-E10 (3,3'-((2- hydroxyethyl)azanediyl)bis(N-(3-(bis(2-hydroxydecyl)amino) propyl)propanamide)); OF- 02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5- piperazinedione); OF-C4-Deg-Lin (9,12-octadecadienoic acid, (9Z,12Z)-1,1′,1′′,1′′′-[(3,6- dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi-4,1-butanediyl)] ester); OF-Deg-Lin (9Z,12Z-octadecadienoic acid, 1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1- butanediylnitrilodi-2,1-ethanediyl)] ester); PL1 (di(octan-3-yl) 9,9'-((3-((3- ((diethoxyphosphoryl)oxy)propyl)(9-(octan-3-yloxy)-9- oxononyl)amino)propyl)azanediyl) dinonanoate); PPPDA-O16B (bis(2- (dodecyldisulfaneyl) ethyl) 3,3'-(propylazanediyl)dipropionate); PPZ-A10 (N,N'- (piperazine-1,4-diylbis(propane-3,1-diyl))bis(3-(didecylamino)propanamide)); Q1-SM- 102 (((2-((4-(dimethylamino)butanoyl) oxy)ethyl)azanediyl)bis(octane-8,1-diyl) bis(2- hexyldecanoate)); SAINT C-18 (1-methyl-4-[(10Z)-1-(9Z)-9-octadecen-1-yl-10- nonadecen-1-yl]-pyridinium, monochloride); SIL Lipid (N,N′,N′′-(nitrilotri-2,1- ethanediyl)tris[N-methyl-β-alanine, 1,1′,1′′-tridodecyl ester); SM-102 (8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester) and SM-102 Analogue; TCL053 (2-(((4-(dimethylamino) butanoyl)oxy)methyl)-2-((((Z)- tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)-bis(tetradec-9-enoate)); THP1 or tetrahydropyrimidine 1 Lipid (diethyl 1,3-dioctyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylate); TT3 (N1,N3,N5-tris[3-(didodecylamino)propyl]-1,3,5- benzenetricarboxamide); YK-009 (6-[[4-(decyloxy)-4-oxobutyl](2-hydroxyethyl) amino]- hexanoic acid, 2-octyldecyl ester); YSK05 (1-methyl-4,4-bis[(9Z,12Z)-9,12- octadecadien-1-yloxy]-piperidine); YSK13-C3 (3-(dimethylamino)propyl (12Z,15Z)-3- ((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trienoate); ZA3-Ep10 (10-[2-[bis(2- hydroxydecyl) amino]ethyl]-15-hydroxy-7,13-bis(2-hydroxydexyl)-N,N-dimethyl-4-oxo- B-(3-sulfopropyl)-3,7,10,13-tetraazatricosan-1-aminium, inner salt), and combinations thereof.

[0013] In some aspects, the LNP of the pHLIP®-LNPs described herein comprises from about 10% to about 60% of cholesterol and / or its naturally occurring analogs. In some aspects, the LNP of the pHLIP®-LNPs described herein comprises cholesterol. In some aspects, the cholesterol is from plant origin (e.g., BotaniChol®).

[0014] In other aspects, pHLIP®-LNPs described herein comprise at least one naturally occurring analog of cholesterol. In some aspects, the naturally occurring analogs of cholesterol are selected from vitamin D3, vitamin D2, calcipotriol, stigmasterol, β- sitosterol, sitostanol, betulin, lupeol, ursolic acid, oleanolic acid, or chemical alkyl derivatives (stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, dehydroergosterol), and combinations thereof.

[0015] In some aspects, the LNP of the pHLIP®-LNPs described herein additionally comprises about 5% to about 25% of other lipid ingredients. In additional aspects, the other lipid ingredients include DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine), DOPE (1,2-dioleoyl-sn- glycero-3-phosphoethanolamine), DMPC (1,2-Dimyristoyl-sn-glycero-3- phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), PS (Phosphatidylserine) and / or other diacyl phospholipids, and combinations thereof.

[0016] In some aspects, the LNP additionally comprises PEG-lipids. In other aspects, the LNP additionally comprises PEG-free polymers (e.g. pSar-lipids).

[0017] In some aspects, the compositions of Formula (I) described herein further encompasses one or more nucleic acid payloads (pHLIP®-LNPs). In the pHLIP®-LNPs, the one or more nucleic acid payloads are encapsulated within the LNP to which the pHLIP®peptide is covalently bonded or linked with a non-cleavable linker.

[0018] In some aspects, the nucleic acid payloads of the pHLIP®-LNPs are RNA and / or DNA molecules. In some aspects, the nucleic acid payloads of the pHLIP®-LNPs areselected mRNA (messenger RNA), miRNA (micro RNA), saRNA (self-Amplyfing RNA), saRNA (small activating RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), piRNA (piwi-interacting RNA), crRNA (CRISPR RNA), gRNA (guide RNA), tracrRNA (trans-activating CRISPR RNA), ncRNA (non-coding RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), regions of lncRNA (long non-coding RNA), and other RNAs from protein-nucleic acid complexes such as signal recognition particles, aptamers, antisense oligonucleotides, and CpG oligonucleotides, pDNA(plasmid DNA), ceDNA (closed ends DNA), cDNA (circular DNA), and combinations thereof.

[0019] In some aspects, the pHLIP®peptide and LNP of Formula (I) are linked by a covalent bond or a non-cleavable linker. In some aspects, the pHLIP®peptide and LNP of Formula (I) described herein are linked by a covalent bond.

[0020] In some aspects, the N-terminus of the pHLIP®peptide is covalently bonded to the LNP of Formula (I).

[0021] In some aspects, pharmaceutical compositions comprising any one of the compositions described herein and one or more pharmaceutically acceptable excipients are provided herein.

[0022] In some aspects, provided herein are methods of delivering a nucleic acid payload to a cell and / or a diseased tissue comprising administering to the cell and / or diseased tissue a composition or a pharmaceutical composition described herein.

[0023] In some aspects, methods of treating diseases in a subject in need thereof comprising administering a composition or a pharmaceutical composition described herein to the subject are provided herein. In some aspects, methods of prevention diseases by vaccination in a subject in need thereof comprising administering a composition or a pharmaceutical composition described herein to the subject are provided. DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows a schematic presentation of the pHLIP®-LNP structure.

[0025] Figure 2 shows a DSPE-PEG-pHLIP®structure using a pHLIP®peptide having the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).

[0026] Figure 3 shows an exemplary reaction pathway for the synthesis of DMG- PEG2000.

[0027] Figure 4 shows a HPLC chromatogram indicating the presence of the pHLIP®- DSPE-PEG2000 conjugate after the completion of coupling reaction.

[0028] Figure 5 shows a HPLC chromatogram indicating the presence of the pHLIP®- DSPE-PEG2000 conjugate in a final sample after purification. DETAILED DESCRIPTION

[0029] The present disclosure provides compositions comprising multiple pHLIP®peptides linked to an LNP described herein. In some aspects, the LNP comprises one or more nucleic acids. As described herein, an advantage of the disclosed compositions comprising pHLIP®peptides linked to an LNP and pHLIP®-LNPs is targeted delivery of, e.g., nucleic acid therapeutics to diseased tissue(s) and enhanced delivery of the nucleic acid payloads to the cells of the diseased tissue(s). Surprisingly, compositions comprising pHLIP®peptides linked to an LNP, as described herein, provide stable and homogeneous formulations without diminishing encapsulation efficiency of nucleic acid payloads. Definitions

[0030] A lipid nanoparticle (“LNP”) is a liposome-like structure composed primarily of cationic lipids, along with other lipid ingredients. The LNP can encapsulate nucleic acids.

