Compositions and methods for biological delivery vehicles

Abstract

Provided are delivery vehicles containing lipid nanoparticles for the administration of therapeutic agents, and methods for making and using the same for delivering therapeutic agents to epithelial cells, such as those in mucus-containing environments. The provided nanoparticles contain ionizable lipids and / or cationic lipids, such as MVL5, MC2, CL1H6, and DODMA, phospholipids, and bile salts. Also provided are methods for using the delivery vehicles to deliver therapeutic agents, particularly nucleic acid therapeutic agents, to epithelial cells in the gastrointestinal tract.

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for biological delivery vehicles. [Background technology]

[0002] Despite advances in gene therapy over the last 50 years, there are still many diseases that are refractory to traditional methods, particularly when the target location of the gene therapy presents delivery challenges, such as in the gastrointestinal tract. The present disclosure addresses this need and also provides several advantages. Summary of the Invention

[0003] Provided herein is a delivery vehicle comprising (i) a cargo and (ii) a lipid nanoparticle, wherein the lipid nanoparticle comprises at least one saturated lipid and a bile salt, and the at least one saturated lipid is a saturated cationic lipid, or the lipid nanoparticle further comprises at least one cationic lipid. In some cases, the lipid nanoparticle further comprises at least one unsaturated cationic lipid or unsaturated non-cationic lipid, and optionally, the concentration of the at least one unsaturated cationic lipid or unsaturated non-cationic lipid in the lipid nanoparticle is less than 50 mol% of the total lipid concentration of the lipid nanoparticle. In some cases, the saturated cationic lipid has a phase transition temperature of at least about 37°C. In some cases, the saturated lipid comprises a saturated non-cationic lipid having a phase transition temperature of at least about 37°C. In some cases, the lipid nanoparticle further comprises at least one of a non-cationic lipid, a multivalent cationic lipid, a permanently charged cationic lipid, or any combination thereof. In some cases, the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, GL67, or any combination thereof. In some cases, the polyvalent cationic lipid comprises MVL5. In one embodiment, the polyvalent cationic lipid is about 25 mol% or less of the total lipid concentration. In some cases, the permanently charged cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol·HCl), or any combination thereof.In some cases, the saturated cationic lipid is 1,2-stearoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-distearoyl-3-dimethylammonium-propane, dimethyldioctadecylammonium, 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammoniumpropane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl) -N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-distearoyl-3-dimethylammonium-propane (DSDAP), or any combination thereof.In some cases, the saturated non-cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, The compound includes at least one of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof.In some cases, the unsaturated cationic lipid is dimethyldioctadecylammonium, 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammoniumpropane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N- [2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]benzamide, 1,2-dialkyloxy-N,N-dimethylaminopropane, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkylethylphosphocholine, MC3, MC2, MC4, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, N4-cholesteryl-spermine, 7-(4-(dimethylamino)butyl)-7-hydroxytridecane-1,13-diyldioleate (CL1H6), or any combination thereof. In some cases, the unsaturated cationic lipid comprises at least MC2 or CL1H6.In some cases, the unsaturated non-cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, In some cases, the at least one saturated or cationic lipid is a polyvalent cationic lipid, and the at least one saturated or cationic lipid is a polyvalent cationic lipid.

[0004] In one embodiment, the delivery vehicle further comprises a non-cationic lipid. In some cases, the polyvalent cationic lipid, the non-cationic lipid, or the polyvalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C. In some cases, the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, and GL67, or any combination thereof. In some cases, the non-cationic lipid comprises a saturated non-cationic lipid. In some cases, the saturated non-cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, The delivery vehicle comprises at least one of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof. In some cases, the delivery vehicle is stable in a high bile salt environment compared to an otherwise identical delivery vehicle that does not contain bile salts. In some cases, the high bile salt environment includes a gastrointestinal environment. In some cases, the delivery vehicle (i) exhibits increased stability in a solution containing at least about 5 g / L of bile salts compared to an otherwise identical delivery vehicle without bile salts, the stability being measured by the relative fluorescence intensity of fluorescent lipids incorporated into the lipid nanoparticles in a Förster resonance energy transfer (FRET) assay.In some cases, the delivery vehicle (i) exhibits increased stability in a solution containing at least about 5 g / L of a mixture of about 50% cholic acid and about 50% deoxycholate, compared to an otherwise identical delivery vehicle that does not contain bile salts, where the stability is measured by the relative fluorescence intensity of fluorescent lipids incorporated into the lipid nanoparticles in a Förster resonance energy transfer (FRET) assay.

[0005] In one embodiment, the delivery vehicle is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3 dioleyloxy)propyl)-N,N,N trimethylammonium chloride (DOTMA), N,N distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydromoxyethylammonium bromide (DMRIE), 1,2 dioleoyl-sn-3-phosphoethanolamine (DOPE), N-(1-(2,3 dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), diocmdecylamidoglycylcarboxyspermine (DO GS), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLin-K-C2-DMA, DLin-M-C3-DMA, 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}- and at least one of {Ci}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienyloxyl]propan-1-amine (CLinDMA), MC4, O-alkylethylphosphocholine, didodecyldimethylammonium bromide (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), or any combination thereof.

[0006] In one embodiment, the delivery vehicle comprises at least one of diacylphosphatidylcholine, diacylphosphatidylevanolamine, ceramide, sphingomyelin, cephalin, cerebroside, diacylglycerol, or any combination thereof.

[0007] In one embodiment, the delivery vehicle comprises at least one of phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), or any combination thereof.

[0008] In one embodiment, the delivery vehicle is distearoylphosphatidylcholine (DSPC), phosphatidylcholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (OPEC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloromyl-phosphatidylethanolamine ... and at least one of dioleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(4-maleimidomethyl) dioleoylhexane-1-carboxylate (DOPE-teal), diphosphatidylethanolamine (DPPE), dimyristoylphosphoevanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPS), 1,2-dielideyl-sn-glycero-3-phosphatidylethanolamine (transDOPE), or any combination thereof.

[0009] In one embodiment, the delivery vehicle comprises at least DSPC or DMPC. In one embodiment, the delivery vehicle further comprises a conjugated lipid, wherein the conjugated lipid comprises a lipid conjugated to a stabilizing component. In some cases, the stabilizing component comprises a hydrophilic polymer. In some cases, the hydrophilic polymer comprises polyethylene glycol, poly(2-alkyl-2-oxazoline), polyvinyl alcohol, or any combination thereof. In some cases, the hydrophilic polymer has a molecular weight of about 50 kDa to about 500 kDa. In some cases, the hydrophilic polymer comprises polyethylene glycol (PEG), and the conjugated lipid comprises a PEGylated lipid. In some cases, the PEGylated lipid comprises DSPE-PEG, DSG-PEG, DMG-PEG, or DPPE-PEG. In some cases, the PEGylated lipid comprises DSPE-PEG or DMG-PEG. In some cases, the concentration of the conjugated lipid is less than 25 mol%. In some cases, the concentration of the conjugated lipid is less than 5 mol%. In some cases, the concentration of the conjugated lipid is about 0.5 mol% to about 20 mol%. In some cases, the delivery vehicle includes a non-cationic lipid, and the concentration of the non-cationic lipid is about 5 mol% to about 75 mol%. In some cases, the lipid nanoparticles have a net positive or near-neutral charge.

[0010] In one embodiment, the delivery vehicle further comprises cholesterol. Provided herein is a delivery vehicle comprising a cargo and a nanoparticle, wherein the nanoparticle comprises a first region that is positively charged at a pH of about 5.5-8.0 and a second region that is negatively charged at a pH of about 5.5-8.0, the first region and the second region being separated such that the positive and negative charges are not dispersed, and the nanoparticle is capable of passing through the mucus barrier to reach epithelial cells. In one embodiment, reaching an epithelial cell comprises the delivery vehicle approaching within 20 microns of the cell surface, binding to the epithelial cell surface, or absorption by the epithelial cell. In one embodiment, the nanoparticle comprises a lipid, a polymer, or a combination thereof. In one embodiment, the first region is contained in a first phase and the second region is contained in a second phase, and the first and second phases are physically separated from each other. In one embodiment, the first phase is a liquid. In one embodiment, the second phase is a gel. In one embodiment, the first phase is a gel. In one embodiment, the second phase is a liquid. In some cases, the delivery vehicle further comprises a stabilizing component. In some cases, the stabilizing component is polyethylene glycol (PEG). In some cases, the first moiety comprises an unsaturated lipid or a short-tail lipid. In some cases, the unsaturated lipid comprises a cationic lipid or an ionizable cationic lipid. In some cases, the cationic lipid comprises a polyvalent cationic lipid or a monovalent cationic lipid. In some cases, the cationic lipid is selected from the group consisting of N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N4-cholesteryl-spermine HCl (GL67), a salt of any of these, and any combination thereof. In one embodiment, one or more lipids in the first phase are PEGylated.In one embodiment, the first moiety further comprises at least one of 1,2-dioleyloxy-3-(dimethylamino)propane (DODMA), 6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propanoate (MC2), or any combination thereof. In one embodiment, the second moiety comprises at least one of 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), depot medroxyprogesterone acetate (DMPA), diphenylphosphoryl azide (DPPA), 1,2-distearoyl-sn-glycero-3-sodium phosphatidate (DSPA), 1,2-dipalmitoylphosphatidylglycerol (DPPG), or 2,4-diacetylphloroglucinol (DAPG). In one embodiment, the second moiety further comprises at least one of 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis(dimethylphosphino)ethane (DMPE), 1,2-bis(diphenylphosphino)ethane (DPPE), 1,2-distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine 20:0 PC (DAPC), or 1,2-diradyl-3-phosphatidylethanolamine 20:0 PE (DAPE). In one embodiment, the second moiety comprises deoxycholate and at least one of 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis(dimethylphosphino)ethane (DMPE), 1,2-bis(diphenylphosphino)ethane (DPPE), 1,2-distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine 20:0 PC (DAPC), or 1,2-diradyl-3-phosphatidylethanolamine 20:0 PE (DAPE). In one embodiment, the first phase has a transition temperature less than 37°C and the second phase has a transition temperature greater than 37°C.In one embodiment, the first phase has a transition temperature above 37°C, and the second phase has a transition temperature below 37°C. In one embodiment, the phase having a transition temperature below 37°C comprises DODMA, MVL5, MC2, a cationic lipid, or an ionizable cationic lipid. In one embodiment, the phase having a transition temperature above 37°C comprises DSPC. In some cases, the ratio of cationic charge in the first portion to anionic charge in the second portion at pH 7.4 is about 0.25 to about 3.0. In some cases, this ratio is about 0.75 to about 1.25. In some cases, the first phase comprises MVL5 and an ionizable cationic lipid. In some cases, the ionizable cationic lipid is selected from the group consisting of DODMA, MC2, MC3, and KC2. In some cases, the ionizable cationic lipid is DODMA or MC2, and the molar ratio of MVL5 to ionizable cationic lipid in the delivery vehicle is about 6.25%:18.75%, 12.5%:12.5%, or 18.75%:6.25%. In some cases, the ratio of MVL5 to ionizable cationic lipid in the delivery vehicle is about 12.5%:12.5%. In some cases, the second phase comprises deoxycholate.

[0011] In one embodiment, the delivery vehicle further comprises 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) or a salt thereof. In some cases, the delivery vehicle further comprises DMPE-PEG or a salt thereof. In some cases, the first moiety comprises a cationic lipid and the second moiety comprises an anionic compound. In some cases, the cationic lipid is MVL5. In some cases, the anionic compound comprises a bile salt. In some cases, the bile salt is selected from the group consisting of cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, isolithocholic acid, isolithocholate, lithocholic acid, and lithocholate. In some cases, the bile salt is selected from the group consisting of lithocholate, deoxycholate, and isolithocholate. In some cases, the bile salt is deoxycholate. In some cases, the bile salt is isolithocholate. In some cases, the bile salt is at a concentration of about 10 mol% to about 80 mol%. In some cases, the cargo is at least partially surrounded by the lipid nanoparticle. In some cases, the cargo comprises a therapeutic agent. In some cases, the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, or any combination thereof. In some cases, the cargo is a nucleic acid, and the nucleic acid comprises DNA, modified DNA, RNA, modified RNA, miRNA, siRNA, antisense RNA, or any combination thereof. In some cases, the delivery vehicle further comprises a component for cellular internalization. In one embodiment, the component is a peptide, a carbohydrate, or a ligand.

[0012] In one aspect, the delivery vehicle further comprises a cell-penetrating peptide, a ligand, a mucus-penetrating polymer, a mucus-penetrating peptide, a non-mucus-adherent cell-penetrating peptide, or any combination thereof.

[0013] Provided herein are pharmaceutical compositions that include a delivery vehicle. Provided herein are methods for delivering a cargo to the gastrointestinal tract, comprising administering a delivery vehicle or pharmaceutical composition, wherein the delivery vehicle reaches the gastrointestinal tract and protects the cargo from bile salts present in the gastrointestinal tract. In some cases, the delivery vehicle easily passes through the mucus barrier. In some cases, the delivery vehicle is capable of reaching epithelial cells in the gastrointestinal tract. In some cases, reaching the epithelial cells involves the delivery vehicle approaching within 20 microns of the cell surface. In some cases, the delivery vehicle contacts the surface of the epithelial cells. In one embodiment, after the delivery vehicle contacts the epithelial cells, the cargo is absorbed by the epithelial cells. In some cases, the delivery vehicle or pharmaceutical composition is administered orally or parenterally to a subject in need thereof. In some cases, the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, or a biologic. In some cases, the nucleic acid encodes a therapeutic agent, and the epithelial cells express the therapeutic agent after absorbing the cargo. In some cases, the therapeutic agent is secreted by the epithelial cells.

[0014] Incorporation by Reference All publications, patents, and patent applications herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control. [Brief explanation of the drawings]

[0015] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure may be utilized and the accompanying drawings. [Figure 1] 1 shows the results of an exemplary assay measuring the transfection efficiency of an exemplary delivery vehicle of the present disclosure carrying DNA as cargo in HEK cells. [Figure 2] 1 shows the results of an exemplary assay measuring the stability of an exemplary delivery vehicle of the present disclosure. [Figure 3] 1 shows the results of an exemplary assay measuring the stability of an exemplary delivery vehicle of the present disclosure. [Figure 4] 1 shows the results of an exemplary assay measuring the stability of an exemplary delivery vehicle of the present disclosure. [Figure 5] 1 shows agarose gel electrophoresis of an exemplary delivery vehicle of the present disclosure (formulation number 5 in Table 1). Lanes are as follows from left to right: Lane 1 shows ladder; Lane 2 shows untreated delivery vehicle; Lane 3 shows delivery vehicle treated with 7% Triton-X 100; and Lane 4 shows delivery vehicle treated with 7% Triton-X and heat (70°C for 30 minutes). [Figure 6] Figure 1 shows a mouse colon section from a mouse administered 30 micrograms of DNA encapsulated in a DiI- and DiO-labeled delivery vehicle. The distribution of a 1% PEG-containing vehicle (Particle 5 in Table 3) labeled with DiI and DiO is observed, as shown by fluorescent imaging from DiI overlaid on bright field. See Example 5, Table 3 for a description of Particle 5 and other referenced particles in the figure. [Figure 7] A mouse colon section from a mouse administered 30 micrograms of DNA encapsulated in a DiI- and DiO-labeled delivery vehicle is shown, showing the distribution of DiI- and DiO-labeled 2% PEG-containing vehicle (particle 6 in Table 3) as shown by fluorescent imaging from DiI overlaid on bright field. [Figure 8] A mouse colon section from a mouse administered 30 micrograms of DNA encapsulated in a DiI- and DiO-labeled delivery vehicle (particle 7 in Table 3) is shown, showing the distribution of the DiI- and DiO-labeled 3% PEG-containing vehicle as shown by fluorescent imaging from DiI overlaid on bright field. [Figure 9]A mouse colon section from a mouse administered 30 micrograms of DNA encapsulated in a DiI- and DiO-labeled delivery vehicle is shown, showing the distribution of DiI- and DiO-labeled 5% PEG-containing vehicle (particle 8 in Table 3) as shown by fluorescent imaging from DiI overlaid on bright field. [Figure 10] A mouse colon section from a mouse administered 30 micrograms of DNA encapsulated in a DiI- and DiO-labeled delivery vehicle is shown, showing the distribution of DiI- and DiO-labeled 10% PEG-containing vehicle (particle 9 in Table 3) as shown by fluorescent imaging from DiI overlaid on bright field. [Figures 11A-11B] 1 shows the distribution of delivery vehicle in a representative colon section obtained from a mouse administered particles at a 0% / 25% MVL5 / DODMA percent molar ratio (particle 1 in Table 3). [Figures 12A-12B] 1 shows the distribution of delivery vehicles in a representative colon section obtained from a mouse administered a 6.25% / 18.75% (MVL5 / DODMA) percent molar ratio of particles (particle 2 in Table 3). [Figures 13A-13B] 1 shows the distribution of delivery vehicles in a representative colon section obtained from a mouse administered a 12.5% / 12.5% ​​(MVL5 / DODMA) percent molar ratio of particles (particle 3 in Table 3). [Figures 14A-14B] 1 shows the distribution of delivery vehicles in a representative colon section obtained from a mouse administered an 18.75% / 6.25% (MVL5 / DODMA) % molar ratio of particles (Particle 4). [Figures 15A-15B] 1 shows the distribution of delivery vehicle in a representative colon section obtained from a mouse administered particles at a 25% / 0% MVL5 / DODMA % molar ratio (particle 10 in Table 3). [Figures 16A-16D]Figures 16A and 16B show Swiss roll images of sections of the colon from the first mouse, and Figures 16C and 16D show Swiss roll images of sections of the colon from the second mouse, each administered MVL5 / DODMA / DOPC / deoxycholate / DMG-PEG with DiI and DiO (particle 11 in Table 3) using BioTek Citation software. Figures 16A and 16C show the DiI channel, and Figures 16B and 16D show the DiI channel overlaid on bright field. [Figures 17A-17D] Figures 17A and 17B show Swiss roll images of colon sections from a first mouse (Figure 17A) and a second mouse (Figure 17C and Figure 17D) administered MVL5 / DODMA / GMO / deoxycholate / DMG-PEG with DiI and DiO (particle 12 in Table 3), using BioTek Citation software. Figures 17A and 17C show the DiI channel, and Figures 17B and 17D show the DiI channel overlaid on bright field. [Figures 18A-18D] Figures 18A and 18B show Swiss roll images of colon sections from a first mouse (Figure 18A) and a second mouse (Figure 18C and Figure 18D) administered MVL5 / DODMA / DSPC / deoxycholate / DMG-PEG with DiI and DiO (particle 5 in Table 3), using BioTek Citation software. Figures 18A and 18C show the DiI channel, and Figures 18B and 18D show the DiI channel overlaid on bright field. [Figures 19A-19D] Figures 19A and 19B show Swiss roll images of a section of the colon from the first mouse administered PBS with DiI and DiO, respectively, using BioTek Cytation software, and Figures 19C and 19D show Swiss roll images of a section of the colon from the second mouse. Figures 19A and 19C show the DiI channel, and Figures 19B and 19D show the DiI channel overlaid on bright field. [Figure 20]Figure 1 shows a bar graph comparing the stability of different bile salt incorporating lipid structures in 10 g / L bile salts (cholate:deoxycholate mixture) by measuring perturbations in the lipid structures using FRET between DiI and DiO. FRET values ​​are normalized to untreated. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following description and examples will explain in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein, and as such may vary. Those skilled in the art will recognize that there are many variations and modifications of the present disclosure that fall within the scope of the present disclosure.

[0017] overview Delivery of drugs, such as therapeutic agents, to epithelial tissues and cells in the gastrointestinal (GI) tract, vagina, and lungs, presents particular challenges. In these tissues, epithelial cells are covered by a mucus layer, so therapeutic agents must penetrate and move through the mucus to reach the epithelial cells. Furthermore, once in or through the mucus layer, the therapeutic agent must come into proximity with the intended target cells and, in some cases, must interact with the cell membrane and / or enter the cells. Thus, delivery of drugs (also referred to herein as "cargo") is improved by a delivery vehicle that not only penetrates and crosses the mucus layer but also comes within reach of the intended epithelial cell target. Additionally, with respect to the GI tract and other tissues, the harsh environment, such as naturally occurring bile acids in the gastrointestinal tract, can present challenges for delivery stability and successful delivery of cargo to the intended target cells.

[0018] Provided herein are compositions ("delivery vehicles") and methods for delivering cargo using the delivery vehicles provided herein. In some aspects, the delivery vehicles can be further modified to provide stability and / or reach target epithelial cells in challenging environments. In embodiments, the delivery vehicles provided herein (also referred to herein as "mucosal epithelial-reaching" and "charge-separated" delivery vehicles) include those with separation of positive and negative charges to distinct sites within the vehicle, such that positively and negatively charged molecules are separated from each other rather than interspersed. The charge-separated delivery vehicles herein provide both epithelial-reaching functionality, reducing or preventing capture of the delivery vehicle in epithelial mucus by penetrating the mucus, and bringing the delivery vehicle into proximity with epithelial cells, such as within a distance of 20 microns or less.

[0019] Also disclosed herein are delivery vehicles comprising lipid-based delivery vehicles comprising lipid structures, such as lipid nanoparticles, and cargo, which have improved stability in high bile salt environments, such as those in the gastrointestinal tract. In some embodiments, the delivery vehicle can provide stability in the harsh environment of the gastrointestinal tract and may be more suitable for mucosal environments. Thus, the delivery vehicle may be suitable for delivering cargo (e.g., nucleic acids) to mucosal epithelial cells, such as intestinal epithelial cells, lung epithelial cells, cervical epithelial cells, rectal epithelial cells, and endometrial cells. Furthermore, the delivery vehicle may also be suitable for delivery to organs such as the skin.

[0020] In some cases, the delivery vehicles provided herein may include additional mucus-penetrating features that may aid in the penetration and movement of the delivery vehicle through the mucus surrounding epithelial cells. Such additional features include the incorporation of polymers, such as polyethylene glycol (PEG), methyl-bearing polyoxazoline polymers (PMOZ), ethyl-bearing polyoxazoline polymers (PEOZ), into the surface of the delivery vehicle and / or the inclusion of a mucus-penetrating peptide (MPP) linked to the surface of the delivery vehicle. In other cases, the vehicle does not have any PEG coating or low-density PEG coating (or low-density coating of another polymer).

[0021] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising." The terms "about" or "approximately" may mean within an acceptable error range for a particular value, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" may mean within ±10% of a given value. Where particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be inferred to mean an acceptable error range for the particular value.

[0022] As used herein, the term "about" and its grammatical equivalents in connection with a reference numerical value and its grammatical equivalents can include values ​​that are 10% above or below that value. For example, the amount "about 10" includes the amounts 9 to 11. The term "about" in connection with a reference numerical value can also include values ​​that are 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above or below that value.

[0023] The term "administering" and its grammatical equivalents may refer to any method of providing a structure described herein to a subject. Such methods are well known to those of skill in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration may be continuous or intermittent. In various embodiments, the structures disclosed herein may be administered therapeutically. In some cases, the structures may be administered to treat an existing disease or condition. In further various embodiments, the structures may be administered prophylactically to prevent a disease or condition.

[0024] The term "biodegradable" and its grammatical equivalents may refer to polymers, compositions, and formulations, such as those described herein, that are intended to degrade during use. The term "biodegradable" is intended to include materials and processes that are also referred to as "bioerodible."

[0025] The term "cancer" and its grammatical equivalents as used herein may refer to the hyperproliferation of cells whose distinctive characteristic - loss of normal control - results in uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis.In the context of the methods of the present invention, cancer includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, The cancer may be any cancer, including hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, e.g., of a malignant or benign type.

[0026] As used herein, the term "cargo" may refer to one or more molecules or structures contained in a delivery vehicle for delivery to or into a cell or tissue. Non-limiting examples of cargo may include nucleic acids, dyes, drugs, proteins, liposomes, small chemical molecules, large biological molecules, and any combination thereof.

[0027] As used herein, the term "cell" and its grammatical equivalents may refer to the structural and functional unit of an organism. Cells may be microscopic in size and consist of a cytoplasm and a nucleus enclosed within a membrane. A cell may refer to a small intestinal crypt cell. A crypt cell may refer to a crypt of Lieberkuhn, a pit-like structure that surrounds the base of a villi in the intestine. A cell may be of human or non-human origin. As used herein, a "conjugate" may refer to a covalent or non-covalent association of two or more molecules or structures, including, but not limited to, an association of a peptide, such as a mucus-penetrating peptide (MPP), with a delivery vehicle, a polymer, a surface modification, or any combination thereof.

[0028] As used herein, the term "functional" and its grammatical equivalents may refer to the ability to operate, have, or serve an intended purpose. The term "functional" may include any percentage from baseline to 100% of the intended purpose. For example, the term "functional" may include or include about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or up to about 100% of the intended purpose. In some cases, the term "functional" may mean greater than or greater than about 100% of normal function, such as 125, 150, 175, 200, 250, 300%, 400%, 500%, 600%, 700%, or up to about 1000% of the intended purpose.

[0029] As used herein, the term "gastrointestinal disorder" may refer to a disorder involving the digestive tract, including, but not limited to, the esophagus, stomach, small intestine, large intestine, and rectum, as well as the accessory organs of digestion, liver, gallbladder, and pancreas, and any combination thereof.

[0030] As used herein, the term "hydrophilic" and its grammatical equivalents refer to a substance or structure that has polar groups that readily interact with water. As used herein, the term "hydrophobic" and its grammatical equivalents refer to substances or structures that have polar groups that do not readily interact with water.

[0031] As used herein, the term "mucus" and its grammatical equivalents may refer to a viscoelastic natural substance, primarily comprising mucin glycoproteins and other materials, that protects the epithelial surfaces of various organs / tissues, including but not limited to the respiratory, nasal, cervicovaginal, gastrointestinal, rectal, visual, and auditory systems.

[0032] The term "lipid structure" as used herein refers to a lipid composition for delivery to cells or tissues, such as for delivering a therapeutic product such as a nucleic acid. As used herein, the term "lipid structure" and its grammatical equivalents can refer to a nanoparticle or a delivery vehicle. The structure can be a liposomal structure. The lipid structure can also refer to a particle. The lipid structure or particle can be a nanoparticle or a delivery vehicle. The lipid particle or structure can be of any shape, with a diameter from about 1 nm to about 1 micron. The nanoparticles or nanostructures can be 100-200 nm or about 100-200 nm. The nanoparticles or nanostructures can be up to 500 nm. Nanoparticles or nanostructures having a spherical shape can be referred to as "nanospheres."

[0033] As used herein, the term "structure" and its grammatical equivalents can refer to a nanoparticle or a delivery vehicle. A structure can be a liposome structure. A structure can also refer to a particle. A structure or particle can be a nanoparticle or a delivery vehicle. A particle or structure can be of any shape with a diameter from about 1 nm to about 1 micron. A nanoparticle or nanostructure can be 100-200 nm or about 100-200 nm. A nanoparticle or nanostructure can be up to 500 nm. A nanoparticle or nanostructure having a spherical shape can be referred to as a "nanosphere."

[0034] The terms "nucleic acid," "polynucleotide," and "oligonucleotide," and their grammatical equivalents, may be used interchangeably and may refer to deoxyribonucleotide and / or ribonucleotide polymers in either single- or double-stranded form, in a linear or circular conformation. For purposes of this disclosure, these terms should not be construed as limiting with respect to length. The terms may also encompass known analogs of natural nucleotides as well as nucleotides that are modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of adenine "A" can pair with thymine "T."

[0035] The term "pharmaceutically acceptable carrier" and its grammatical equivalents may refer to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These solutions, dispersions, suspensions, or emulsions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).

[0036] As used herein, the term "predisposed" may be understood to mean an increased probability (e.g., at least a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more increase in probability) that a subject will suffer from a disease or condition.

[0037] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to situations characterized by the supervision (e.g., continuous or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, hospital janitor, or hospice worker). A subject can be a mammal. A subject can be a human male or a human female. A subject can be of any age. A subject can be an embryo. A subject can be a newborn or up to about 100 years old. A subject can be in need thereof. A subject can have a disease such as cancer.

[0038] As used herein, the term "sequence" and its grammatical equivalents can refer to a nucleotide sequence, which can be DNA and / or RNA, which can be linear, circular, or branched, and which can be single-stranded or double-stranded. The sequence can be of any length, e.g., from 2 to 1,000,000 or more nucleotides in length (or any integer value therebetween or greater), e.g., from about 100 to about 10,000 nucleotides or from about 200 to about 500 nucleotides. In some cases, where indicated, "sequence" as used herein can refer to an amino acid sequence, such as a protein, polypeptide, and / or peptide sequence.

[0039] As used herein, the term "stem cell" may refer to an undifferentiated cell of a multicellular organism that is capable of giving rise to an unlimited number of cells of the same type. Stem cells can also give rise to other types of cells by differentiation. Stem cells may be found in crypts. Stem cells may be progenitor cells of epithelial cells found on the villous surface of the intestine. Stem cells may be cancerous. Stem cells may be totipotent, unipotent, or pluripotent. Stem cells may be artificial stem cells.

[0040] The terms "treatment" or "treating" and their grammatical equivalents may refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. Treatment may include active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder. Treatment may include causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. Additionally, treatment may include palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder. Treatment may include preventative treatment, i.e., treatment directed at minimizing or partially or completely inhibiting the occurrence of a disease, condition, or disorder. Treatment may include supportive treatment, i.e., treatment used to supplement another specific therapy directed at ameliorating a disease, condition, or disorder. In some cases, a condition may be pathological. In some cases, a treatment may not completely cure, ameliorate, stabilize, or prevent a disease, condition, or disorder.

[0041] When used in the context of chemical groups, "hydrogen" means -H, "hydroxy" means -OH, and "halogen" means independently -F, -Cl, -Br, or -I. In the structures provided herein, the following bracketed subscripts further define groups as follows: "(C n ) defines the exact number (n) of carbon atoms in the group. For example, "(C 2~10 )"Alkyl" refers to an alkyl group having 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a derivable range therein (e.g., 3 to 10 carbon atoms).

[0042] An "alkyl" group may refer to an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, meaning that it does not contain an alkene or alkyne moiety. The alkyl moiety may also be an "unsaturated alkyl" group, meaning that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight-chain, or cyclic. Furthermore, the alkyl moiety, whether saturated or unsaturated, may contain branched, straight-chain, and / or cyclic moieties. Depending on the structure, an alkyl group may be a monoradical or a diradical (i.e., an alkylene group). A "heteroalkyl" group is as described for "alkyl" in which at least one C atom is replaced by an N, S, or O atom. A "heteroalkyl" group may contain straight-chain, branched, and / or cyclic moieties. In certain embodiments, a "lower alkyl" is an alkyl group having 1 to 6 carbon atoms (i.e., a C1-C6 alkyl group). In specific cases, a "lower alkyl" can be straight-chain or branched-chain.