[0031] A pHLIP®peptide (also known as a pH Low Insertion Peptide) is a linear, water- soluble membrane peptide that interacts weakly with a cell membrane at neutral pH, without insertion into the lipid bilayer, but inserts into the cell membrane and forms a stable transmembrane alpha-helix at a slightly acidic pH (<7.0). A pHLIP®peptide senses acidity or low pH and targets it to the surfaces of cancer and stromal cells, activated macrophages, and some other immune cells in tumors, inflamed tissues, or any naturally or artificially created acidic tissues. When pHLIP®peptides are exposed to a low pH environment at the surfaces of acidic cells, its negatively charged residues are protonated to become neutral, which enhances the pHLIP®hydrophobicity and triggers insertion of the peptides into the plasma membranes of targeted cells. Multiple insertion events of multiple pHLIP®peptides into cellular membrane promotes membrane fusion and release of an encapsulated payload in the cytoplasm.

[0032] Even if some pHLIP®-LNPs described herein, are taken up by endocytosis, in the environment of the late endosome, where the pH is low (pH 5.0-5.5) the one or morepHLIP®peptides of the pHLIP®-LNPs described herein readily insert into the endosomal membrane, promoting fusion and cytoplasmic release of encapsulated payloads. By binding pHLIP®peptides, or a pHLIP®equivalent, to an LNP encapsulated with nucleic acids, pHLIP®peptides will target the LNP(s) to acidic diseased tissues or artificially created acidic tissues and promote intracellular delivery of nucleic acid payloads into the cytosols of targeted cells. Delivering therapeutic nucleic acid payloads encapsulated within the pHLIP®-LNPs described herein activates and / or blocks synthesis of specific proteins for treatment and / or prevention of diseases.

[0033] As used herein, “effective” when referring to an amount of a composition described herein, refers to the quantity of the composition that is sufficient to yield a desired response. For example, the amount of cargo, e.g., nucleic acid in the LNP, yields a desired response, e.g., therapeutic effect in targeted tissue, without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of this disclosure.

[0034] In some aspects, a subject administered the composition of Formula (I) or pHLIP®-LNPs described herein is a mammal. In certain aspects, the subject is a mammal. In some aspects, the mammal is a rodent (e.g., a mouse or a rat), a primate (e.g., a chimpanzee, a gorilla, a monkey, a gibbon, a baboon), a cow, a camel, a dog, a cat, a horse, a llama, a sheep, a goat, or a pig. In some aspects, the subject is a human.

[0035] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0036] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease,” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.

[0037] As used herein, “treating” encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the prevention or amelioration ofany symptom or symptoms of the disorder. As used herein, “inhibition” of disease progression or a disease complication in a subject means preventing or reducing the disease progression and / or disease complication in the subject.

[0038] As used herein, a “pharmaceutically acceptable” carrier or excipient refers to a carrier or excipient that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.

[0039] As used herein, a “pHLIP®-LNP” refers to one or more nucleic acid payloads encapsulated within a lipid nanoparticle to which one or more peptides (e.g., pHLIP®peptide) is covalently bonded or linked with a non-cleavable linker. The pHLIP®peptide can be linked (i.e., covalently bonded or linked with a non-cleavable linker) to the LNP either before or after the one or more nucleic acid payloads is encapsulated. In some aspects, the one or more nucleic acid payloads is encompassed in the LNP prior to linking the pHLIP®peptide to the LNP.

[0040] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0041] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. LNP encapsulating one or more nucleic acids payloads

[0042] In some aspects, the LNP of the pHLIP®-LNPs described herein comprises from about 10% to about 60% of one or more cationic or ionizable lipids. In some aspects, the ionizable lipids have a pK of about 5.5 to about 9.5. In some aspects, the ionizable lipids are propargylamine-based lipids. In an additional aspect, the ionizable / cationic lipids are selected from the following lipids: 11-10-8 (((2-(pyrrolidin-1- yl)ethyl)azanediyl)bis(decane-1,2-diyl) dioctanoate); 119-23 (9Z-octadecenoic acid, 6-[[3-(dimethylamino)propyl][(9Z)-1-oxo-9-octadecen-1-yl]amino]-7-oxo-7- (tricyclo[3.3.1.13,7]dec-1-ylamino)heptyl ester); 244cis (4-[2-[bis[9-[(2Z)-2-nonen-1- yloxy]-9-oxononyl]amino]ethyl]-1-piperazinenonanoic acid, (2Z)-2-nonen-1-yl ester); 304-O13 (tri-N-tridecyl 3-(ethyl(methyl)amino)propanoate or trimidin); 306-N16B (3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)) tetrakis(N-(2- (dodecyldisulfaneyl) ethyl)propanamide) or disulpax); 306-O12B (tetrakis(2- (octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl)) tetrapropionate or triscormin); 306Oi9-cis2 (tetra((Z)-non-2-en-1-yl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate); 80- O18 (N-[3-(dimethylamino)propyl]-N-[3-(octadecyloxy)-3-oxopropyl]-β-alanine, octadecyl ester); 98N12-5 (^^1,^^16-didodecyl-4,7,13-tris[3-(dodecylamino)-3- oxopropyl]-4,7,10,13-tetraazahexadecanediamide); 9A1P9 (2-(dioctylamino)ethyl nonyl hydrogen phosphate or lidophos); A18-ISO5-2DC18 (1H -Imidazole-2-carboxylic acid, 1-[3-(2-ethyl-1-piperidinyl)propyl]-5,5-di-(8Z )-8-heptadecen-1-yl-2,5-dihydro-, ethyl ester (ACI) or pimidol); AA3-Dlin (piperazine-1,4-diylbis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); ALC-0315 (2-hexyl-decanoic acid, 1,1'- [[(4-hydroxybutyl)imino]di-6,1-hexanediyl]); AMAP-O16B (bis(2- (dodecyldisulfaneyl)ethyl) 3,3'-((4-methyl-11-oxo-12-oxa-15,16-dithia-4,8- diazaoctacosyl)azanediyl)dipropionate) and analogues; ATX-100 (4,4′-[[[[3- (dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1′-bis(1-heptyloctyl) ester); BAmP-O16B (bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-(4-(3-((3-(2- (dodecyldisulfaneyl)ethoxy)-3-oxopropyl)amino)propyl)piperazin-1- yl)propyl)azanediyl)dipropionate); C12-200 (1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl) amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2- dodecanol); C13-112-tetra-tail (14,23-bis(2-hydroxytridecyl)-17,20-dioxa-14,23- diazahexatriacontane-12,25-diol); C14-A1 (1,1'-((5-(4-(2-((5-(bis(2-hydroxytetradecyl) amino)pentyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1- yl)pentyl)azanediyl)bis(tetradecan-2-ol)); C3-K2-E14 (3,3'-(propylazanediyl)bis(N-(2- (bis(2-hydroxytetradecyl)amino) ethyl)propanamide); Cationic Lipid A or Lipid III-45 (2- butyl-octanoic acid, 1,1′-[[(3-hydroxypropyl)imino]di-9,1-nonanediyl] ester); Cho-Arg (cholest-5-en-3-ol (3β)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl)amino]-1- oxopentyl]amino]hexanoate], 2,2,2-trifluoroacetate (1:2)); cKK-E12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione); cKK-E15 (3,6-bis[4-[bis(2- hydroxypentadecyl)amino]butyl]-2,5-piperazinedione); CL15F6 (1-methyl-4- piperidinecarboxylic acid, 11-[(2-hexyl-1-oxodecyl)oxy]-5-[6-[(2-hexyl-1- oxodecyl)oxy]hexyl]-5-hydroxyundecyl ester); CL4F8-6 (7-(4-(dipropylamino)butyl)-7- hydroxytridecane-1,13-diyl bis(2-hexyloctanoate)); CP-LC-0743 (3-[[4-[[2- (dimethylamino)ethyl]amino]-3-[(2-hexyl-1-oxodecyl)amino]-4-oxobutyl]thio]-propanoic acid, 9Z-octadecen-1-yl ester); CP-LC-0867 (3-[[4-[[2-(dimethylamino)ethyl]amino]-3- [(2-octyl-1-oxododecyl)amino]-4-oxobutyl]thio]-propanoic acid, 2-ethylhexyl ester); CP- LC-1254 (2-[[[2-(dimethylamino)ethyl]amino]carbonyl]-4-[[3-[(2-hexyldecyl)oxy]-3- oxopropyl]thio]-butanoic acid, 2-hexyldecyl ester); CP-LC-1428 (6-methylheptyl 7-(2- ((3-((2-butyloctyl)oxy)-3-oxopropyl)thio)ethyl)-2-methyl-14-((3-((6-methylheptyl)oxy)- 3-oxopropyl)thio)-6,9-dioxo-17-thia-2,5,8-triazaicosan-20-oate); DC-1-16 (N-hexadecyl- N,N-dimethyl-1-hexadecanaminium, monobromide); DC-Cholesterol ((3^^)-3-[N-[2- (dimethylamino)ethyl]carbamate cholest-5-en-3-ol hydrochloride); DDAB (dimethyldioctadecylammonium (Bromide Salt)); DGTS (diacylglyceryl-N,N,N- trimethylhomoserine); Dios-Arg ((3β,25R)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl) amino]-1-oxopentyl]amino]hexanoate] spirost-5-en-3-ol, bis(2,2,2-trifluoroacetate)); DLin-DMA (N,N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-1- propanamine); DLin-KC2-DMA (N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl- 1,3-dioxolane-4-ethanamine); DLin-KC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DLin- MC3 DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien- 1-yl-10,13-nonadecadien-1-yl ester); Dlin-MC4-DMA (5-(dimethylamino)-pentanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DMT7 (1-(2-(dimethylamino)ethyl)-3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl)thiourea); DMTAP (N,N,N-trimethyl-2,3-bis[(1-oxotetradecyl)oxy]-1- propanaminium, monochloride); DOBAQ (N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium); DODAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9- octadecen-1-aminium, monochloride); DODAP (1,2-dioleoyl-3-dimethylammonium propane); DODMA (N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanamine); DODMAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9-octadecen-1- aminium, monochloride); DOEPC (4-ethoxy-N,N,N-trimethyl-10-oxo-7R-[[(9Z)-1-oxo-9-octadecenyl] oxy]-3,5,9-trioxa-4-phosphaheptacos-18Z-en-1-aminium 4-oxide, monochloride); DOG-IM4 (N-[(25Z)-14-[(9Z)-9-octadecen-1-yloxy]-3,6,9,12,16- pentaoxatetratriacont-25-en-1-yl]-1H-imidazole-5-carboxamide); DOSPA (N-[2-[[2,5- bis[(3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(9Z)-9- octadecen-1-yloxy]-1-propanaminium, chloride, hydrochloride (1:1:4)); DOTAP (N,N,N- trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanaminium, monochloride) and analogues; DOTMA (N,N,N-trimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanaminium, monochloride); DPDAP (hexadecanoic acid, 1,1′-[1- [(dimethylamino)methyl]-1,2-ethanediyl] ester); E12CA1A3 (2-hexyl-decanoic acid, 2- [4-(dimethylamino)-1-oxobutoxy]dodecyl ester); FTT5 (9,9′,9′′,9′′′,9′′′′,9′′′′′-[1,3,5- benzenetriyltris(carbonylimino-3,1-propanediylnitrilo)]hexakis-nonanoic acid, 1,1′,1′′,1′′′,1′′′′,1′′′′′-hexakis(1-ethylhexyl) ester); GL67 (3β-[(3-aminopropyl)[4-[(3- aminopropyl)amino]butyl]carbamate] cholest-5-en-3-ol); H1L1A1B3 (7- (cyclohexylamino)-6-(N-(3-(dimethylamino)propyl)-4-methylnonanamido)-7-oxoheptyl dodecanoate); Hexa Lipid 114 (ditetradecyl 4,8,13,17-tetrakis(3-oxo-3- (tetradecyloxy)propyl)-4,8,13,17-tetraazaicosanedioate); IZ-Cholesterol ([3-(1H- imidazol-1-yl)propyl]carbamate, cholest-5-en-3β-ol); L319 (9-[4-(dimethylamino)-1- oxobutoxy]-heptadecanedioic acid, 1,17-di-(2Z)-2-nonen-1-yl ester); Lipid 14 (((2-((4- (dimethylamino)butanoyl)oxy)ethyl)azanediyl) bis(octane-8,1-diyl) bis(2- hexyldecanoate)); Lipid 16 (7-(((4-(dimethylamino)butanoyl)oxy) ((9Z,12Z)-octadeca- 9,12-dien-1-yl)amino)heptyl decanoate); Lipid 2-10 (10-[[3-(dimethylamino)propyl](1- oxooctyl)amino]-nonadecanedioic acid, 1,19-bis(1-hexylheptyl) ester); Lipid 29 (8-[[8- [(1-ethylnonyl)oxy]-8-oxooctyl][3-[[2-(methylamino)-3,4-dioxo-1-cyclobuten-1- yl]amino]propyl]amino]-octanoic acid, 1-octylnonyl ester); Lipid 369 (9-[4- (dimethylamino)-1-oxobutoxy]-heptadecanedioic acid, 1,17-bis(3-pentyloctyl) ester); Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1- octylnonyl ester); Lipid 50 (2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy) decanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)); Lipid A (2-hexyl-decanoic acid, 1,1′-[[[1-(hydroxymethyl)propyl]imino]di-6,1-hexanediyl] ester); Lipid A4 (1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxydodecyl) amino)ethoxy)ethyl)(2- hydroxydodecyl)amino) ethyl) piperazin-1-yl)ethoxy)ethyl) azanediyl)bis(dodecan-2-ol)); Lipid A9 (bis(2-butyloctyl) 10-(N-(3-(dimethylamino) propyl)nonanamido)nonadecanedioate); Lipid C2 (N1,N4-bis[2-[bis(2-hydroxytetradecyl)amino]ethyl]-1,4-piperazinedipropanamide); Lipid DIM1(((((3R,3aS,6S,6aS)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(propane-3,1- diyl))bis(azanetriyl))tetrakis(dodecan-2-ol)); Lipid OA2 (di((Z)-octadec-9-en-1-yl) L- lysyl-L-glutamate, dihydrochloride); Lipid R6 (2-((3-(diethylamino)propyl)amino)-N- undecylheptadecanamide); Lipid U105 (di(heptadecan-9-yl) 6,6'-((3-(bis(2- hydroxyethyl)amino)propyl)azanediyl)dihexanoate); LP-01 (9,12-Octadecadienoic acid (9Z,12Z)-, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy] carbonyl]oxy]methyl]propyl ester); MVL5 (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4- [di(3-amino-propyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); ND98 (N1,N16-didodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13- tetraazahexadecanediamide); O12-D3-I3 (didodecyl 3,3'-((3-(2-amino-3-(1H-imidazol-4- yl)propanamido)propyl)azanediyl) dipropionate); OC2-K3-E10 (3,3'-((2- hydroxyethyl)azanediyl)bis(N-(3-(bis(2-hydroxydecyl)amino) propyl)propanamide)); OF- 02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5- piperazinedione); OF-C4-Deg-Lin (9,12-octadecadienoic acid, (9Z,12Z)-1,1′,1′′,1′′′-[(3,6- dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi-4,1-butanediyl)] ester); OF-Deg-Lin (9Z,12Z-octadecadienoic acid, 1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1- butanediylnitrilodi-2,1-ethanediyl)] ester); PL1 (di(octan-3-yl) 9,9'-((3-((3- ((diethoxyphosphoryl)oxy)propyl)(9-(octan-3-yloxy)-9- oxononyl)amino)propyl)azanediyl) dinonanoate); PPPDA-O16B (bis(2- (dodecyldisulfaneyl) ethyl) 3,3'-(propylazanediyl)dipropionate); PPZ-A10 (N,N'- (piperazine-1,4-diylbis(propane-3,1-diyl))bis(3-(didecylamino)propanamide)); Q1-SM- 102 (((2-((4-(dimethylamino)butanoyl) oxy)ethyl)azanediyl)bis(octane-8,1-diyl) bis(2- hexyldecanoate)); SAINT C-18 (1-methyl-4-[(10Z)-1-(9Z)-9-octadecen-1-yl-10- nonadecen-1-yl]-pyridinium, monochloride); SIL Lipid (N,N′,N′′-(nitrilotri-2,1- ethanediyl)tris[N-methyl-β-alanine, 1,1′,1′′-tridodecyl ester); SM-102 (8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester) and SM-102 Analogue; TCL053 (2-(((4-(dimethylamino) butanoyl)oxy)methyl)-2-((((Z)- tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)-bis(tetradec-9-enoate)); THP1 or tetrahydropyrimidine 1 Lipid (diethyl 1,3-dioctyl-1,2,3,6-tetrahydropyrimidine-4,5- dicarboxylate); TT3 (N1,N3,N5-tris[3-(didodecylamino)propyl]-1,3,5- benzenetricarboxamide); YK-009 (6-[[4-(decyloxy)-4-oxobutyl](2-hydroxyethyl) amino]- hexanoic acid, 2-octyldecyl ester); YSK05 (1-methyl-4,4-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-piperidine); YSK13-C3 (3-(dimethylamino)propyl (12Z,15Z)-3- ((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trienoate); ZA3-Ep10 (10-[2-[bis(2- hydroxydecyl) amino]ethyl]-15-hydroxy-7,13-bis(2-hydroxydexyl)-N,N-dimethyl-4-oxo- B-(3-sulfopropyl)-3,7,10,13-tetraazatricosan-1-aminium, inner salt), and combinations thereof.