[0043] "Aryl" refers to a group derived from an aromatic monocyclic or aromatic polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or aromatic polycyclic hydrocarbon ring system contains only hydrogen and carbon, and has 5 to 18 carbon atoms, and at least one ring in the ring system is aromatic, i.e., it contains a cyclic delocalized (4n+2) π-electron system consistent with Hückel theory. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene. In some embodiments, the term "aryl" can refer to an aromatic ring in which each of the atoms forming the ring is a carbon atom. An aryl ring can be formed by 5, 6, 7, 8, 9, or 10 or more carbon atoms. An aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (ie, an arylene group).

[0044] "Heteroaryl" refers to a group derived from a 3- to 12-membered aromatic ring group containing 2 to 11 carbon atoms and at least one heteroatom, each heteroatom being selected from N, O, and S. As used herein, a heteroaryl ring can be selected from monocyclic or bicyclic fused or bridged ring systems in which at least one ring in the ring system is aromatic, i.e., it contains a cyclic delocalized (4n+2) π-electron system consistent with Hückel theory. Heteroatoms in a heteroaryl group can be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. A heteroaryl can be attached to the remainder of the molecule through any moiety of the heteroaryl, such as a carbon or nitrogen atom of the heteroaryl, where valence permits. Examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[ 1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxazolyl Soazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl , pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4 Examples include, but are not limited to, 5-thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e., thienyl). "X-membered heteroaryl" refers to the number of ring atoms in the ring, i.e., X. For example, a 5-membered heteroaryl ring or a 5-membered aromatic heterocycle has 5 ring atoms, such as triazole, oxazole, thiophene, etc.

[0045] In some embodiments, the term "heteroaryl" when used without the "substituted" modifier refers to a monovalent group having an aromatic carbon or nitrogen atom as its point of attachment, said carbon or nitrogen atom forming part of an aromatic ring structure, at least one ring atom being nitrogen, oxygen, or sulfur, and the monovalent group consisting of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. Non-limiting examples of heteroaryl groups include acridinyl, furanyl, imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl, pyrroloimidazolyl, chromenyl (when the point of attachment is at one of the aromatic atoms), and chromanyl (when the point of attachment is at one of the aromatic atoms). Substituted heteroaryl refers to a monovalent group having an aromatic carbon or nitrogen atom as its point of attachment, said carbon or nitrogen atom forming part of an aromatic ring structure, at least one ring atom being nitrogen, oxygen, or sulfur, and the monovalent group further having at least one atom independently selected from the group consisting of non-aromatic nitrogen, non-aromatic oxygen, non-aromatic sulfur F, Cl, Br, I, Si, and P.

[0046] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbon or substitutable heteroatoms, e.g., NH, of the structure. It will be understood that "substituted" or "substituted by" includes the implicit proviso that such substitution is consistent with the allowed valences of the replaced atom and substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, and the like. In certain embodiments, the term "substituted" refers to a moiety having substituents replacing two hydrogen atoms on the same carbon atom, such as replacing two hydrogen atoms on a single carbon with an oxo, imino, or thioxo group. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.

[0047] In some embodiments, substituents can include any substituent described herein, for example, halogen, hydroxy, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO), imino (=NH), oximo (=N-OH), hydrazino (=N-NH), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -Rb -OR c -C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (wherein t is 1 or 2), -R b -S(O) t R a (wherein t is 1 or 2), -R b -S(O) t OR a (wherein t is 1 or 2), and -R b -S(O) t N(R a )2 (wherein t is 1 or 2), and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which may be alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c-C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (wherein t is 1 or 2), -R b -S(O) t R a (wherein t is 1 or 2), -R b -S(O) t OR a (wherein t is 1 or 2), and -R b -S(O) t N(R a )2 (wherein t is 1 or 2), and each R a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl; and each R a is, if valence permits, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c-C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (wherein t is 1 or 2), -R b -S(O) t R a (wherein t is 1 or 2), -R b -S(O) t OR a (wherein t is 1 or 2), and -R b -S(O) t N(R a )2 (wherein t is 1 or 2), and each R b are independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain; and each R c is a straight or branched alkylene, alkenylene, or alkynylene chain.

[0048] Delivery vehicles with charge separation In some cases, the delivery vehicles provided herein have separated positive and negative charges in different regions within the particle, with each region being composed of a different polymer (which provides the charge to the region). In some cases, the delivery vehicles provided herein contain positively charged lipids and negatively charged lipids, and the regions are separated by phases, such as in a liquid phase and a gel phase. In some cases, the delivery vehicle may contain a positively charged liquid phase and a negatively charged gel phase, or a positively charged gel phase and a negatively charged liquid phase.

[0049] The delivery vehicles provided herein can efficiently deliver cargoes, such as nucleic acids, proteins, peptides, and / or small molecules, to epithelial cells in mucosal tissues. The delivery vehicles provided herein are useful for treating diseases and conditions that affect and / or occur in mucosal tissues, such as mucosal tissues in the digestive tract. Non-limiting examples include familial adenomatous polyposis (FAP), mild FAP, colon cancer, chronic inflammatory bowel disease, chronic inflammatory bowel disease, microvillus inclusion disease, and congenital diarrheal disease. The delivery vehicles provided herein are also useful for providing therapeutic agents and / or nucleic acids that express the therapeutic agent in mucosal tissues, where such agents can remain in the targeted epithelial cells and / or be transported to other disease-affected cells and tissues within the subject. In some cases, the delivery vehicles provide close proximity to epithelial cells. In some embodiments, such proximity is less than about 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron. In some cases, the delivery vehicle herein contacts an epithelial cell. In some cases, the delivery vehicle is absorbed into the cell and the cargo carried by the delivery vehicle is released inside the cell. In some cases, the delivery vehicle contacts an epithelial cell and the cargo from the delivery vehicle is released outside the cell.

[0050] The delivery vehicles provided herein can be lipid structures. The lipid structures can be used to deliver cargo to cells or tissues. In some cases, the cargo can include a therapeutic product such as a nucleic acid. Lipid structures include, but are not limited to, lipid particles, lipid nanoparticles, liposomes, or vesicles, such as vesicles in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single, unilamellar, or multiple, multilamellar) or at least partially coated with lipids, containing a therapeutic product, or lipid aggregates or micelles in which a lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.

[0051] The delivery vehicles (e.g., lipid nanoparticles, liposomes, and micelle-like structures) herein have at least two sites, with positive and negative charges that are not interspersed but instead located in separate sites. For example, the negative and positive charges can be located in opposing sites on the lipid structures provided herein at a pH of about 5.5 to 8.0, such as a pH of about 7.4.

[0052] In one embodiment, the positive and negative charges are in two separate regions, each region being a different phase of the lipid structure, e.g., the liquid phase or the solid (gel) phase. In one embodiment, the positive charge can be in the liquid phase and the negative charge can be in the solid phase, e.g., the gel phase, or vice versa. Charge separation can allow for both attractive and repulsive forces. In some cases, positive lipids can be attracted to target cells due to their high negative potential. In another embodiment, the repulsive force of the negative face can prevent the positive face from being dynamically trapped in mucus. In some cases, for example, cationic charges on lipids on a delivery vehicle can be attracted to mucus on the way to the target cell, causing dynamic trapping in the mucus, thereby trapping the delivery vehicle. The mucus eventually sloughs off, removing the delivery vehicle. In another embodiment, anionic delivery vehicles can be repelled by mucus and may not be able to navigate through it. Zwitterionic particles can act like neutral particles, with no net force. Zwitterionic particles may follow the water currents, similar to PEGylated systems, and may not be trapped in mucus, but may not reach epithelial cells.

[0053] In certain embodiments, the lipid structure may comprise one or more of anionic or cationic lipids, neutral lipids, sterols, and lipids selected to reduce aggregation of lipid particles during formation. Aggregation may occur from steric stabilization of the lipid structure, which may prevent charge-induced aggregation during formation. The lipid structure may comprise two or more cationic lipids. In one aspect, the cationic lipid may be in a first phase and the anionic lipid may be in a second phase, so that the lipid structure comprises two phases with lipids of different charges. Lipids may be selected to contribute different advantageous properties. For example, amine pK aCationic lipids with different properties, such as chemical stability, circulation half-life, tissue half-life, net tissue accumulation, or toxicity, can be used in the lipid structure. In particular, cationic lipids can be selected so that the properties of the lipid structure of the mixed lipids are more desirable than those of the single lipid structure of the individual lipids. The net tissue accumulation and long-term toxicity (if any) of the cationic lipids can be advantageously controlled by selecting a mixture of cationic lipids instead of a single cationic lipid in a given formulation. Such a mixture may also provide better encapsulation and / or release of cargo such as nucleic acids. Combinations of cationic lipids can also affect systemic stability compared to a single entity in a formulation.

[0054] In some cases, cationic lipids can acquire a positive charge due to one or more amines present in the polar head group. In some cases, the lipid structure can be a cationic liposome. In some cases, the liposome can be a cationic liposome used to carry negatively charged polynucleic acids such as DNA. The presence of positively charged amines can facilitate binding with anions such as those found in DNA. The liposomes formed in this manner can be the result of energetic contributions from van der Waals forces and electrostatic binding to the DNA cargo, which can partially contribute to the liposome shape. In some cases, cationic (and neutral) lipids can be used for gene delivery. In other cases, anionic liposomes can be used to deliver other therapeutic agents.

[0055] In some embodiments, the delivery vehicles provided herein further comprise a cargo. In some cases, the cargo comprises a therapeutic agent. In some cases, the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, or any combination thereof. In some embodiments, the delivery vehicles provided herein comprise a component for cellular internalization. In some cases, the component is a peptide, a carbohydrate, or a ligand. In some embodiments, the delivery vehicles provided herein also comprise a stability component. In some cases, the stability component is polyethylene glycol (PEG).

[0056] In some embodiments of the delivery vehicle, the first moiety comprises an unsaturated or short-tail lipid. In some cases, the unsaturated lipid comprises a cationic or ionizable cationic lipid. In some embodiments, the cationic lipid comprises a polyvalent cationic lipid or a monovalent cationic lipid.

[0057] In some cases, charge separation can result in superior and / or unexpected performance of the subject delivery vehicle. For example, the use of PEG is believed to increase transport to target cells, such as intestinal epithelial cells, as provided in Maisel K et al., "Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distribution in the gastrointestinal tract following oral and rectal administration in the mouse," Journal of Controlled Release, incorporated herein by reference. In some cases, increasing PEGylation reduces distribution within or at intestinal tissues, thereby supporting the use of delivery vehicles with reduced PEGylation compared to conventional vehicles. One mechanism by which reduced PEGylation can improve transport and / or distribution to and near target cells is by increasing the exposure of positive charges on the surface of the subject vehicle by reducing the shielding properties of PEGylation.

[0058] In some cases, delivery vehicles comprising charge separation provided herein may have improved transport, target cell transduction, epithelial reach, or a combination thereof, compared to a comparable delivery vehicle lacking charge separation. In some cases, the improvement is from about 1-fold to 50-fold, 99-fold, 148-fold, 197-fold, 246-fold, 295-fold, 344-fold, 393-fold, 442-fold, 491-fold, 540-fold, 589-fold, 638-fold, 687-fold, 736-fold, 785-fold, 834-fold, 883-fold, 932-fold, 981-fold, or up to about 1000-fold, compared to a comparable delivery vehicle lacking charge separation.

[0059] In some cases, the delivery vehicle is MVL5 / MC2 / DSPC / deoxycholate / DMG-PEG, MVL5 / MC2 / DSPC / deoxycholate / DMPE-PEG, MVL5 / CL1H6 / DSPC / deoxycholate / DMG-PEG, MVL5 / CL4H6 / DSPC / deoxycholate / DMG-PEG, MVL5 / MC2 / DSPC / chenodeoxycholate / DMG-PEG, MVL5 / MC2 / DMPC / deoxycholate / DMG-PEG, MVL5 / M It may comprise any one of C2 / DMPC / deoxycholate / DMPE-PEG, MVL5 / CL1H6 / DMPC / deoxycholate / DMG-PEG, MVL5 / MC2 / DSPC / deoxycholate / lithocholate / DMG-PEG, MVL5 / CL1H6 / DSPC / deoxycholate / lithocholate / DMG-PEG, MVL5 / MC2 / DSPC / alloisolithocholate / DMG-PEG, or MVL5 / MC2 / DSPC / dehydrolithocholate / DMG-PEG.

[0060] The delivery vehicle can be produced using various molar ratios. In some cases, the pharmaceutical formulation includes MVL5, MC2, deoxycholate, DSPC, and DMG-PEG in a molar ratio of about 0.96:0.96:2.592:3.168:0.0768:0.0384:0.0384. In some cases, the ratio of cationic charge in the first portion to anionic charge in the second portion at pH 7.4 is about 0.25, 0.45, 0.65, 0.85, 1.05, 1.25, 1.45, 1.65, 1.85, 2.05, 2.25, 2.45, 2.65, or 2.85. In some cases, the ratio of cationic charge in the first portion to anionic charge in the second portion at pH 7.4 is about 0.25 to about 1.05, 0.75 to about 1.25, 1.05 to about 1.45, or 0.85 to about 1.85. In another embodiment, the ratio of polyvalent lipid to ionizable cationic lipid in the delivery vehicle is about (6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, or 8%) to (8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%), (12%, 12.25%, 12.5%), or (13%). %, 12.75%, or 13%) to (12%, 12.25%, 12.5%, 12.75%, or 13%), or (18%, 18.25%, 18.5%, 18.75%, 19%, 19.25%, 19.5%, 19.75%, 20%) to (6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, or 8%). In some embodiments, the bile salts are at a concentration of about 10 molar%, 15 molar%, 20 molar%, 25 molar%, 30 molar%, 35 molar%, 40 molar%, 45 molar%, 50 molar%, 55 molar%, 60 molar%, 65 molar%, 70 molar%, 75 molar%, or about 80 molar%. In some cases, the bile salts are about 10 mol% to 30 mol%, 20 mol% to 50 mol%, 30 mol% to 60 mol%, or 40 mol% to 80 mol%.Suitable alternative formulations may include multivalent lipids, ionizable cationic lipids, bile salts, structural lipids, and / or lipid-PEGs in molar ratios that are about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% more or less than those provided herein.

[0061] Delivery Vehicle Stability In some embodiments, delivery vehicle stability may be increased by the incorporation of bile acids or bile salts. The terms "bile acid," "bile salt," and "bile acid / salt" are used interchangeably herein unless otherwise specified. Any reference to bile acid as used herein may include a reference to a bile acid or its salt. As used herein, the terms "bile acid" (and "bile salt," "bile acid / salt") may include, by non-limiting example, steroid acids (and their anions) found in animal (e.g., human) bile, and salts thereof, including cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, lithocholic acid, lithocholate, etc., or salts thereof. In some embodiments, the bile acid is ursodiol, isolithocholate, alloisolithocholate, dehydrolithocholate, or 5-β-cholanic acid. Taurocholic acid and taurocholate are referred to herein as TCAs. Any reference to a bile acid as used herein may include a reference to a bile acid, the only bile acid, one or more bile acids, or at least one bile acid. Additionally, pharmaceutically acceptable bile acid esters, such as bile acids conjugated to amino acids (e.g., glycine or taurine), may be used as "bile acids" as described herein. Other bile acid esters may include, for example, substituted or unsubstituted alkyl esters, substituted or unsubstituted heteroalkyl esters, substituted or unsubstituted aryl esters, substituted or unsubstituted heteroaryl esters, etc. For example, the term "bile acid" may include cholic acid, glycocholate, and taurocholate (and salts thereof) conjugated to either glycine or taurine, respectively. Any reference to a bile acid as used herein may include a reference to the same compound whether naturally or synthetically prepared. Furthermore, it should be understood that any single reference to a component (bile acid or otherwise) as used herein may include a reference to only one, more than one, or at least one such component.Similarly, any plural reference to an ingredient as used herein may include a reference to only one, one or more, or at least one such ingredient, unless otherwise specified.