[0043] In some aspects, the LNP of Formula (I) or the pHLIP®-LNPs described herein comprises from about 10% to about 60% of cholesterol and / or its naturally occurring analogs. In some aspects, the LNP of Formula (I) or the pHLIP®-LNPs described herein comprises cholesterol. In some aspects, the cholesterol is from plant origin (e.g., BotaniChol®). In other aspects, the LNP of Formula (I) or pHLIP®-LNPs described herein comprise at least one naturally occurring analog of cholesterol. In some aspects, the naturally occurring analogs of cholesterol are selected from vitamin D3, vitamin D2, calcipotriol, stigmasterol, β-sitosterol, sitostanol, betulin, lupeol, ursolic acid, oleanolic acid, or chemical alkyl derivatives (stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, dehydroergosterol), and combinations thereof.

[0044] In some aspects, the LNP of Formula (I) or the pHLIP®-LNPs described herein additionally comprises about 5% to about 25% of other lipid ingredients. In additional aspects, the other lipid ingredients include DOPC (1,2-dioleoyl-sn-glycero-3- phosphatidylcholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DMPC (1,2-Dimyristoyl-sn-glycero-3- phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), PS (Phosphatidylserine) and / or other diacyl phospholipids, and combinations thereof.

[0045] In some aspects, the LNP of Formula (I) or the pHLIP®-LNPs described herein additionally comprises PEG-lipids or PEG free polymers (e.g. pSar-lipids).

[0046] In some aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a polar flanking sequence at the membrane non-inserting end of the pHLIP®peptide, which is linked to a lipid, e.g., PEGylated (polyethylene glycol, e.g., PEG2000) lipid or pSar (polysarcosine) lipid. In some aspects, the pHLIP®polar flanking sequence comprises 2 to 10 negatively-charged and / or polar residues. In some aspects, the pHLIP®polar flanking sequence consists of one or more of the following residues: Asp, Glu, Asn, Gln, Tyr, Ser, and Thr. In an additional aspect, the pHLIP®polar flanking sequence comprises a sequence consisting of 2 to 10 of the sequence repeats Asp-Asn.

[0047] In some aspects, compositions of Formula (I) described herein further encompasses one or more nucleic acid payloads (pHLIP®-LNPs). In some aspects, the nucleic acid payloads are RNA and / or DNA molecules. In some aspects, the nucleic acid payloads are selected from mRNA (messenger RNA), miRNA (micro RNA), saRNA (self-Amplyfing RNA), saRNA (small activating RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), piRNA (piwi-interacting RNA), crRNA (CRISPR RNA), gRNA (guide RNA), tracrRNA (trans-activating CRISPR RNA), ncRNA (non-coding RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), regions of lncRNA (long non-coding RNA), and other RNAs from protein-nucleic acid complexes such as signal recognition particles, aptamers, antisense oligonucleotides, and CpG oligonucleotides, pDNA(plasmid DNA), ceDNA (closed ends DNA), cDNA (circular DNA), and combinations thereof. pHLIP®peptides

[0048] In some aspects, the one or more pHLIP®peptides of the compositions of Formula (I) or pHLIP®-LNPs described herein comprise the following sequence: XnYm; YmXn; Xn- YmXj; YmXnYi; YmXnYiXj; XnYmXjYi; YmXnYiXjYl; XnYmXjYiXl; YmXnYiXjYlXh; XnYmXjYiXhYg; YmXnYiXjYlXhYg; XnYmXjYiXhYgXf; (XY)n; (YX)n; (XY)nYm; (YX)nYm; (XY)nXm; (YX)nXm; Ym(XY)n; Ym(YX)n; Xn(XY)m; Xn(YX)m; (XY)nYm(XY)i; (YX)nYm(YX)i; (XY)nXm(XY)i; (YX)nXm(YX)i; Ym(XY)n; Ym(YX)n; Xn(XY)m; or Xn(YX)m, wherein, (i) each Y is, individually, a non-polar amino acid with solvation energy, DGXcor> +0.50, or Gly; (ii) each X is, individually, a protonatable amino acid, (iii) n, m, i, j, l, h, g, f are each, individually, an integer from 1 to 8.

[0049] In some aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence comprising the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids. In other aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence consisting of the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected fromlysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids. In some aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence comprising the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227). In other aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence consisting of the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).

[0050] Other exemplary pHLIP®peptides that can be included in the pHLIP®-LNPs described herein are shown in the Tables below.

[0051] The sequences are provided N-terminus to C-terminus. Table 1Table 2* Ac means Acetylated N-terminus; and Am means Amidated C-terminus Table 3. Coded and exemplary non-coded amino acids including L-isomers, D-isomers, alpha-isomers, beta-isomers, glycol-, and methyl-modifications.Table 4. Non-limiting examples of protonatable residues and their substitutions including L-isomers, D- isomers, alpha-isomers, and beta-isomers.Table 5. Examples of coded amino acid substitutionsTable 6. Non-limiting examples of membrane-inserting sequences belonging to different groups of pHLIP®peptides. Each protonatable residue (shown in bold) could be replaced by its substitution from Table 4. Each non-polar residue could be replaced by its coded amino acid substitution from Table 5, and / or non-coded amino acid substitutions from Table 3.Table 7. Non-limiting examples of pHLIP®sequences. A cysteine, a lysine, an azido- modified amino acid, or an alkynyl modified amino acid can be incorporated at the N- terminal (first 6 residues) or C-terminal (last 6 residues) parts of the peptides for conjugation with a cargo, and a linker.

[0052] In some aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a sequence selected from the group consisting of any one of SEQ ID NOs.1-225. In some aspects, the one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein consists of a sequence selected from the group consisting of any one of SEQ ID NOs.1-225.

[0053] In some aspects, one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein comprises a polar flanking sequence at the membrane non-inserting end of the pHLIP®peptide, which is linked to a lipid, e.g., PEGylated (polyethylene glycol, e.g., PEG2000) lipid or pSar (polysarcosine) lipid. In some aspects, the pHLIP®polar flanking sequence comprises 2 to 10 negatively-charged and / or polar residues. In some aspects, the pHLIP®polar flanking sequence consists of one or more of the following residues: Asp, Glu, Asn, Gln, Tyr, Ser, and Thr. In an additional aspect, the pHLIP®polar flanking sequence comprises a sequence consisting of 2 to 10 of the sequence repeats Asp-Asn.

[0054] In some aspects, the one or more pHLIP®peptides and LNP of Formula (I) described herein are linked by a covalent bond or a non-cleavable linker. In some aspects, the one or more pHLIP®peptides and LNP of Formula (I) described herein are linked by a covalent bond. The one or more pHLIP®peptides can be linked (i.e., covalently bonded or linked with a non-cleavable linker) to the LNP either before or after the one or morenucleic acid payloads is encapsulated. In some aspects, the one or more nucleic acid payloads is encompassed in the LNP prior to linking the pHLIP®peptide to the LNP.

[0055] In some aspects, the membrane non-inserting end of the one or more pHLIP®peptides is linked by a covalent bond or a non-cleavable linker to the LNP. In some aspects, the membrane non-inserting end of the one or more pHLIP®peptides is linked by a covalent bond or a non-cleavable linker to the LNP. In some aspects, the N-terminus of the pHLIP®peptide is covalently bonded to the LNP of Formula (I) described herein. In some aspects, the C-terminus of the pHLIP®peptide is covalently bonded to the LNP of Formula (I) described herein.

[0056] In some aspects, 1 or 2 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 5 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 10 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 50 pHLIP®peptides are independently covalently bonded and / or linked with a non- cleavable linker to a single LNP. In some aspects, 1 to 100 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 500 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 1000 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 5000 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. In some aspects, 1 to 10,000 pHLIP®peptides are independently covalently bonded and / or linked with a non-cleavable linker to a single LNP. Methods of Treatment

[0057] Methods of using the compositions described herein for treating a disease or disorder in a subject in need thereof comprising administering an effective amount of a composition or a pharmaceutical composition described herein are also provided. In some aspects, the subject is a human. In some aspects the disease or disorder comprises one or more of liver disease, cancer, heart disease, ocular disease, and muscular dystrophy. In some aspects, the disease or disorder comprises one or more of hepatitis, cirrhiosis, liver fibrosis, nonalcoholic fatty liver disease, and non-alcoholic steatohepatitis. In someaspects the disease or disorder comprises one or more of breast cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, bladder cancer, heart cancer, pancreatic cancer, nervous system cancer, ocular cancer, and melanoma. In some aspects the disease or disorder comprises one or more of myocardial infarction, heart failure, and hypercholesterolemia. In some aspects the disease or disorder comprises one or more of glaucoma, corneal disease, choroidal disease, and retinal disease.

[0058] The methods provided herein also include use of the compositions described herein to deliver a nucleic acid payload to a cell and / or a diseased tissue. In some aspects, the methods comprise administering a composition comprising one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein or a pharmaceutical composition comprising one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein to a cell and / or a diseased tissue. In some aspects, administering comprises contacting a cell with a composition comprising one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein or a pharmaceutical composition comprising one or more pHLIP®peptides of Formula (I) or pHLIP®-LNPs described herein. In some aspects the contacting occurs in vitro. In some aspects the contacting occurs in vivo. In some aspects the contacting occurs ex vivo. Preparation of pHLIP®-LNP formulation

[0059] The following steps were taken to synthesize 1,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000-OH) (Figure 3): Step 1: Benzyl-protected PEG2000 was coupled with IPC-tosylate. Step 2: Cleavage of the acetal was performed in acidic conditions. Step 3: Esterification of the diol was achieved using myristic anhydride in the presence of polypyrrole (PPY) as catalyst. Step 4: Deprotection of the Bn group was achieved by hydrogenation with Pd / C and H2. Step 5: Direct coupling of the alcohol DMG-PEG2000-OH with an unprotected maleimide was challenging. To overcome the issues of instability the maleimide function was protected by a Diels-Alder reaction with furane. This furane protected maleimide was very stable and was isolated in high purity by standard chromatographic purification.Step 6: To deprotect the furane protected product a retro-Diels-Alder reaction was performed in toluene at 90°C and the product was obtained in very high yield without degradation.

[0060] The mol% lipid composition of pHLIP®-LNP was the following: 50 mol% of ionizable lipids SM-102, 10 mol% of DSPC natural phospholipids, 38.5 mol% of Botanichol (cholesterol) and 1.5 mol% of pegylated lipids among them were 1.35 mol% of DSPE-PEG2000 and 0.15 mol% of DSPE-PEG2000-Mal for conjugation with pHLIP®. LNP formulation was prepared using the Automated Nanoparticle System by Dolomite Microfluidics. All lipids were dissolved in ethanol as an organic phase, with a final lipid concentration of 12.5 mM. The composition of lipids is shown in Table 8. Table 8: Composition of pHLIP®-LNP formulation

[0061] mRNA was solubilized in 100 mM acetate buffer (pH 4) to reach a final concentration of 0.12 mg / mL. The aqueous phase and organic phase were injected into sample loops within the system later combined on a microfluidic chip. The Flow Rate Ratio (FRR) was 3:1 and the Total Flow Rate (TFR) was 21 mL / min including an in-line dilution step with 1X PBS pH 7.4. After preparation of the LNP formulations an-offline dilution step (1:4) with Tris buffer 100 mM pH 8.0 was immediately performed to reduce the percentage of ethanol. Reaction parameters are presented in Table 9. Table 9. Reaction parameters

[0062] For coating of LNPs with pHLIP®we used the following sequence: pHLIP®peptide: ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (Seq. ID NO 227)

[0063] pHLIP®peptide (1.2 eq) was dissolved in DMSO for conjugation with the surface of LNPs through the coupling of SH group of single Cys residue at the pHLIP®peptide with the maleimide group at the distal end of DSPE-PEG2000-Mal. Reaction was complete in 5 min. To ensure pHLIP®coupling with LNPs, the HPLC protocol was developed: Chromatographic Conditions: • Mobile phase A: H2O / ACN 80 / 20 +0.06% TFA • Mobile phase B: MeOH / ACN 80 / 20 +0.05% TFA • Instrument: UHPLC ThermoFisher Vanquish • Column Temperature: 50°C • Wavelength: 220 nm • Column: Waters Acquity UPLC CSH C181.7mm 2.1X150mm - (C-127) • Autosampler temperature: 5°C • Flow: 0.2 mL / min • Injection Volume: 10uL Sample preparation LNP: • 1:2 MeOH / LNP solution • Vortex 10 sec

[0064] HPLC analysis was performed to establish conjugation of pHLIP®peptide with LNPs (Figure 4).