[0062] In some embodiments of the delivery vehicle herein, the bile salt may be cholic acid. In some embodiments, the bile salt may be deoxycholate. In some embodiments, the bile salt incorporation may be cholic acid and deoxycholate. In some embodiments, the bile salt may include cholate, deoxycholate, conjugates or derivatives thereof, or combinations thereof. In further embodiments, the bile salt may be chenodeoxycholic acid, lithocholic acid, taurodeoxycholic acid, or combinations thereof.

[0063] In some embodiments, the bile salt concentration in the lipid nanoparticles of the delivery vehicle (or in a composition comprising the lipid nanoparticles) is about 80 mol% to about 70 mol%, about 65 mol% to about 55 mol%, about 60 mol% to about 50%, about 55 mol% to about 45 mol%, about 50 mol% to about 40 mol%, about 45 mol% to about 35 mol%, about 40 mol% to about 30 mol%, about 35 mol% to about 25 mol%, or %, about 30 mol% to about 20 mol%, about 25 mol% to about 15 mol%, about 20 mol% to about 10 mol%, about 15 mol% to about 10 mol%, about 60 mol% to about 20 mol%, about 25.9 mol%, about 30.4 mol%, about 34.9 mol%, about 39.4 mol%, about 37.1 mol%, about 43.9 mol%, or about 45 mol%, or about 80 mol% to about 10 mol%, etc. In some cases, the bile salt concentration in the lipid nanoparticles of the delivery vehicle (or a composition comprising the lipid nanoparticles) may comprise about 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, or 85 mol%.

[0064] The efficiency of cellular uptake by structures such as the compositions described herein having bile salts contained in lipid nanoparticles of a delivery vehicle can allow efficient penetration and passage through the mucus layer to target cells, thereby having efficient uptake by target cells, for example, uptake can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 18 In some embodiments, the composition may have a higher percent cellular uptake than a comparable delivery vehicle that does not contain bile salts. The improvement can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or up to about 80% or better. In some cases, the transfection or integration efficiency of polynucleic acid cargo delivered to cells by the delivery vehicle compositions described herein can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% better than a comparable delivery vehicle that does not include additional features such as bile salts and specific compositions of MPPs and / or lipids. In some cases, the transfection or integration efficiency of polynucleic acid cargo delivered to cells by the delivery vehicle compositions described herein may be about 5%, to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or even 65% better than an equivalent delivery vehicle that does not contain bile salts.

[0065] In some embodiments, the stability of a delivery vehicle can be measured by a bile salt stability assay in a high bile salt mimicking environment. For example, bile salt stability can be measured by fluorescence spectroscopy, such as the relative fluorescence of a delivery vehicle containing various concentrations of bile salts in a Förster resonance energy transfer (FRET) assay. In some embodiments, the incorporated bile salts may increase the stability of the delivery vehicle by about 80% to about 10%, such as about 80% to about 70%, about 65% to about 55%, about 60% to about 50%, about 55% to about 45%, about 50% to about 40%, about 45% to about 35%, about 40% to about 30%, about 35% to about 25%, about 30% to about 20%, about 25% to about 15%, about 20% to about 10%, about 15% to about 10%, about 60% to about 20%, about 25.9%, about 30.4%, about 34.9%, about 39.4%, about 37.1%, about 43.9%, or about 45%. In some embodiments, the incorporated bile salts may increase the stability of the delivery vehicle compared to an equivalent delivery vehicle lacking bile salts. In some cases, delivery vehicles comprising bile salts provided herein may have improved transport, target cell transduction, epithelial penetration, or a combination thereof, compared to an equivalent delivery vehicle lacking bile salts. In some cases, the improvement is about 1-fold, 50-fold, 99-fold, 148-fold, 197-fold, 246-fold, 295-fold, 344-fold, 393-fold, 442-fold, 491-fold, 540-fold, 589-fold, 638-fold, 687-fold, 736-fold, 785-fold, 834-fold, 883-fold, 932-fold, 981-fold, or up to about 1000-fold, compared to an equivalent delivery vehicle lacking bile salts. In some examples, the percent increase in stability can be measured by increased relative fluorescence units or relative luminescence units in an assay such as in vivo or ex vivo FRET.

[0066] In some embodiments, a delivery vehicle of the present disclosure can comprise a cationic lipid and a bile salt, where the lipid can be a saturated cationic lipid or an unsaturated cationic lipid, and the saturated cationic lipid can have a phase transition temperature that is at least about 20° C. In some embodiments, a delivery vehicle of the present disclosure can comprise at least one saturated cationic lipid and at least a bile salt, where the at least one saturated cationic lipid can have a phase transition temperature of at least about 37° C. In some embodiments, the saturated cationic lipid has a phase transition temperature of at least about 20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 52° C., 54° C., 56° C., 58° C., and / or up to about 60° C. For example, saturated cationic lipids may have a phase transition temperature of 30°C to 60°C, 35°C to 60°C, 37°C to 60°C, 37°C to 55°C, 37°C to 50°C, 37°C to 45°C, or 37°C to 40°C. In some embodiments, a delivery vehicle of the present disclosure may comprise at least one saturated cationic lipid and at least a bile salt, wherein the at least one saturated cationic lipid has a phase transition temperature of at least about 37°C. The lipid delivery vehicle may further comprise a saturated non-cationic lipid. The saturated non-cationic lipid may have a phase transition temperature of at least about 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, and / or up to about 60°C. For example, saturated noncationic lipids may have a phase transition temperature of about 30°C to 60°C, 35°C to 60°C, 37°C to 60°C, 37°C to 55°C, 37°C to 50°C, 37°C to 45°C, or 37°C to 40°C. The lipid delivery vehicle, in some cases, may further comprise a lipid conjugated to a hydrophilic polymer such as polyethylene glycol (PEG). The delivery vehicle, in some cases, may be conjugated to at least one of a cell-penetrating peptide, a ligand, a mucus-penetrating polymer, a peptide enabling mucus penetration, a cell-penetrating peptide that is not substantially mucus-adhesive, or any combination thereof.

[0067] In some embodiments, the delivery vehicle comprises a cargo in a lipid structure, e.g., lipid nanoparticle, wherein the lipid nanoparticle comprises a bile salt and (a) a saturated cationic lipid and a non-cationic lipid having a phase transition temperature of at least about 37°C, or (b) a saturated cationic lipid, an unsaturated cationic lipid, and a non-cationic lipid, wherein the unsaturated cationic lipid, the non-cationic lipid, or the unsaturated cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C, or (c) a polyvalent cationic lipid, a non-cationic lipid, wherein the polyvalent cationic lipid, the non-cationic lipid and (iii) a delivery vehicle that is stable in a high bile salt environment compared to an otherwise identical delivery vehicle that comprises at least one of a bile salt, a polycationic lipid, and a non-cationic lipid having a phase transition temperature of at least about 37°C, and that (i) does not comprise lipid nanoparticles comprising bile salts and at least one of (a), (b), or (c), or (ii) comprises lipid nanoparticles comprising at least one of (a), (b), or (c) but does not comprise bile salts, or (iii) comprises bile salts but does not comprise at least one of (a), (b), or (c). The saturated cationic lipids, unsaturated cationic lipids, non-cationic lipids, and / or polyvalent cationic lipids may have a phase transition temperature of at least about 20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 52° C., 54° C., 56° C., 58° C., and / or up to about 60° C. For example, the saturated cationic lipids, unsaturated cationic lipids, non-cationic lipids, and / or polyvalent cationic lipids may have a phase transition temperature of about 30° C. to 60° C., 35° C. to 60° C., 37° C. to 60° C., 37° C. to 55° C., 37° C. to 50° C., 37° C. to 45° C., or 37° C. to 40° C.

[0068] In some embodiments, a delivery vehicle comprising a cargo and a lipid structure, such as a lipid nanoparticle, wherein the lipid nanoparticle comprises a bile salt and either (a) a saturated cationic lipid having a phase transition temperature of at least about 37°C, or (b) a saturated cationic lipid, an unsaturated cationic lipid, and a non-cationic lipid, wherein the unsaturated cationic lipid, the non-cationic lipid, or the unsaturated cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C, or (c) a polyvalent cationic lipid and a non-cationic lipid, wherein the polyvalent cationic lipid, the non-cationic lipid, or the polyvalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C. The delivery vehicle exhibits increased stability in a solution containing at least about 5 g / L of cholic acid and deoxycholate compared to otherwise identical lipid nanoparticles that (i) do not contain lipid nanoparticles comprising bile salts and at least one of (a), (b), or (c), or (ii) contain lipid nanoparticles comprising at least one of (a), (b), or (c) but do not contain bile salts, or (iii) contain bile salts but do not contain at least one of (a), (b), or (c), wherein the stability is measured by the relative fluorescence intensity of fluorescent lipids incorporated into the lipid nanoparticles in a Förster resonance energy transfer (FRET) assay. In some cases, the delivery vehicle (i) exhibits increased stability in a solution containing at least about 0.5 g / L, 1 g / L, 5 g / L, 7 g / L, 9 g / L, 11 g / L, 13 g / L, 15 g / L, 17 g / L, 19 g / L, 21 g / L, 23 g / L, or up to about 25 g / L of bile acids, e.g., a mixture of about 40%, 45%, 50%, or up to about 55% cholic acid and about 40%, 45%, 50%, 55%, or up to about 60% deoxycholate, compared to an otherwise identical delivery vehicle that does not contain bile salts, as measured by the relative fluorescence intensity of fluorescent lipids incorporated into the lipid nanoparticles in a Förster resonance energy transfer (FRET) assay.

[0069] In some embodiments, a delivery vehicle is provided comprising (i) a cargo and (ii) a lipid structure, such as a lipid nanoparticle, wherein the lipid nanoparticle comprises at least one saturated cationic lipid and a bile salt, wherein the at least one saturated cationic lipid has a phase transition temperature of at least about 37° C. In some embodiments, a delivery vehicle is provided comprising (i) a cargo and (ii) a lipid nanoparticle, wherein the lipid nanoparticle comprises at least one saturated lipid, at least one unsaturated cationic lipid, and a bile salt, wherein the concentration of the at least one unsaturated cationic lipid in the lipid nanoparticle is less than 50 mol %.

[0070] Exemplary delivery vehicles are described herein and are provided, for example, in Tables 1, 2, 3, and 4. Any one of the delivery vehicles exemplified in Tables 1-4 can be further modified. For example, additional lipids, cargo, modifications, additions, or subtractions can be made. In some cases, any one of the delivery vehicles in Table 1 can further comprise a lipid-PEG.

[0071] [Table 1]

[0072] Lipids for use in delivery vehicles The delivery vehicles herein, including those bearing cargo, comprise one or more lipids, such as in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise at least one saturated lipid, at least one of an unsaturated cationic lipid or an unsaturated non-cationic lipid, and a bile salt. In some embodiments, the lipid nanoparticles comprise at least one saturated lipid, wherein the saturated lipid comprises a saturated cationic lipid having a phase transition temperature of at least about 37°C or a saturated non-cationic lipid having a phase transition temperature of at least about 37°C. In some aspects, the lipid nanoparticles further comprise at least one of a non-cationic lipid, a polyvalent cationic lipid, a permanently charged cationic lipid, or any combination thereof. In some embodiments, the lipid nanoparticles comprise a bile salt and a polyvalent cationic lipid and a non-cationic lipid, wherein the polyvalent cationic lipid, the non-cationic lipid, or the polyvalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C. In some embodiments, the lipid nanoparticles comprise a bile salt and a saturated cationic lipid and a non-cationic lipid having a phase transition temperature of at least about 37°C. In some embodiments, the lipid nanoparticles comprise bile salts and saturated cationic lipids, unsaturated cationic lipids, and non-cationic lipids, wherein the unsaturated cationic lipids, non-cationic lipids, or the unsaturated cationic lipids and non-cationic lipids have a phase transition temperature of at least about 37°C. In some embodiments, the delivery vehicle comprises a first region that is positively charged at a pH of about 5.5-8.0 and a second region that is negatively charged at a pH of about 5.5-8.0, the first region and the second region being separated so that the positive and negative charges are not interspersed, and one or both regions comprise a lipid. In some embodiments, the first region comprises an unsaturated or short-tail lipid, such as a cationic or ionizable cationic lipid, e.g., a polyvalent cationic lipid or a monovalent cationic lipid.

[0073] In one aspect, cationic lipids for use in the lipid nanoparticles of the delivery vehicles herein may include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP), dimethyldioctadecylammonium (DDA), 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), and dioctadecylamidoglycylspermine (DOGS). Dioleoylphosphatidylethanolamine (DOPE), polyethyleneimine (PEI), and neutral lipids are often used in conjunction with cationic lipids due to their membrane-destabilizing effect at low pH, which may aid in endolysosomal escape. In some embodiments, saturated cationic lipids may be utilized in the delivery vehicles provided herein. Saturated cationic lipids may have a positive charge at or above pH 4. In some embodiments, the saturated cationic lipid is 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammonium propane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzoyl)-

[0033] The compound may comprise at least one of N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, or any combination thereof.In examples where the saturated cationic lipid comprises an alkyl group, the alkyl group can be at least one conjugated derivative of myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, lauroyl, tridecanoyl, nonadecanoyl, arachidoyl, heneicassanoyl, behenoyl, tricosanoyl, lignoceroyl, or any combination thereof. In some embodiments, the saturated cationic lipid can comprise at least one saturated cationic lipid having a phase transition temperature of at least about 37 ° C, including at least one of 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-distearoyl-3-dimethylammonium-propane (DSDAP), or any combination thereof. In one aspect, the cationic lipid can be in the gel phase of the lipid structure, and the anionic lipid can be in the liquid phase.

[0074] In some embodiments, the lipid nanoparticles of the delivery vehicle may comprise at least one unsaturated cationic lipid. In some embodiments, the unsaturated cationic lipid may have a positive charge at pH 4, or at a pH greater than about pH 4 and less than about pH 8. In some embodiments, the unsaturated cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammoniumpropane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminium] N-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, 1,2-dialkyloxy-N,N-dimethylaminopropane, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkylethylphosphocholine, MC3, MC2, MC4, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, N4-cholesteryl-spermine, or a salt thereof, or any combination thereof. In examples where the unsaturated cationic lipid comprises an alkyl, the alkyl can be at least one conjugated derivative of oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, paulic acid, vaccenic acid, palmitoleic acid, docosatetraenoic acid, arachidonic acid, dihomo-γ-linolenic acid, γ-linolenic acid, linoelaidic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, stearidonic acid, α-linolenic acid, or any combination thereof.In some embodiments, the unsaturated cationic lipid can include at least one of 1,2-dialkyloxy-N,N-dimethylaminopropane, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkylethylphosphocholine, MC3, MC2, MC4, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, N4-cholesteryl-spermine, or a salt thereof, or any combination thereof. In some cases, the lipid can be selected from the group consisting of those described in U.S. Patent Application Publication No. 20200129431A1 and Sato Y et al., "Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo," both of which are incorporated herein by reference. As described in "In vivo," Journal of Controlled Release, 2019, Vol. 295, pp. 140-152, the lipid structure may include or be 7-(4-(dimethylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL1H6), CL1A6, CL1A6, CL3A6, CL4A6, CL5A6, CL6A6, CL7A6, CL8A6, CL9A6, CL10A6, CL11A6, CL12A6, CL13A6, CL14A6, CL15A6, YSK12-C4. In one embodiment, the cationic lipid may be in the liquid phase of the lipid structure, and the anionic lipid may be in the gel or solid phase of the lipid structure.