[0065] pHLIP®-LNP was purified by ultracentrifugation in Amicon® Ultra Centrifugal Filter devices (100 kDa MWCO, Millipore at 2000 RCF 4°C) following diafiltration (3 diavolumes) against Tris buffer 100 mM (pH 8.0). The presence of pHLIP®in pHLIP®-LNP formulation was confirmed by HPLC (Figure 5). At the final step, sucrose was added to cryo-preserve samples (the final sucrose concentration in the sample was 10%).

[0066] pHLIP®-LNP formulation was characterized by measuring the amount of encapsulated mRNA, particle size and polydispersity index (PDI). At each step of the process, the particle size and PDI were determined by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern), the data are presented in Table 10. Encapsulated mRNA was measured by a fluorescence-based Quant-iT™ RiboGreen kit (Thermofisher) according to the manufacturer’s protocol. The efficiency of mRNA encapsulation was 77% after off-line dilution and 74% after addition of sucrose. Table 10: Size and polydispersity index (PDI) measured at different stages of pHLIP®- LNP preparation

[0067] In conclusion, the stable homogeneous formulation of pHLIP®-LNPs (of about 120−150 nm in size) was developed with about 74%−77% encapsulation efficiency of mRNA.

Claims

What is claimed is.

1. A composition comprising Formula (I): (pHLIP®peptides)n–LNP (I) wherein n is 1 to 10,000; wherein LNP is a lipid nanoparticle comprising (a) at least 10% molar ratio of one or more cationic or ionizable lipids; and (b) at least 10% molar ratio of cholesterol and / or its naturally occurring analogs; and wherein each “–” is a is a covalent bond or a non-cleavable linker.

2. The composition of claim 1, wherein the pHLIP®peptides comprises the following sequence: XnYm; YmXn; XnYmXj; YmXnYi; YmXnYiXj; XnYmXjYi; YmXnYiXjYl; XnYmXjYiXl; YmXnYiXjYlXh; XnYmXjYiXhYg; YmXnYiXjYlXhYg; XnYmXjYiXhYgXf; (XY)n; (YX)n; (XY)nYm; (YX)nYm; (XY)nXm; (YX)nXm; Ym(XY)n; Ym(YX)n; Xn(XY)m; Xn(YX)m; (XY)nYm(XY)i; (YX)nYm(YX)i; (XY)nXm(XY)i; (YX)nXm(YX)i; Ym(XY)n; Ym(YX)n; Xn(XY)m; or Xn(YX)m, wherein, (i) each Y is, individually, a non-polar amino acid with solvation energy, DGXcor> +0.50, or Gly; (ii) each X is, individually, a protonatable amino acid, (iii) n, m, i, j, l, h, g, f are each, individually, an integer from 1 to 8.

3. The composition of claim 1 or 2, wherein the pHLIP®peptides comprises the sequence comprising the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids.

4. The composition of claim 1 or 2, wherein the pHLIP®peptides comprises the sequence comprising the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).

5. The composition of claims 1 or 2, wherein the pHLIP®peptides comprises the sequence consisting of the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids.

6. The composition of claim 1 or 2, wherein the pHLIP®peptides comprises the sequence consisting of the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).

7. The composition of claims 1 or 2, wherein the pHLIP®peptides comprises any one of the sequences selected from the group consisting of any one of SEQ ID NOs.1-225.

8. The composition of claims 1 or 2, wherein the pHLIP®peptides consists of any one of the sequences selected from the group consisting of any one of SEQ ID NOs.1-225.

9. The composition of any one of claims 1 to 8, wherein the pHLIP®peptides comprises a polar flanking sequence at the membrane non-inserting end of the pHLIP®peptides.

10. The composition of claim 9, wherein the polar flanking sequence is linked to a lipid.

11. The composition of claim 10, wherein the lipid is a PEGylated lipid or pSar (polysarcosine) lipid.

12. The composition of any one of claims 9to 11, wherein the pHLIP®polar flanking sequence comprises 2 to 10 negatively-charged and / or polar residues.

13. The composition of any one of claims 9 to 12, wherein the pHLIP®polar flanking sequence comprises one or more of the following residues: Asp, Glu, Asn, Gln, Tyr, Ser, and Thr.

14. The composition of any one of claims 9 to 13, wherein the polar flanking sequence of the pHLIP®peptide comprises a sequence comprising 2 to 10 Asp-Asn repeats.

15. The composition of any one of claims 1 to 14, wherein all amino acids in the one or more pHLIP®peptides are D-amino acids.

16. The composition of any one of claims 1to 15, wherein the LNP comprises from about 10% to about 60% of one or more cationic or ionizable lipids.

17. The composition of claim 16, wherein the cationic or ionizable lipids have a pK of about 5.5 to about 9.

5.

18. The composition of claim 16 or 17, wherein the cationic or ionizable lipids comprise propargylamine-based lipids.

19. The composition of any one of claims 16 to 18, wherein the cationic or ionizable lipids comprise 11-10-8 (((2-(pyrrolidin-1-yl)ethyl)azanediyl)bis(decane-1,2-diyl) dioctanoate); 119-23 (9Z-octadecenoic acid, 6-[[3-(dimethylamino)propyl][(9Z)-1-oxo-9-octadecen-1- yl]amino]-7-oxo-7-(tricyclo[3.3.1.13,7]dec-1-ylamino)heptyl ester); 244cis (4-[2-[bis[9- [(2Z)-2-nonen-1-yloxy]-9-oxononyl]amino]ethyl]-1-piperazinenonanoic acid, (2Z)-2- nonen-1-yl ester); 304-O13 (tri-N-tridecyl 3-(ethyl(methyl)amino)propanoate or trimidin); 306-N16B (3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl)) tetrakis(N-(2-(dodecyldisulfaneyl) ethyl)propanamide) or disulpax); 306-O12B (tetrakis(2-(octyldisulfaneyl)ethyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl)) tetrapropionate or triscormin); 306Oi9-cis2 (tetra((Z)-non-2-en-1-yl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate); 80- O18 (N-[3-(dimethylamino)propyl]-N-[3-(octadecyloxy)-3-oxopropyl]-β-alanine, octadecyl ester); 98N12-5 (^^1,^^16-didodecyl-4,7,13-tris[3-(dodecylamino)-3- oxopropyl]-4,7,10,13-tetraazahexadecanediamide); 9A1P9 (2-(dioctylamino)ethyl nonyl hydrogen phosphate or lidophos); A18-ISO5-2DC18 (1H -Imidazole-2-carboxylic acid, 1-[3-(2-ethyl-1-piperidinyl)propyl]-5,5-di-(8Z )-8-heptadecen-1-yl-2,5-dihydro-, ethyl ester (ACI) or pimidol); AA3-Dlin (piperazine-1,4-diylbis(ethane-2,1-diyl)(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); ALC-0315 (2-hexyl-decanoic acid, 1,1'- [[(4-hydroxybutyl)imino]di-6,1-hexanediyl]); AMAP-O16B (bis(2- (dodecyldisulfaneyl)ethyl) 3,3'-((4-methyl-11-oxo-12-oxa-15,16-dithia-4,8- diazaoctacosyl)azanediyl)dipropionate) and analogues; ATX-100 (4,4′-[[[[3- (dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1′-bis(1-heptyloctyl) ester); BAmP-O16B (bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-(4-(3-((3-(2- (dodecyldisulfaneyl)ethoxy)-3-oxopropyl)amino)propyl)piperazin-1- yl)propyl)azanediyl)dipropionate); C12-200 (1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl) amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2- dodecanol); C13-112-tetra-tail (14,23-bis(2-hydroxytridecyl)-17,20-dioxa-14,23- diazahexatriacontane-12,25-diol); C14-A1 (1,1'-((5-(4-(2-((5-(bis(2-hydroxytetradecyl) amino)pentyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-1- yl)pentyl)azanediyl)bis(tetradecan-2-ol)); C3-K2-E14 (3,3'-(propylazanediyl)bis(N-(2- (bis(2-hydroxytetradecyl)amino) ethyl)propanamide); Cationic Lipid A or Lipid III-45 (2- butyl-octanoic acid, 1,1′-[[(3-hydroxypropyl)imino]di-9,1-nonanediyl] ester); Cho-Arg (cholest-5-en-3-ol (3β)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl)amino]-1- oxopentyl]amino]hexanoate], 2,2,2-trifluoroacetate (1:2)); cKK-E12 (3,6-bis[4-[bis(2- hydroxydodecyl)amino]butyl]-2,5-piperazinedione); cKK-E15 (3,6-bis[4-[bis(2- hydroxypentadecyl)amino]butyl]-2,5-piperazinedione); CL15F6 (1-methyl-4- piperidinecarboxylic acid, 11-[(2-hexyl-1-oxodecyl)oxy]-5-[6-[(2-hexyl-1- oxodecyl)oxy]hexyl]-5-hydroxyundecyl ester); CL4F8-6 (7-(4-(dipropylamino)butyl)-7- hydroxytridecane-1,13-diyl bis(2-hexyloctanoate)); CP-LC-0743 (3-[[4-[[2- (dimethylamino)ethyl]amino]-3-[(2-hexyl-1-oxodecyl)amino]-4-oxobutyl]thio]-propanoic acid, 9Z-octadecen-1-yl ester); CP-LC-0867 (3-[[4-[[2-(dimethylamino)ethyl]amino]-3- [(2-octyl-1-oxododecyl)amino]-4-oxobutyl]thio]-propanoic acid, 2-ethylhexyl ester); CP- LC-1254 (2-[[[2-(dimethylamino)ethyl]amino]carbonyl]-4-[[3-[(2-hexyldecyl)oxy]-3- oxopropyl]thio]-butanoic acid, 2-hexyldecyl ester); CP-LC-1428 (6-methylheptyl 7-(2- ((3-((2-butyloctyl)oxy)-3-oxopropyl)thio)ethyl)-2-methyl-14-((3-((6-methylheptyl)oxy)- 3-oxopropyl)thio)-6,9-dioxo-17-thia-2,5,8-triazaicosan-20-oate); DC-1-16 (N-hexadecyl- N,N-dimethyl-1-hexadecanaminium, monobromide); DC-Cholesterol ((3^^)-3-[N-[2- (dimethylamino)ethyl]carbamate cholest-5-en-3-ol hydrochloride); DDAB (dimethyldioctadecylammonium (Bromide Salt)); DGTS (diacylglyceryl-N,N,N-trimethylhomoserine); Dios-Arg ((3β,25R)-3-[6-[[(2S)-2-amino-5-[(aminoiminomethyl) amino]-1-oxopentyl]amino]hexanoate] spirost-5-en-3-ol, bis(2,2,2-trifluoroacetate)); DLin-DMA (N,N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien-1-yloxy]-1- propanamine); DLin-KC2-DMA (N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl- 1,3-dioxolane-4-ethanamine); DLin-KC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DLin- MC3 DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien- 1-yl-10,13-nonadecadien-1-yl ester); Dlin-MC4-DMA (5-(dimethylamino)-pentanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester); DMT7 (1-(2-(dimethylamino)ethyl)-3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl)thiourea); DMTAP (N,N,N-trimethyl-2,3-bis[(1-oxotetradecyl)oxy]-1- propanaminium, monochloride); DOBAQ (N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium); DODAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9- octadecen-1-aminium, monochloride); DODAP (1,2-dioleoyl-3-dimethylammonium propane); DODMA (N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanamine); DODMAC (N,N-dimethyl-N-(9Z)-9-octadecen-1-yl-9-octadecen-1- aminium, monochloride); DOEPC (4-ethoxy-N,N,N-trimethyl-10-oxo-7R-[[(9Z)-1-oxo- 9-octadecenyl] oxy]-3,5,9-trioxa-4-phosphaheptacos-18Z-en-1-aminium 4-oxide, monochloride); DOG-IM4 (N-[(25Z)-14-[(9Z)-9-octadecen-1-yloxy]-3,6,9,12,16- pentaoxatetratriacont-25-en-1-yl]-1H-imidazole-5-carboxamide); DOSPA (N-[2-[[2,5- bis[(3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(9Z)-9- octadecen-1-yloxy]-1-propanaminium, chloride, hydrochloride (1:1:4)); DOTAP (N,N,N- trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanaminium, monochloride) and analogues; DOTMA (N,N,N-trimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1- propanaminium, monochloride); DPDAP (hexadecanoic acid, 1,1′-[1- [(dimethylamino)methyl]-1,2-ethanediyl] ester); E12CA1A3 (2-hexyl-decanoic acid, 2- [4-(dimethylamino)-1-oxobutoxy]dodecyl ester); FTT5 (9,9′,9′′,9′′′,9′′′′,9′′′′′-[1,3,5- benzenetriyltris(carbonylimino-3,1-propanediylnitrilo)]hexakis-nonanoic acid, 1,1′,1′′,1′′′,1′′′′,1′′′′′-hexakis(1-ethylhexyl) ester); GL67 (3β-[(3-aminopropyl)[4-[(3- aminopropyl)amino]butyl]carbamate] cholest-5-en-3-ol); H1L1A1B3 (7- (cyclohexylamino)-6-(N-(3-(dimethylamino)propyl)-4-methylnonanamido)-7-oxoheptyl dodecanoate); Hexa Lipid 114 (ditetradecyl 4,8,13,17-tetrakis(3-oxo-3- (tetradecyloxy)propyl)-4,8,13,17-tetraazaicosanedioate); IZ-Cholesterol ([3-(1H-imidazol-1-yl)propyl]carbamate, cholest-5-en-3β-ol); L319 (9-[4-(dimethylamino)-1- oxobutoxy]-heptadecanedioic acid, 1,17-di-(2Z)-2-nonen-1-yl ester); Lipid 14 (((2-((4- (dimethylamino)butanoyl)oxy)ethyl)azanediyl) bis(octane-8,1-diyl) bis(2- hexyldecanoate)); Lipid 16 (7-(((4-(dimethylamino)butanoyl)oxy) ((9Z,12Z)-octadeca- 9,12-dien-1-yl)amino)heptyl decanoate); Lipid 2-10 (10-[[3-(dimethylamino)propyl](1- oxooctyl)amino]-nonadecanedioic acid, 1,19-bis(1-hexylheptyl) ester); Lipid 29 (8-[[8- [(1-ethylnonyl)oxy]-8-oxooctyl][3-[[2-(methylamino)-3,4-dioxo-1-cyclobuten-1- yl]amino]propyl]amino]-octanoic acid, 1-octylnonyl ester); Lipid 369 (9-[4- (dimethylamino)-1-oxobutoxy]-heptadecanedioic acid, 1,17-bis(3-pentyloctyl) ester); Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1- octylnonyl ester); Lipid 50 (2-((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy) decanoyl)oxy)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)); Lipid A (2-hexyl-decanoic acid, 1,1′-[[[1-(hydroxymethyl)propyl]imino]di-6,1-hexanediyl] ester); Lipid A4 (1,1'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxydodecyl) amino)ethoxy)ethyl)(2- hydroxydodecyl)amino) ethyl) piperazin-1-yl)ethoxy)ethyl) azanediyl)bis(dodecan-2-ol)); Lipid A9 (bis(2-butyloctyl) 10-(N-(3-(dimethylamino) propyl)nonanamido)nonadecanedioate); Lipid C2 (N1,N4-bis[2-[bis(2- hydroxytetradecyl)amino]ethyl]-1,4-piperazinedipropanamide); Lipid DIM1 (1,1',1'',1'''- (((((3R,3aS,6S,6aS)-hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(propane-3,1- diyl))bis(azanetriyl))tetrakis(dodecan-2-ol)); Lipid OA2 (di((Z)-octadec-9-en-1-yl) L- lysyl-L-glutamate, dihydrochloride); Lipid R6 (2-((3-(diethylamino)propyl)amino)-N- undecylheptadecanamide); Lipid U105 (di(heptadecan-9-yl) 6,6'-((3-(bis(2- hydroxyethyl)amino)propyl)azanediyl)dihexanoate); LP-01 (9,12-Octadecadienoic acid (9Z,12Z)-, 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy] carbonyl]oxy]methyl]propyl ester); MVL5 (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4- [di(3-amino-propyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); ND98 (N1,N16-didodecyl-4,7,13-tris[3-(dodecylamino)-3-oxopropyl]-4,7,10,13- tetraazahexadecanediamide); O12-D3-I3 (didodecyl 3,3'-((3-(2-amino-3-(1H-imidazol-4- yl)propanamido)propyl)azanediyl) dipropionate); OC2-K3-E10 (3,3'-((2- hydroxyethyl)azanediyl)bis(N-(3-(bis(2-hydroxydecyl)amino) propyl)propanamide)); OF- 02 (3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-1-yl]amino]butyl]-2,5- piperazinedione); OF-C4-Deg-Lin (9,12-octadecadienoic acid, (9Z,12Z)-1,1′,1′′,1′′′-[(3,6- dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi-4,1-butanediyl)] ester); OF-Deg-Lin(9Z,12Z-octadecadienoic acid, 1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1- butanediylnitrilodi-2,1-ethanediyl)] ester); PL1 (di(octan-3-yl) 9,9'-((3-((3- ((diethoxyphosphoryl)oxy)propyl)(9-(octan-3-yloxy)-9- oxononyl)amino)propyl)azanediyl) dinonanoate); PPPDA-O16B (bis(2- (dodecyldisulfaneyl) ethyl) 3,3'-(propylazanediyl)dipropionate); PPZ-A10 (N,N'- (piperazine-1,4-diylbis(propane-3,1-diyl))bis(3-(didecylamino)propanamide)); Q1-SM- 102 (((2-((4-(dimethylamino)butanoyl) oxy)ethyl)azanediyl)bis(octane-8,1-diyl) bis(2- hexyldecanoate)); SAINT C-18 (1-methyl-4-[(10Z)-1-(9Z)-9-octadecen-1-yl-10- nonadecen-1-yl]-pyridinium, monochloride); SIL Lipid (N,N′,N′′-(nitrilotri-2,1- ethanediyl)tris[N-methyl-β-alanine, 1,1′,1′′-tridodecyl ester); SM-102 (8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester) and SM-102 Analogue; TCL053 (2-(((4-(dimethylamino) butanoyl)oxy)methyl)-2-((((Z)- tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)-bis(tetradec-9-enoate)); THP1 or tetrahydropyrimidine 1 Lipid (diethyl 1,3-dioctyl-1,2,3,6-tetrahydropyrimidine-4,5- dicarboxylate); TT3 (N1,N3,N5-tris[3-(didodecylamino)propyl]-1,3,5- benzenetricarboxamide); YK-009 (6-[[4-(decyloxy)-4-oxobutyl](2-hydroxyethyl) amino]- hexanoic acid, 2-octyldecyl ester); YSK05 (1-methyl-4,4-bis[(9Z,12Z)-9,12- octadecadien-1-yloxy]-piperidine); YSK13-C3 (3-(dimethylamino)propyl (12Z,15Z)-3- ((9Z,12Z)-octadeca-9,12-dien-1-yl)henicosa-2,12,15-trienoate); ZA3-Ep10 (10-[2-[bis(2- hydroxydecyl) amino]ethyl]-15-hydroxy-7,13-bis(2-hydroxydexyl)-N,N-dimethyl-4-oxo- B-(3-sulfopropyl)-3,7,10,13-tetraazatricosan-1-aminium, inner salt), and any combinations thereof.