[0075] In some cases, the lipid nanoparticles of the delivery vehicle may comprise a multivalent cationic lipid. The multivalent cationic lipid may be selected from N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), a salt thereof, and any combination thereof. In one embodiment, the delivery vehicle provided herein may be produced using MVL5. In one embodiment, MVL5, GL67, or a combination thereof is in the liquid phase of the delivery vehicle. Any of the polyvalent cationic lipids provided herein can be incorporated into a provided vehicle or particle at less than about 50 mol%, 48 mol%, 46 mol%, 44 mol%, 42 mol%, 40 mol%, 38 mol%, 36 mol%, 34 mol%, 32 mol%, 30 mol%, 28 mol%, 26 mol%, 24 mol%, 22 mol%, 20 mol%, 18 mol%, 16 mol%, 14 mol%, 12 mol%, 10 mol%, 8 mol%, 6 mol%, 4 mol%, 2 mol%, or 0 mol%. Any of the polyvalent cationic lipids provided herein can be incorporated into a provided vehicle or particle at about 50 mol%, 48 mol%, 46 mol%, 44 mol%, 42 mol%, 40 mol%, 38 mol%, 36 mol%, 34 mol%, 32 mol%, 30 mol%, 28 mol%, 26 mol%, 24 mol%, 22 mol%, 20 mol%, 18 mol%, 16 mol%, 14 mol%, 12 mol%, 10 mol%, 8 mol%, 6 mol%, 4 mol%, 2 mol%, or 0 mol%. In some embodiments, the polyvalent cationic lipids provided herein can be incorporated into a provided vehicle or particle at a concentration of 5-50 mol%, 5-40 mol%, 5-30 mol%, 5-25 mol%, 5-20 mol%, 5-15 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 15-50 mol%, 15-40 mol%, 15-30 mol%, and 15-25 mol%.

[0076] In some embodiments, the lipid nanoparticles of the delivery vehicles provided herein may also contain anionic lipids. The anionic lipids may contain any of a wide range of fatty acid chains in the hydrophobic region. The particular fatty acid incorporated is responsible for the fluid properties of the lipid structure in terms of phase behavior and elasticity. In some cases, divalent cations are incorporated into the anionic lipid structure to allow condensation of the nucleic acid prior to encapsulation by the anionic lipid. Ca 2+ , Mg 2+ , Mn 2+ , and Ba 2+ Several divalent cations can be used in anionic lipoplexes, such as Ca 2+ Suitable anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), or any combination thereof.

[0077] In some embodiments, the anionic lipid in the lipid nanoparticles is phosphatidylglycerol, cardiolipin, dialkylphosphatidylserine, dialkylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), glycerophosphoinositol monophosphate, glycerophosphoinositol bisphosphate, glycerophosphoinositol triphosphate, or the like. The glycosylglycerol-based glycerols include at least one of phosphate, glycerophosphate, glyceropyrophosphate, glycerophosphoglycerophosphoglycerol, cytidine-5'-diphosphate-glycerol, glycosylglycerophospholipid, glycerophosphoinositol glycan, 1,2-dialkyl-sn-glycero-3-phosphate, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphopropanol, and / or 1,2-dialkyl-sn-glycero-3-phosphobutanol. In some embodiments where the anionic lipid is conjugated to an alkyl and the anionic lipid is in the liquid phase, the alkyl is at least one conjugated derivative of oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, paulic acid, vaccenic acid, palmitoleic acid, docosatetraenoic acid, arachidonic acid, dihomo-γ-linolenic acid, γ-linolenic acid, linoelaidic acid, linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, stearidonic acid, α-linolenic acid, or a salt thereof, or any combination thereof. In other cases, the alkyl is at least one conjugated derivative of myristic acid, pentadecylic acid, palmitic acid, heptadecanoic acid, stearic acid, lauric acid, tridecylic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, and / or salts thereof, or any combination thereof. In the above, if the alkyl has a phase transition temperature above 37° C., it is considered to be in the gel phase; otherwise, it is in the liquid phase.

[0078] In one embodiment, the anionic lipid may be a saturated lipid having a phase transition temperature above 37° C., such lipid may be used in the solid phase, and a cationic lipid may be used in the liquid phase. If the anionic lipid is an unsaturated or short-chain lipid having a transition temperature below 37° C., it may be utilized in the liquid phase, and a cationic lipid may be used in the gel or solid phase.

[0079] In one aspect, the concentration of at least one unsaturated cationic lipid and / or unsaturated non-cationic lipid in the lipid nanoparticle may be less than 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, 5 mol%, or 2 mol% of the total lipid concentration of the lipid nanoparticle. In some embodiments, the concentration of at least one unsaturated cationic lipid and / or unsaturated non-cationic lipid in the lipid nanoparticle may be about 50 mol%, 45 mol%, 40 mol%, 35 mol%, 30 mol%, 25 mol%, 20 mol%, 15 mol%, 10 mol%, 5 mol%, or 2 mol% of the total lipid concentration of the lipid nanoparticle. In some embodiments, the concentration of at least one unsaturated cationic lipid and / or unsaturated non-cationic lipid in the lipid nanoparticles can be 5-50 mol%, 5-40 mol%, 5-30 mol%, 5-25 mol%, 5-20 mol%, 5-15 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 15-50 mol%, 15-40 mol%, 15-30 mol%, and 15-25 mol%.

[0080] In some cases, the delivery vehicle may contain a high-temperature phase transition lipid, such as a high-temperature phase transition neutral lipid such as DSPC, and a bile salt such as deoxycholate, cholic acid, or a conjugate thereof. Deoxycholate can function as a solid phase (gel phase), while deoxycholate provides a negative charge. In the same delivery vehicle, cationic lipids can exist as unsaturated or short-tail lipids and can exist in the liquid phase. Multivalent cationic lipids such as MVL5 can be used to create a sufficient ratio of positive and negative charges to provide a balance of attraction and repulsion in the system, thereby producing a delivery vehicle that includes charge separation.

[0081] In some embodiments, the delivery vehicle may further comprise a conjugated lipid, and the conjugated lipid may comprise a lipid conjugated to a stabilizing component. In some embodiments, the stabilizing component may comprise a hydrophilic polymer. In some embodiments, the hydrophilic polymer may comprise polyethylene glycol, poly(2-alkyl-2-oxazoline), polyvinyl alcohol, or any combination thereof. In some embodiments, the hydrophilic polymer may comprise a molecular weight of at least about 500 Da to about 500 kDa. In some embodiments, the hydrophilic polymer may comprise polyethylene glycol (PEG), and the conjugated lipid comprises a PEGylated lipid. In some embodiments, the PEGylated lipid may comprise DSPE-PEG, DSG-PEG, DPG-PEG, DAG-PEG, DMG-PEG, DPPE-PEG, DMPE-PEG, or any combination thereof.

[0082] In some cases, the concentration of the conjugated lipid is about 0 mol%, 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%, 15.5 mol%, 16 mol%, 16.5 mol%, 17 mol%, 17.5 mol%, 18 mol%, 18.5 mol%, 19 mol%, 19.5 mol%, 20 mol%, 20.5 mol%, 21 mol%, 21.5 mol%, 22 mol%, 22.5 mol%, 23 mol%, 23.5 mol%, 24 mol%, 24.5 mol%, 25 mol%, 25.5 mol%, 26 mol%, 26.5 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, or 30 mol%. In some cases, the concentration of the conjugated lipid is about 0.5 mol% to about 20 mol%, 0.5 mol% to about 5 mol%, 0.5 mol% to about 10 mol%, 5 mol% to about 10 mol%, or 10 mol% to about 20 mol%.

[0083] In some cases, bile salts can be used as anionic components in delivery vehicles. In other cases, non-bile salts can be used as anionic components. In some embodiments, delivery vehicle stability can be increased by incorporating bile salts (also referred to herein as bile acids), such as cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, lithocholic acid, and lithocholate. In some embodiments, the bile salt can be cholic acid. In further embodiments, the bile salt can be deoxycholate. In some embodiments, the bile salt incorporated can be cholic acid and deoxycholate. In some embodiments, the stability of the delivery vehicle can be measured by a bile salt stability assay in a high bile salt mimicking environment. For example, bile salt stability can be measured by fluorescence spectroscopy, such as the relative fluorescence of delivery vehicles containing varying concentrations of bile salts in a Forster resonance energy transfer (FRET) assay. In some embodiments, the incorporated bile salts may increase the stability of the delivery vehicle by about 80% to about 10%, such as about 80% to about 70%, about 65% to about 55%, about 60% to about 50%, about 55% to about 45%, about 50% to about 40%, about 45% to about 35%, about 40% to about 30%, about 35% to about 25%, about 30% to about 20%, about 25% to about 15%, about 20% to about 10%, about 15% to about 10%, about 60% to about 20%, about 25.9%, about 30.4%, about 34.9%, about 39.4%, about 37.1%, about 43.9%, or about 45%. In some examples, the percent increase in stability can be measured by increased relative fluorescence units or relative luminescence units in an assay such as FRET.

[0084] In some cases, the delivery vehicles provided herein can include at least one of a multivalent lipid, a cationic lipid, a structured lipid, a bile salt, or a lipid-PEG. Any or all of the lipids provided herein can be present in an amount of, for example, 0 mol%, 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%, 15.5 mol%, or any combination thereof. mol%, 16 mol%, 16.5 mol%, 17 mol%, 17.5 mol%, 18 mol%, 18.5 mol%, 19 mol%, 19.5 mol%, 20 mol%, 20.5 mol%, 21 mol%, 21.5 mol%, 22 mol%, 22.5 mol%, 23 mol%, 23.5 mol%, 24 mol%, 24.5 mol%, 25 mol%, 25.5 mol%, 26 mol%, 26.5 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5 mol%, 43 mol%, 43.5 mol%, 44 mol%, 44.5 mol%, 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol %, 48.5 mol%, 49 mol%, 49.5 mol%, 50 mol%, 50.5 mol%, 51 mol%, 51.5 mol%, 52 mol%, 52.5 mol%, 53 mol%, 53.5 mol%, 54 mol%, 54.5 mol%, 55 mol%, 55.5 mol%, 56 mol%, 56.5 mol%, 57 mol%, 57.5 mol%, 58 mol%, 58.5 mol%, 59 mol%, 59.5 mol%, 60 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.It may be formulated in any mole percent, including, but not limited to, 5 mole%, 65 mole%, 65.5 mole%, 66 mole%, 66.5 mole%, 67 mole%, 67.5 mole%, 68 mole%, 68.5 mole%, 69 mole%, 69.5 mole%, 70 mole%, 70.5 mole%, 71 mole%, 71.5 mole%, 72 mole%, 72.5 mole%, 73 mole%, 73.5 mole%, 74 mole%, 74.5 mole%, 75 mole%, 75.5 mole%, 76 mole%, 76.5 mole%, 77 mole%, 77.5 mole%, 78 mole%, 78.5 mole%, 79 mole%, 79.5 mole%, or 80 mole%.

[0085] In some embodiments, the delivery vehicle herein may contain additional components. For example, the lipid structure of the delivery vehicle may include a lipid bilayer. In certain cases, the lipid bilayer may be formed of one or more compositions selected from the group consisting of phospholipids, phosphatidylcholine, phosphatidylserine, phosphatidyldiethanolamine, phosphatidylinositol, sphingolipids, and ethoxylated sterols, or mixtures thereof. In illustrative examples of such embodiments, the phospholipid may be lecithin, the phosphatidylinositol may be derived from soybean, rapeseed, cottonseed, egg, or mixtures thereof, the sphingolipid may be ceramide, cerebroside, sphingosine, sphingomyelin, or mixtures thereof, and the ethoxylated sterol may be phytosterol, PEG-(polyethylene glycol)-5 rapeseed sterol. In certain embodiments, the phytosterol comprises a mixture of at least two of the following compositions: schistosterol, camposterol, and stigmasterol. In yet other embodiments, the lipid bilayer may be composed of one or more phosphatidyl groups selected from the group including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylinositol. In other cases, the lipid bilayer may be composed of a phospholipid selected from monoacyl or diacylphosphoglycerides.In still other cases, the lipid bilayer is composed of phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-4-phosphate (PI-4-P), phosphatidylinositol-5-phosphate (PI-5-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), phosphatidylinositol-3,5-bisphosphate (PI-3,5-P2), phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2), phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3), lysophosphatidylinositol-3-phosphate (LPI-3-P), lysophosphatidylinositol The phosphoinositides may be composed of one or more phosphoinositides selected from the group including lysophosphatidylinositol-4-phosphate (LPI-4-P), lysophosphatidylinositol-5-phosphate (LPI-5-P), lysophosphatidylinositol-3,4-bisphosphate (LPI-3,4-P2), lysophosphatidylinositol-3,5-bisphosphate (LPI-3,5-P2), lysophosphatidylinositol-4,5-bisphosphate (LPI-4,5-P2), and lysophosphatidylinositol-3,4,5-triphosphate (LPI-3,4,5-P3), phosphatidylinositol (PI), or lysophosphatidylinositol (LPI).

[0086] The lipid structures used as delivery vehicles can be modified. The modification can be a surface modification. The surface modification can increase the average speed at which the lipid structures move through mucus compared to a comparable lipid structure. The comparable lipid structure can be unsurface-modified, or the comparable lipid structure can be modified with polyethylene glycol (PEG) polymers. The modification can promote protection from in vivo degradation. The modification can also aid in the transport of the lipid structure. For example, the modification can enable the lipid structure to be transported in the gastrointestinal (GI) tract, which has an acidic pH, through pH-sensitive modification. The surface modification can also improve the average speed at which the lipid structures move through mucus. For example, the modification may increase the rate by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 300-fold, 500-fold, 700-fold, 900-fold, or up to about 1000-fold compared to an equivalent lipid structure without the modification or a lipid structure with a modification containing PEG. In some cases, the modification to the lipid structure occurs through a bond. The bond may be covalent, non-covalent, polar, ionic, hydrogen, or any combination thereof. The bond may be considered to be an association between two groups or portions of a group. For example, the lipid structure may be linked to PEG by a linker containing a covalent bond. In some cases, the bond may occur between two adjacent groups. The bond may be dynamic. A dynamic bond may occur when one group temporarily associates with a second group. For example, a polynucleic acid suspended within a liposome may become associated with part of the lipid bilayer during suspension.

[0087] In some cases, the modification can be polyethylene glycol (PEG) addition. Methods for modifying the lipid structure surface with PEG can include its physical adsorption to the lipid structure surface, its covalent attachment to the lipid structure, its coating on the lipid structure, or any combination thereof. In some cases, PEG can be covalently attached to the lipid particle before the lipid structure is formed. PEGs of various molecular weights can be used. PEG can range from about 10 to about 100 units of ethylene PEG moieties that can be conjugated via amine groups to phospholipids comprising 1% to 20%, preferably 5% to 15%, or 10% of the weight of lipids contained in the lipid structure.