20. The composition of any one of claims 1 to 19, wherein the LNP comprises from about 10% to about 60% of cholesterol and / or its naturally occurring analogs.

21. The composition of claim 20, wherein the LNP comprises from about 10% to about 60% of cholesterol.

22. The composition of claim 21, wherein the cholesterol is from plant origin (e.g., BotaniChol®).

23. The composition of claim 20, wherein the LNP comprises from about 10% to about 60% of at least one naturally occurring analog of cholesterol.

24. The composition of claim 23, wherein the at least one naturally occurring analog of cholesterol are selected from vitamin D3, vitamin D2, calcipotriol, stigmasterol, β- sitosterol, sitostanol, betulin, lupeol, ursolic acid, oleanolic acid, or chemical alkyl derivatives (stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, dehydroergosterol), and combinations thereof.

25. The composition of any one of claims 1 to 24, wherein the LNP further comprises about 5 to about 25% of other lipid ingredients.

26. The composition of claim 25, wherein the other lipid ingredients include DOPC (1,2- dioleoyl-sn-glycero-3-phosphatidylcholine), DSPC (1,2-distearoyl-sn-glycero-3- phosphatidylcholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DMPC (1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine), PS (Phosphatidylserine) and / or other diacyl phospholipids, and combinations thereof.

27. The composition of claim 25, wherein the other lipid ingredients comprise PEG-lipids.

28. The composition of claim 25, wherein the other lipid ingredients comprise polysarcosine lipids.

29. The composition of any one of claims 1 to 28, wherein the LNP encapsulate one or more nucleic acid payloads.

30. The composition of claim 29, wherein the one or more nucleic acid payloads is an RNA and / or DNA molecule.

31. The composition of claim 29 or 30, wherein the one or more nucleic acid payloads is selected from mRNA (messenger RNA), miRNA (micro RNA), saRNA (self-Amplyfing RNA), saRNA (small activating RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), piRNA (piwi-interacting RNA), crRNA (CRISPR RNA), gRNA (guide RNA), tracrRNA (trans-activating CRISPR RNA), ncRNA (non-coding RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), regions of lncRNA (long non-coding RNA), and other RNAs from protein-nucleic acid complexes such as signal recognition particles, aptamers, antisense oligonucleotides, and CpG oligonucleotides, pDNA(plasmid DNA), ceDNA (closed ends DNA), cDNA (circular DNA), and combinations thereof.

32. The composition of any one of claims 1 to 31, wherein the one or more pHLIP®peptide and LNP of Formula (I) are linked by a covalent bond.

33. The composition of any one of claims 1 to 32, wherein the one or more pHLIP®peptide and LNP of Formula (I) are linked by a non-cleavable bond.

34. A pharmaceutical composition comprising the composition of any one of claims 1 to 33 and one or more pharmaceutically acceptable excipients.

35. A method of delivering a nucleic acid payload to a cell and / or a diseased tissue comprising administering to the cell and / or diseased tissue the composition of any one of claims 1 to 33 or the pharmaceutical composition of claim 34.

36. A method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of the composition of any one of claims 1 to 33 or the pharmaceutical composition of claim 34.

37. A method of preventing a disease in a subject in need thereof comprising immunizing the subject with an effective amount of the composition of any one of claims 1 to 33 or the pharmaceutical composition of claim 34.

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