[0088] In some cases, the lipid structure may include phosphatidylcholine. Exemplary phosphatidylcholines include, but are not limited to, dilauroylphophatidylcholine, dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine, distearoylphophatidylcholine, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl-phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, egg phosphatidylcholine, myristoyl-palmitoylphosphatidylcholine, palmitoyl-myristoyl-phosphatidylcholine, myristoyl-stearoylphosphatidylcholine, palmitoyl-stearoyl-phosphatidylcholine, stearoyl-palmitoylphosphatidylcholine, stearoyl-oleoyl-phosphatidylcholine, stearoyl-linoleoylphosphatidylcholine, and palmitoyl-linoleoyl-phosphatidylcholine. Asymmetric phosphatidylcholine may be referred to as 1-acyl, 2-acyl-sn-glycero-3-phosphocholine, but the acyl groups are different from each other. Symmetric phosphatidylcholine may be referred to as 1,2-diacyl-sn-glycero-3-phosphocholine. As used herein, the abbreviation "PC" refers to phosphatidylcholine. Phosphatidylcholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine may be abbreviated herein as "DMPC." Phosphatidylcholine 1,2-dioleoyl-sn-glycero-3-phosphocholine may be abbreviated herein as "DOPC." Phosphatidylcholine 1,2-dipalmitoyl-sn-glycero-3-phosphocholine may be abbreviated herein as "DPPC." Saturated acyl groups commonly present in various lipids include groups having the names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heneicosanoyl, behenoyl, torcisanoyl, and lignoceroyl.The corresponding IUPAC names for saturated acyl groups are triane, tetrane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, 3,7,11,15-tetramethylhexadecane, heptadecane, octadecane, nonadecane, eicosane, heneicosane, docosane, trocosane, and tetracosane. Unsaturated acyl groups found in both symmetrical and asymmetrical phosphatidylcholines include myristoleyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl, and arachidonoyl. The corresponding IUPAC names of the unsaturated acyl groups are 9-cis-tetradecane, 9-cis-hexadecane, 9-cis-octadecane, 9-trans-octadecane, 9-cis-12-cis-octadecadiene, 9-cis-12-cis-15-cis-octadecatriene, 11-cis-eicosene, and 5-cis-8-cis-11-cis-14-cis-eicosatetraene. Exemplary phosphatidylethanolamines include dimyristoyl-phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoyl-phosphatidylethanolamine, and egg phosphatidylethanolamine. Phosphatidylethanolamine can be referred to as 1,2-diacyl-sn-glycero-3-phosphoethanolamine or 1-acyl-2-acyl-sn-glycero-3-phosphoethanolamine in the IUPAC nomenclature system, depending on whether the lipid is symmetric or asymmetric. Exemplary phosphatidic acids include dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, and dioleoylphosphatidic acid. Phosphatidic acid can be referred to as 1,2-diacyl-sn-glycero-3-phosphate or 1-acyl-2-acyl-sn-glycero-3-phosphate in the IUPAC nomenclature system, depending on whether the lipid is symmetric or asymmetric. Exemplary phosphatidylserines include dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, distearoylphosphatidylserine, palmitoyl-oleylphosphatidylserine, and brain phosphatidylserine.Phosphatidylserine may also be referred to in the IUPAC nomenclature system as 1,2-diacyl-sn-glycero-3-[phospho-L-serine] or l-acyl-2-acyl-sn-glycero-3-[phospho-L-serine], depending on whether it is a symmetric or asymmetric lipid. As used herein, the abbreviation "PS" refers to phosphatidylserine. Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl-phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl-phosphatidylglycerol, and egg phosphatidylglycerol. Phosphatidylglycerol may also be referred to in the IUPAC nomenclature system as 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] or 1-acyl-2-acyl-sn-glycero-3-[phospho-rac-(1-glycerol)], depending on whether it is a symmetric or asymmetric lipid. Phosphatidylglycerol 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] is abbreviated herein as "DMPG." Phosphatidylglycerol 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (sodium salt) is abbreviated herein as "DPPG." Suitable sphingomyelins may include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin. Other suitable lipids include glycolipids, such as glycolipids, sphingolipids, ether lipids, cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol.

[0089] In some cases, the lipid structure may comprise cholesterol or a derivative thereof, a phospholipid, a mixture of phospholipids and cholesterol or a derivative thereof, or a combination. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. When the lipid structure comprises a mixture of phospholipids and cholesterol or a cholesterol derivative, the lipid structure may comprise up to about 40, 50, or 60 mol% of the total lipid present in the lipid structure. One or more phospholipids and / or cholesterol may constitute about 10 mol% to about 60 mol%, about 15 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 10 mol% to about 55 mol%, about 15 mol% to about 55 mol%, about 20 mol% to about 55 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 55 mol%, about 13 mol% to about 50 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol% of the total lipids in the lipid structure.

[0090] In some embodiments, the delivery vehicles herein are designed to be absorbed into epithelial cells, such as epithelial cells in the gastrointestinal tract. Peptides, particularly cell-penetrating peptides (CPPs) and cell-penetrating peptides (MPPs) with mucus-penetrating functionality, confer cellular absorption. Delivery vehicles herein, such as the lipid structures described herein for such purposes, further comprise a mucus-penetrating peptide (MPP), a cell-penetrating peptide (CPP), or both. In some embodiments, the cell-penetrating peptide (CPP) can be a short polypeptide that can enable increased cellular uptake of the delivery vehicle and / or cargo. The cell-penetrating peptide (CPP) can be a peptide sequence that facilitates efficient crossing of the cell membrane. Exemplary CPPs and MPPs include those disclosed in PCT / US17 / 61111 and PCT / US2019 / 032484, which are incorporated herein by reference.

[0091] In some embodiments, mucus-penetrating cell-penetrating peptides (MPPs) are used in conjunction with the delivery vehicles described herein. MPPs have cell-penetrating properties and further enable penetration through mucus layers, such as the natural mucus layers in the colon, lungs, eyes, and cervix. MPPs can also be used to target structures to intracellular components of cells. They can also be designed to specifically target specific cell types. To achieve increased penetration or cellular targeting, MPPs can be conjugated to delivery vehicles to enable the particles to penetrate through mucus layers and interact with cells. In some embodiments, lipid structures with MPPs can be absorbed into cells with at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% efficiency compared to comparable particles without MPPs. In some embodiments, the delivery vehicle may comprise a mucus-penetrating peptide (MPP). The MPP may be conjugated to a lipid structure, such as a lipid nanoparticle, a surface modification of a lipid nanoparticle, or conjugated to a cargo, such that the MPP is exposed so that it can contact, in whole or in part, the mucus layer, mucus-containing tissue, organ, or extracellular surface. The presence of the MPP may confer improved penetration of the delivery vehicle through (diffusion and / or movement therethrough) the mucus. In some embodiments, penetration may be improved by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 100-fold, or more, compared to delivery of a delivery vehicle and / or cargo without the MPP. In some embodiments, an MPP can have an amino acid sequence having about 3 to 100 amino acids, including, but not limited to, about 3 to 5, 5 to 10, 10 to 20, 20 to 40, 30 to 60, or 80 to 100 amino acids. An MPP can have about 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, or up to about 100 amino acids.In some embodiments, MPPs may have the ability to penetrate the mucus layer covering or surrounding target cells or tissues. MPPs can be used to penetrate the mucus layer of target tissues, such as the intestinal epithelium, colon, lung, eye, or cervix of a mammal. MPPs can be conjugated to delivery vehicles, including nanoparticles, to enable the delivery vehicle to penetrate the mucus layer and also interact with cells, resulting in increased penetration or cellular targeting. In some embodiments, particles with MPPs penetrate the mucus layer with at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% efficiency compared to comparable particles without MPPs. Many methods for measuring penetration of mucus layers can be used to assess penetration by MPPs or MPPs directly or indirectly conjugated to delivery vehicles.

[0092] In one aspect, the lipid structure may be a mucus-penetrating particle, or MPP, as used herein, which may refer to a particle coated with a mucosal permeation-enhancing coating. In some cases, the particle may be or deliver an active agent, such as a therapeutic, diagnostic, preventive, and / or nutraceutical agent (i.e., drug particle), which may be coated with a mucosal permeation-enhancing coating. In other cases, the particle may be formed of a matrix material, such as a polymeric material, in which the therapeutic, diagnostic, preventive, and / or nutraceutical agent may be encapsulated, dispersed, and / or associated therewith.

[0093] In certain cases, the delivery vehicle may further comprise at least one targeting agent. The term targeting agent may refer to a moiety, compound, antibody, etc. that specifically binds to a specific type or class of cells and / or other specific type of compound (e.g., a moiety that targets a specific cell or type of cell). The targeting agent may be specific for (e.g., have affinity for) the surface of a specific target cell, a target cell surface antigen, a target cell receptor, or a combination thereof. In some cases, the targeting agent may refer to an agent that has a specific action (e.g., cleavage) when exposed to a specific type or class of substance and / or cell, and this action may drive the delivery vehicle to target a specific type or class of cell. Thus, the term targeting agent may refer to an agent that may be part of the delivery vehicle and plays a role in the targeting mechanism of the delivery vehicle, although the agent itself may or may not be specific for a specific type or class of cell itself. In certain cases, the efficiency of cellular uptake of polynucleic acids delivered by a delivery vehicle can be increased and / or made more specific by incorporating a targeting agent into the delivery vehicle. In certain embodiments, the delivery vehicles described herein can include one or more small molecule targeting agents (e.g., carbohydrate moieties). Suitable targeting agents include, by way of non-limiting example, peptides such as antibodies, antibody-like molecules, or integrin-binding peptides such as RGD-containing peptides, or small molecules such as vitamins, e.g., folic acid, sugars such as lactose and galactose, or other small molecules. Cell surface antigens include cell surface molecules such as proteins, sugars, lipids, or other antigens on the cell surface. In particular embodiments, cell surface antigens undergo absorption. Examples of cell surface antigens targeted by the targeting agents of embodiments of the delivery vehicle include, but are not limited to, transferrin receptors types 1 and 2, EGF receptor, HER2 / Neu, VEGF receptor, integrins, NGF, CD2, CD3, CD4, CD5, CD19, CD20, CD22, CD33, CD43), CD56, CD69, and leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Targeting agents can also include artificial affinity molecules, such as peptidomimetics or aptamers.Peptidomimetics can refer to compounds in which at least a portion of a peptide, such as a therapeutic peptide, has been modified, such that the three-dimensional structure of the peptidomimetic remains substantially the same as that of the peptide. Peptidomimetics (both peptides and non-peptidyl analogs) can have improved properties (e.g., reduced proteolysis, increased retention, or increased bioavailability). Peptidomimetics generally have improved oral availability, making them particularly suitable for the treatment of human or animal disorders. It should be noted that peptidomimetics may or may not have a similar two-dimensional chemical structure, but share common three-dimensional structural features and shapes.

[0094] In some embodiments, the targeting agent can be a proteinaceous targeting agent (e.g., a peptide, an antibody, or an antibody fragment). In some specific embodiments, the delivery vehicle can include multiple different targeting agents. In embodiments, lipid structural modifications can impart biocompatibility and can be modified to include targeting species including, for example, antibodies, targeting peptides including aptamers, polyethylene, or combinations thereof. The targeting agent can be a receptor. In some cases, a T cell receptor (TCR), a B cell receptor (BCR), a single-chain variant fragment (scFv), a chimeric antigen receptor (CAR), or a combination thereof is used as the targeting agent.

[0095] In some embodiments, one or more targeting agents may be coupled to the polymer forming the delivery vehicle. In some cases, the targeting agent may be attached to a polymer coating the delivery vehicle. In some cases, the targeting agent may be covalently attached to the polymer. In some cases, the targeting agent may be attached to the polymer such that the targeting agent may be substantially at or near the surface of the resulting delivery vehicle. In certain embodiments, monomers comprising a targeting agent residue (e.g., a polymerizable derivative of a targeting agent, such as an (alkyl)acrylic acid derivative of a peptide) may be copolymerized to form a copolymer that forms the delivery vehicle provided herein. In certain embodiments, one or more targeting agents may be attached to the polymer of the delivery vehicle by a linking moiety. In some embodiments, the linking moiety that connects the targeting agent to the membrane-destabilizing polymer may be a cleavable linking moiety (e.g., comprises a cleavable bond). In some embodiments, the linking moiety may be cleavable and / or comprise a bond that may be cleavable under endosomal conditions. In some embodiments, the linking moiety may be cleavable and / or comprise a bond that may be cleavable by a specific enzyme (e.g., a phosphatase or protease). In some embodiments, the linking moiety may be cleavable and / or comprise a bond that may be cleavable upon a change in an intracellular parameter (e.g., pH, redox potential), and in some embodiments, the linking moiety may be cleavable and / or comprise a bond that may be cleavable upon exposure to a matrix metalloproteinase (MMP) (e.g., an MMP-cleavable peptide linking moiety).

[0096] In certain cases, the targeting mechanism of the delivery vehicle may depend on the cleavage of a cleavable segment in the polymer. For example, the polymer may include a cleavable segment that, upon cleavage, exposes the delivery vehicle and / or the core of the delivery vehicle. In some embodiments, the cleavable segment may be located at either or both ends of the polymer. In some embodiments, the cleavable segment may be located along the length of the polymer, optionally between blocks of the polymer. For example, in certain embodiments, the cleavable segment may be located between a first block and a second block of the polymer, such that the first block can be cleaved from the second block when the delivery vehicle is exposed to a specific cleavable substance. In a specific embodiment, the cleavable segment may be an MMP-cleavable peptide that can be cleaved upon exposure to an MMP.

[0097] The attachment of a targeting agent, such as an antibody or peptide, to a polymer or lipid can be accomplished in any suitable manner, for example, by any one of several conjugate chemistry approaches, including, but not limited to, an amine-carboxyl linker, an amine-sulfhydryl linker, an amine-carbohydrate linker, an amine-hydroxyl linker, an amine-amine linker, a carboxyl-sulfhydryl linker, a carboxyl-carbohydrate linker, a carboxyl-hydroxyl linker, a carboxyl-carboxyl linker, a sulfhydryl-carbohydrate linker, a sulfhydryl-hydroxyl linker, a sulfhydryl-sulfhydryl linker, a carbohydrate-hydroxyl linker, a carbohydrate-carbohydrate linker, and a hydroxyl-hydroxyl linker. In a specific embodiment, "click" chemistry can be used to attach a targeting agent to the polymer of a delivery vehicle provided herein. A wide variety of conjugate chemistries can be optionally used, and in some embodiments, a targeting agent can be attached to a monomer, and then the resulting compound can be used in the polymerization synthesis of a polymer (e.g., copolymer) used in the delivery vehicle described herein. In some embodiments, a targeting agent can be attached to the sense or antisense strand of the siRNA that is bound to the polymer of the delivery vehicle. In certain embodiments, a targeting agent can be attached to the 5' or 3' end of the sense or antisense strand.

[0098] Methods for linking compounds can include, but are not limited to, proteins, labels, and other chemicals, such as nucleotides. Cross-linking reagents such as n-maleimidobutyryloxy-succinimide ester (GMBS) and sulfo-GMBS have reduced immunogenicity. Substituents have been attached to the 5' end of pre-assembled oligonucleotides using amidite or H-phosphonate chemistry. Substituents can also be attached to the 3' end of oligomers. This last method utilizes 2,2'-dithioethanol attached to a solid support, where diisopropylamine is transferred from the 3' phosphonate bearing an acridine moiety, followed by phosphorus oxidation and subsequent deletion. Alternatively, oligonucleotides can contain one or more modified nucleotides with groups attached to the base by a linker arm. For example, biotin can be attached to the C-5 position of dUTP by an allylamine linker arm. Attachment of biotin and other groups to the 5-position of pyrimidines by a linker arm can also be implemented.

[0099] Chemical cross-linking can involve the use of spacer arms, i.e., linkers or tethers. Spacer arms can provide intramolecular flexibility or adjust the intramolecular distance between conjugated moieties, thereby helping to maintain biological activity. Spacer arms can be in the form of peptide moieties containing spacer amino acids. Alternatively, the spacer arm can be part of the cross-linking reagent, such as in "long-chain SPDP."

[0100] Various coupling or cross-linking agents, such as protein A, carbodiimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-5-acetyl-thioacetate (SATA), and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), 6-hydrazinonicotinimide (HYNIC), N3S, and N2S2, can be used in well-known procedures to synthesize targeted constructs. For example, biotin can be conjugated to oligonucleotides with DTPA using the bicyclic anhydride method. Furthermore, sulfosuccinimidyl 6-(biotinamido)hexanoate (NHS-LC-biotin, available from Pierce Chemical Co., Rockford, Illinois), a lysine conjugate of biotin, "biocytin," can be useful for preparing biotin compounds due to the availability of a primary amine. Additionally, the corresponding biotin acid chloride or acid precursor can be coupled to an amino derivative of a therapeutic agent by known methods. By coupling a biotin moiety to the surface of a particle, another moiety can be coupled to avidin and then coupled to the particle via strong avidin-biotin affinity, or vice versa. In certain embodiments in which the polymeric particles contain PEG moieties on their surface, the free hydroxyl groups of the PEG can be used to link or connect (e.g., covalently bond) additional molecules or moieties to the particle.

[0101] In one embodiment, the lipid structures (delivery vehicles) herein have sizes that can range from nanometers to micrometers, such as 20-200 nm, 200 nm to 1 μm, etc. In some cases, polynucleic acids can be condensed and properly encapsulated by the lipid structures. DNA condensation occurs via Mn condensation, which can condense DNA by neutralizing the phosphate groups of the DNA backbone and distorting the B-DNA structure through hydrogen bonding with the bases. 2+ , Ni 2+ , Co 2+ , and Cu 2+This can be achieved by divalent metal ions such as ethanol, which allow both local bending of DNA and interhelical association. In some cases, the concentration of metal ions used for condensation can depend on the dielectric constant of the medium used for condensation. The addition of ethanol or methanol can also reduce the concentration of metal ions required for condensation. In some cases, ethanol can be used to condense DNA at a concentration of about 0.5% to about 60% by volume. In some cases, ethanol can be used to condense DNA at a concentration of about 0.5% to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume. In some cases, calcium can also be used for condensation. Calcium not only binds to DNA phosphates, but can also complex with the nitrogen and oxygen of guanine, disrupting base pairing.

[0102] In some cases, polynucleic acids can be completely encapsulated in lipid structures. Complete encapsulation can indicate that the polynucleic acids in the lipid structures cannot be significantly degraded after exposure to serum or nuclease or protease assays that would significantly degrade free DNA, RNA, or protein. In a completely encapsulated system, preferably, less than about 25% of the polynucleic acids in the lipid structures can be degraded in a treatment that would normally degrade 100% of the free polynucleic acids, more preferably, less than about 10%, and most preferably, less than about 5% of the polynucleic acids in the lipid structures can be degraded. In the context of polynucleic acids, complete encapsulation can be determined by the Oligreen® assay. Oligreen® is an ultrasensitive fluorescent nucleic acid dye that quantifies dissolved oligonucleotides and single-stranded DNA or RNA (available from Invitrogen Corporation, Carlsbad, CA). "Fully encapsulated" may also indicate that the lipid structures may be serum stable, ie, they do not rapidly degrade into their component parts upon in vivo administration.

[0103] In certain applications, it may be desirable to release a moiety once a drug, such as a polynucleic acid, enters a cell. The moiety can be used to identify certain cells that have received the polynucleic acid. The moiety can be an antibody, dye, scFv, peptide, glycoprotein, carbohydrate, ligand, or polymer, to name a few. The moiety can be attached to a linker. The linker can be non-cleavable. Thus, in some cases, the linker can be a cleavable linker. This can allow the moiety to be released from the lipid structure once contact with the target cell. This may be desirable if the moiety has a greater therapeutic effect when separated from the lipid structure. In some cases, the moiety, when separated from the lipid structure, can have a better ability to be absorbed by intracellular components of cells, such as small intestinal crypt cells or small intestinal crypt stem cells. In some cases, the linker can include a disulfide bond, an acylhydrazone, a vinyl ether, an orthoester, or N-PO3.

[0104] Therefore, it may be necessary or desirable to separate the moiety from the lipid structure so that the moiety can enter the intracellular compartment. Cleavage of the linker, releasing the moiety, may be the result of a change in conditions inside the cell compared to outside the cell, for example, due to a change in intracellular pH. Linker cleavage may occur due to the presence of an enzyme inside the cell, which cleaves the linker when a drug such as a polynucleic acid enters the cell. Alternatively, linker cleavage may occur in response to energy or chemicals applied to the cell. Examples of energy types that can be used to induce linker cleavage include, but are not limited to, light, ultrasound, microwave, and radiofrequency energy. In some cases, the linker may be a photolabile linker. The linker used to link the complexes may be an acid-labile linker. Examples of acid-labile linkers include linkers formed using cis-aconitic acid, cis-carboxylic acid alkatrienes, polymaleic anhydride, and other acid-labile linkers.

[0105] In some cases, lipid structures such as liposomes can be biocompatible and biodegradable. For example, in some cases, liposomes can biodegrade after introduction into a subject. Biodegradation can, in some cases, begin immediately after introduction. Biodegradation can occur within the mucosal tract of a subject receiving the liposome or liposomal structure. Biodegradation can result in the release of liposomal cargo, such as polynucleic acids. In other cases, biodegradation can include the degradation of components of the liposomal structure, such as polymers. Biodegradation can occur under standard body conditions, such as from about 36.4°C (97.6°F) to about 37.2°C (99°F). In other cases, biodegradation can occur at temperatures from about 35°C (95°F) to about 41.1°C (106°F). Biodegradation may occur up to about 35°C (95°F), 35.6°C (96°F), 36.1°C (97°F), 36.7°C (98°F), 37.2°C (99°F), 37.8°C (100°F), 38.3°C (101°F), 38.9°C (102°F), 39.4°C (103°F), 40°C (104°F), 40.6°C (105°F), or 41.1°C (106°F). In other embodiments, biodegradation may occur from about 10°C (50°F) to about 65.6°C (150°F).

[0106] In other cases, biodegradation may not occur. If biodegradation does occur, it may take from about 1 minute to about 100 years after administration of the liposome or structure to a subject. Biodegradation may take about 1 minute, 5 minutes, 30 minutes, 1 hour, 3 hours, 7 hours, 10 hours, 15 hours, 20 hours, 25 hours, 2 days, 4 days, 8 days, 12 days, 20 days, 30 days, 1.5 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 3 years, 5 years, 8 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or at least about 100 years. The lipids of a structure such as a liposome may be or may include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, prenol lipids (derived from the condensation of isoprene subunits), or any combination thereof.

[0107] cargo The delivery vehicles herein having charge separation and epithelial-access functionality provided herein can be utilized to deliver any type of cargo to a target, e.g., a target cell. In some cases, the cargo can include a therapeutic agent. Exemplary therapeutic agents can include nucleic acids, proteins, antibodies, peptides, small molecules, biologics, antisense oligonucleotides, peptidomimetics, ribozymes, chemical agents such as chemotherapeutic molecules, or any large molecule, including, but not limited to, viral particles, growth factors, cytokines, immunomodulatory agents, small molecule drugs, fluorescent dyes, including fluorescent dye peptides that can be expressed by DNA incorporated into liposomes, or any combination thereof.

[0108] In one embodiment, the cargo can be a nucleic acid. The nucleic acid can be DNA-based or RNA-based. The nucleic acid can be a vector. DNA-based vectors can be non-viral and include molecules such as plasmids, minicircles, nanoplasmids, closed-end linear DNA (doggybone), linear DNA, and single-stranded DNA. Nucleic acids that can be present in lipid-nucleic acid particles include any known form of nucleic acid. As used herein, nucleic acids can be single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes containing regulatory and termination regions, and self-replicating systems such as viruses or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides. The nucleic acid present in the lipid-nucleic acid particle can include one or more of the oligonucleotide modifications described below. Nucleic acids can generally be of various lengths, depending on the specific form of the nucleic acid. For example, in certain embodiments, a plasmid or gene can be about 1,000 to 100,000 nucleotide residues in length. In certain embodiments, an oligonucleotide can be in the range of about 10 to 100 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triplexed oligonucleotides can be in the range of about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides in length. In certain embodiments, an oligonucleotide can be in the range of about 2 to 10 nucleotides in length.

[0109] DNA-based vectors can also be viral, such as adeno-associated viruses, lentiviruses, and adenoviruses. Vectors can also be RNA. RNA vectors can be unmodified RNA in linear or circular form. They can also contain various nucleotide modifications designed to increase half-life, reduce immunogenicity, and / or increase the level of translation. As used herein, vectors can be composed of either DNA or RNA. In some embodiments, vectors can be composed of DNA. Vectors can be capable of autonomous replication in prokaryotes, such as E. coli, used for growth. In some embodiments, vectors can be stably integrated into the genome of an organism. In other cases, vectors can remain separate in either the cytoplasm or nucleus. In some embodiments, vectors can contain targeting sequences. In some embodiments, vectors can contain antibiotic resistance genes. Vectors can contain regulatory elements to control gene expression. In some cases, minicircles can be packaged in delivery vehicles.

[0110] In one embodiment, minicircle (MC) DNA can be delivered as cargo by the vehicles described herein. MC can be similar to plasmid DNA, as both MC and plasmid DNA can contain expression cassettes that allow high levels of transgene product to be produced immediately after delivery. In some cases, MC can differ in that MC DNA can lack prokaryotic sequence elements (e.g., bacterial origins of replication and antibiotic resistance genes). Removal of prokaryotic sequence elements from backbone plasmid DNA can be achieved by site-specific recombination in Escherichia coli prior to episomal DNA isolation. The absence of prokaryotic sequence elements can reduce the size of MC relative to its parent full-length (FL) plasmid DNA, but it can also result in increased transfection efficiency. The result may be that MC can transfect more cells than its FL plasmid DNA counterpart, allowing for sustained high-level transgene expression upon delivery. In some cases, minicircle DNA may not contain a bacterial origin of replication. For example, minicircle DNA or closed-end linear DNA may lack a bacterial origin of replication, ranging from approximately 50% of the bacterial origin of replication sequence to 100% of the bacterial origin of replication. In some cases, the bacterial origin of replication is truncated or inactive. The polynucleic acid may be derived from a vector that initially encodes a bacterial origin of replication. Methods can be used to completely or partially remove the bacterial origin of replication, leaving the polynucleic acid without the bacterial origin of replication. In some cases, the bacterial origin of replication can be identified by its high adenine and thymine content. Minicircle DNA vectors can be supercoiled, minimal expression cassettes derived from conventional plasmid DNA by in vivo site-specific recombination in Escherichia coli for use in non-viral gene therapy and vaccination. Minicircle DNA may lack or have reduced bacterial backbone sequences, such as antibiotic resistance genes, origins of replication, and / or inflammatory sequences, that are inherent in bacterial DNA. In addition to their improved safety profile, minicircles can greatly increase the efficiency of transgene expression.

[0111] In some cases, a portion of a gene can be delivered by a polynucleic acid cargo. The portion of a gene can be from 3 nucleotides to the entire genomic sequence. For example, the portion of a gene can be from about 1% to about 100% of the endogenous genomic sequence. The portion of a gene can be about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% of the entire genomic sequence of the gene.

[0112] Various proteins and polypeptides can be delivered as cargo by the vehicles described herein, including, but not limited to, proteins for treating metabolic and endocrine disorders. Examples of proteins include phenylalanine hydroxylase, insulin, antidiuretic hormone, and growth hormone. Disorders include phenylketonuria, diabetes, organic aciduria, tyrosinemia, urea cycle disorders, and familial hypercholesterolemia. Genes for any protein or peptide that can correct defects in phenylketonuria, diabetes, organic aciduria, tyrosinemia, urea cycle disorders, and familial hypercholesterolemia can be introduced into stem cells so that the protein or peptide product is expressed by the intestinal epithelium. Clotting factors such as antihemophilic factor (factor 8), Christmas factor (factor 9), and factor 7 can also be produced in the intestinal epithelium. Proteins that can be used to treat circulating protein deficiencies can also be expressed in the intestinal epithelium. Proteins that can be used to treat circulating protein deficiencies can be, for example, albumin for the treatment of albuminemia, alpha-1-antitrypsin, or hormone-binding proteins. Additionally, the intestinal symptoms of cystic fibrosis can be treated by inserting the normal cystic fibrosis transmembrane conductance regulator gene into intestinal epithelial stem cells. Abetalipoproteinemia can be treated by inserting apolipoprotein B. Disaccharide intolerance can be treated by inserting sucrase-isomaltose, lactase-phlorizin hydrolase, and maltase-glucoamylase. Vitamin B 12 Intrinsic factor or vitamin B for absorption 12The receptor for the intrinsic factor / cobalamin complex for absorption of steroids and the bile acid transporter can be inserted into the intestinal epithelium. Furthermore, any drug that can be encoded by nucleic acid can be inserted into the stem cells of the intestinal epithelium and secreted at high concentrations locally for the treatment of cancer. In this regard, those skilled in the art will readily recognize that antisense RNA can be encoded in stem cells after antisense production, which can then be integrated into cancer cells for the treatment of cancer.

[0113] The therapeutic agent or drug may be a small molecule, protein, polysaccharide or sugar, nucleic acid molecule, lipid, peptidomimetic, or a combination thereof. The delivery vehicle may include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such an effect may be, for example, biological, physiological, or cosmetic. The molecule or compound may include, for example, nucleic acids, peptides, and polypeptides, such as antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized™ antibodies), cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and small organic molecules or compounds. In one embodiment, the molecule or compound may be a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may themselves be therapeutically active, or they may be prodrugs that become active upon further modification. Thus, in one embodiment, a molecule or compound derivative may retain some or all of the therapeutic activity compared to the unmodified drug, while in another embodiment, a therapeutic agent derivative lacks therapeutic activity.

[0114] In various embodiments, the therapeutic agent includes any therapeutically effective agent or drug, such as anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, birth control medications, antipyretics, vasodilators, anti-angiogenic agents, cytovascular agents, signal transduction inhibitors, cardiovascular agents, e.g., antiarrhythmic agents, vasoconstrictors, hormones, and steroids. In certain embodiments, the molecule or compound can be an oncology drug, which may also be referred to as an anti-tumor drug, anti-cancer drug, tumor drug, anti-neoplastic agent, etc.Examples of oncology drugs that may be used include adriamycin, alkeran, allopurinal, altretamine, amifostine, anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, intravenous busulfan, oral busulfan, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporine A, Cytarabine, cytosine arabinoside, daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, goserelin acetate , hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen These include, but are not limited to, mustard, paclitaxel, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituximab, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (hycarntin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that may be used are ellipticine and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecin.

[0115] In some embodiments, polynucleic acids for use as cargo in conjunction with the delivery vehicles herein include nucleic acids encoding tumor suppressor genes. Tumor suppressor genes generally encode proteins capable of inhibiting cell growth in some way. Loss of one or more of these "brakes" can contribute to the development of cancer. Five broad classes of proteins can generally be recognized as being encoded by tumor suppressor genes: intracellular proteins such as p16 cyclin kinase inhibitors, which can regulate or inhibit progression through specific stages of the cell cycle; receptors for secreted hormones (e.g., tumor-derived growth factor β) that can function to inhibit cell growth; checkpoint control proteins that halt the cell cycle when DNA is damaged or chromosomes are abnormal; proteins that can promote apoptosis; enzymes involved in DNA repair; or combinations thereof. While DNA repair enzymes may not directly function to inhibit cell growth, cells that have lost the ability to repair errors, gaps, or broken ends in DNA accumulate mutations in many genes, including those critical for controlling cell growth and proliferation. Thus, loss-of-function mutations in genes encoding DNA repair enzymes can promote the inactivation of other tumor suppressor genes and the activation of oncogenes. Generally, one copy of a tumor suppressor gene is sufficient to control cell proliferation, so both alleles of the tumor suppressor gene must be lost or inactivated to promote tumorigenesis. In one embodiment, oncogenic loss-of-function mutations in tumor suppressor genes act recessively. In many cancers, tumor suppressor genes have deletions or point mutations that prevent protein production or lead to the production of non-functional proteins. In some cases, the introduction of a tumor suppressor gene encoding a protein can ameliorate, prevent, or treat the disease in a subject.

[0116] Tumor suppressor genes that can be delivered by the delivery vehicles herein include, for example, APC, ARHGEF12, ATM, BCL11B, BLM, BMPR1A, BRCA1, BRCA2, CARS, CBFA2T3, CDH1, CDH11, CDK6, CDKN2C, CEBPA, CHEK2, CREB1, CREBBP, CYLD, DDX5, EXT1, EXT2, FBXW7, FH, FLT3, FOXP1, GPC3, IDH1, IL2 , JAK2, MAP2K4, MDM4, MEN1, MLH1, MSH2, NF1, NF2, NOTCH1, NPM1, NR4A3, NUP98, PALB2, PML, PTEN, RB1, RUNX1, SDHB, SDHD, SMARCA4, SMARCB1, SOCS1, STK11, SUFU, SUZ12, SYK, TCF3, TNFAIP3, TP53, TSC1, TSC2, VHL, WRN, WT1, and any combination thereof.

[0117] In certain embodiments, the vehicle may comprise an imaging agent that may be further linked to a detectable label (e.g., the label may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The active moiety may be a radioactive heavy metal such as an iron chelate, a radioactive chelate of gadolinium or manganese, a positron emitter of oxygen, nitrogen, iron, carbon, or gallium, 43 K. 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 132 I, or 99The radioisotope may be a radioactive agent such as Tc. Delivery vehicles containing such moieties can be used as imaging agents and administered in amounts effective for diagnostic use in mammals, such as humans. In this manner, localization and accumulation of the imaging agent can be detected. Localization and accumulation of the imaging agent can be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography. As will be apparent to those skilled in the art, the amount of radioisotope to be administered will depend on the radioisotope. Those skilled in the art will be able to readily formulate the amount of imaging agent to be administered based on the specific activity and energy of a given radionuclide used as the active moiety. Typically, 0.1 to 100 millicuries, 1 to 10 millicuries, and 2 to 5 millicuries can be administered per dose of imaging agent. Thus, compositions useful as imaging agents may include a targeting moiety conjugated to a radioactive moiety, which may contain 0.1 to 100 millicuries, in some embodiments, preferably 1 to 10 millicuries, in some embodiments, preferably 2 to 5 millicuries, and in some embodiments, more preferably 1 to 5 millicuries. The detection means used to detect the label depend on the nature of the label used and the nature of the biological sample used and may include fluorescence polarization, high-performance liquid chromatography, antibody capture, gel electrophoresis, differential precipitation, organic extraction, size-exclusion chromatography, fluorescence microscopy, or fluorescence-activated cell sorting (FACS) assays. The targeting moiety may also refer to a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The entity may be, for example, a therapeutic compound such as a small molecule or a diagnostic entity such as a detectable label. The location may be a tissue, a specific cell type, or a subcellular compartment. In one embodiment, the targeting moiety can direct the localization of the active entity. The active entity may be a small molecule, protein, polymer, or metal. Active entities such as liposomes containing nucleic acids may be useful for therapeutic, prophylactic, or diagnostic purposes. In some cases, the moiety may enable the delivery vehicle to penetrate the blood-brain barrier.

[0118] The cargo can be a drug. A drug can be a substance that can cause a physiological change in a subject when administered. The drug can be a pharmaceutical used to treat a disease such as cancer. In some cases, the drug can be completely encapsulated in the liposomal lipid bilayer, in the aqueous compartment, or in both the liposomal lipid bilayer and the aqueous compartment. A highly lipophilic drug can be almost completely encapsulated in the lipid bilayer. A highly hydrophilic drug can be localized exclusively in the aqueous compartment. A drug with an intermediate logP can easily partition between the lipid and aqueous phases in both the bilayer and the aqueous core. Exemplary drugs can include drugs such as adalimumab, anti-TNF, insulin-like growth factor, interleukin, mesalamine, GLP-1 analogs, GLP-2 analogs, and combinations thereof.

[0119] In some cases, the polynucleic acid may encode a heterologous sequence. The heterologous sequence may provide subcellular localization (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a mitochondrial localization signal for targeting the mitochondria, a chloroplast localization signal for targeting the chloroplast, an endoplasmic reticulum retention signal, etc.). In some cases, the polynucleic acid, such as a minicircle DNA or a closed-end linear DNA, may comprise a nuclear localization sequence (NLS).

[0120] The cargo may comprise one or more nuclear localization sequences (NLSs). Some NLS sequences may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the vector comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino terminus, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy terminus, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs, or a combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). When more than one NLS is present, each may be selected independently of the other, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs may include NLS sequences derived from: NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 1), NLS of nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)), c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4), hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5), sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6) of the IBB domain of importin-α, sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the fibroid T protein, sequence POPKKKPL (SEQ ID NO: 9) of human p53, mouse c-abl The sequence SALIKKKKKMAP (SEQ ID NO: 10) of IV, the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12) of influenza virus NS1 16), the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO: 12), the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 13), the sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 14), and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: 15). Generally, one or more NLSs can be strong enough to promote the accumulation of detectable amounts of minicircle DNA vectors or short linear DNA vectors in the nucleus of a eukaryotic cell. The eukaryotic cell can be a human small intestinal crypt cell.

[0121] In some cases, the particles may include a DNAse inhibitor. The DNAse inhibitor may be localized within or on the particle. In other cases, a polynucleic acid encoding the inhibitor may be encapsulated within the particle. In other cases, the inhibitor may be a DNA methyltransferase inhibitor, such as DNA methyltransferase inhibitor-2 (DMI-2). DMI-2 may be produced by Streptomyces sp. strain no. 560. The structure of DMI-2 can be 4'''R,6aR,10S,10aS-8-acetyl-6a,10a-dihydroxy-2-methoxy-12-mefil-10-[4'-[3"-hydroxy-3",5"-dimethyl-4"(Z-2''',4'''-dimethyl-2'''-heptenoyloxy)tetrahydropyran-1"-yloxy]-5'-methylcyclohexan-1'-yloxy]-1,4,6,7,9-pentaoxo-1,4,6,6a,7,8,9,10,10a,11-decahydronaphthacene. Other inhibitors, such as chloroquine, can also be encapsulated on the particles, such as within or on the surface of the particles.

[0122] Detection of nuclear accumulation can be performed by any suitable technique. For example, a detectable marker can be fused to the vector, such as in combination with a means for detecting the location of the nucleus (e.g., a nuclear-specific dye such as DAPI), so that the location within the cell can be visualized. The cell nucleus can be isolated from the cell, and its contents can then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay. Embodiments herein can demonstrate time-dependent pH-triggered release of cargo to a target site. Embodiments herein can include and provide cellular delivery of complex multiple cargoes. The additional cargo can be an inhibitor, such as a small molecule, antibody, DNAse inhibitor, or RNAse inhibitor.

[0123] Lipid structures can carry capacities of up to greater than 100% by weight (defined as (cargo weight / lipid structure weight) x 100). Optimal cargo loading can be between 1% and 100% by weight of the lipid structure, or between about 1% and 100% by weight of the lipid structure. For example, lipid structures can contain between about 1% and about 10%, about 10% and about 20%, about 20% and about 30%, about 30% and about 40%, about 40% and about 50%, about 50% and about 60%, about 60% and about 70%, about 70% and about 80%, about 80% and about 90%, about 90% and about 100%, about 100% and about 200%, about 200% and about 300%, about 300% and about 400%, about 400% and about 500% or more of the weight of the structure of polynucleic acid cargo.

[0124] Polynucleic acids can be delivered to cells of the intestinal tract. For example, polynucleic acids can be delivered to intestinal crypt stem cells by the delivery vehicle described herein. For example, the delivered polynucleic acid can be (1) a polynucleic acid not normally found in intestinal epithelial stem cells, (2) a polynucleic acid normally found in intestinal epithelial stem cells but not expressed at physiologically significant levels, (3) a polynucleic acid normally found in intestinal epithelial stem cells and normally expressed at physiologically desired levels in the stem cells or their progeny, (4) any other DNA that can be modified for expression in intestinal epithelial stem cells, and (5) any combination of the above.

[0125] In some cases, proteins encoded by polynucleic acids contained within lipid structures can be measured and quantified. In some cases, modified cells can be isolated, and Western blots can be performed on the modified cells to determine the presence and relative amount of protein produced compared to unmodified cells. In other cases, intracellular staining of proteins using flow cytometry can be performed to determine the presence and relative amount of protein produced. Additional assays can also be performed to determine whether a protein, such as APC, is functional. For example, modified cells expressing an APC transgene can be measured for cytosolic β-catenin expression and compared to unmodified cells. Decreased expression of β-catenin in the cytosol of modified cells compared to unmodified cells can indicate a functional APC transgene. In other cases, a mouse model of FAP can be used to determine the functionality of a transgene encoding an APC protein. For example, mice with FAP can be treated with modified cells encoding APC, and a reduction in FAP disease can be measured relative to untreated mice.

[0126] Additional treatments that can be performed on the subject receiving the subject delivery vehicle can also be provided herein. The subject can undergo treatments such as blood transfusion, blood sampling, computerized tomography scan (CT), magnetic resonance imaging (MRI), X-ray, radiation therapy, organ transplant, and any combination thereof. In some cases, evaluation of lesions, such as cancerous lesions, can be performed.

[0127] In some cases, non-target lesions may be evaluated. A complete response of non-target lesions may be the disappearance and normalization of tumor marker levels. All lymph nodes must be non-pathological in size (minor axis less than 10 mm). If tumor markers are initially above the upper limit of normal, they must normalize for the patient to be considered in a complete clinical response. Non-CR / non-PD is the persistence of one or more non-target lesions and / or the maintenance of tumor marker levels above normal limits. Progression is the appearance of one or more new lesions and / or a clear worsening of existing non-target lesions. Clear worsening should usually not exceed target lesion status. In some cases, best overall response may be the best response recorded from the start of treatment to disease progression / recurrence.

[0128] Delivery of cargo The delivery vehicles provided herein can be used to deliver cargo to target cells. In some cases, the target cells are found in the gastrointestinal tract, reproductive tract, circulatory system, respiratory system, musculoskeletal system, excretory system, nervous system, ocular system, and combinations thereof. In some cases, suitable target cells can be found in any major organ of the body, including, but not limited to, the skin, lungs, heart, liver, stomach, urinary system, reproductive system, intestine, pancreas, kidney, thymus, thyroid, and / or brain. In some cases, the target cells are part of the gastrointestinal tract, such as the anus, rectum, large intestine, small intestine, liver, stomach, esophagus, or mouth. In some cases, the target cells are enteroendocrine cells, mast cells, enterocytes, brush cells, Paneth cells, or goblet cells. In some cases, the target cells are enteroendocrine cells, such as EC cells, D cells, CCK cells, L cells, P / D1 cells, or G cells. In some cases, the target cells are in the intestinal epithelium and are selected from intestinal stem cells, Paneth cells, goblet cells, enterocytes, transit-amplifying cells, enteroendocrine cells, or any combination thereof. In some cases, the target cells are intestinal stem cells. In some cases, the target cells are crypt cells.

[0129] A delivery vehicle can be used to introduce a cargo into a target cell. In some cases, the introduction involves contacting the target cell with the cargo. In other cases, the introduction involves transducing or transducing the target cell with the cargo. In some cases, the cargo can modify the genome of the cell or can be present extragenomically within the cell.

[0130] In some embodiments, the delivery vehicle utilized may comprise a cargo delivered to a target cell, e.g., for expression in the cell and / or to genetically modify the target cell. The efficiency of such delivery, e.g., nucleic acid transduction by a cargo such as a polynucleic acid described herein, may be, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 16 It may be 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%, or may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%. The efficiency of such delivery, e.g., nucleic acid transfer, by a cargo such as a polynucleic acid described herein, may be, for example, greater than or about 1 fold, 10 fold, 20 fold, 40 fold, 60 fold, 80 fold, 100 fold, 120 fold, 140 fold, 160 fold, 180 fold, 200 fold, 300 fold, 400 fold, 500 fold, or 1000 fold greater than the total number of cells contacted (in vivo or ex vivo) and / or present in the tissue or location.

[0131] The efficiency of cellular uptake by a subject delivery vehicle, such as a composition used herein (including a delivery vehicle having charge separation for epithelial cell delivery, bile salts for stability in harsh environments, and optionally other features such as MPPs or other mucus-penetrating features), can allow for efficient penetration and passage (e.g., through the mucus layer) into target cells, thereby having efficient uptake by target cells, e.g., uptake of 20%, 25%, 30%, 35%, 40%, 45%, or more of the total number of cells contacted. , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%, or may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%. In some embodiments, the compositions may have a higher percent cellular uptake compared to a comparable delivery vehicle that does not include bile salts and / or charge separation, or compared to a delivery vehicle lacking one or more components. The improvement may be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or up to about 80% better. In some cases, the efficiency of transfection or delivery of a cargo delivered to a cell by a delivery vehicle composition described herein (such as the integration or expression of a protein from a polynucleic acid) may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% better compared to a comparable delivery vehicle that does not include bile salts and / or charge separation, or compared to a delivery vehicle lacking one or more components.In some cases, the efficiency of transfection or integration of or expression of a polynucleic acid cargo delivered to a cell by a delivery vehicle composition described herein may be about 5%, to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% better than a comparable delivery vehicle that does not include bile salts and / or charge separation, or compared to a delivery vehicle lacking one or more components.

[0132] In some embodiments, the compositions described herein for delivering cargo may be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days after introduction into a subject in need thereof. The structures may be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after introduction into a subject. The delivery vehicles provided herein may be functional for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after introduction into a subject. In some embodiments, the delivery vehicle can be functional for the lifetime of the recipient. Furthermore, the delivery vehicle can function at 100% of its intended normal operation. The delivery vehicle can also function at 1, 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% functional. Functionality of a delivery vehicle can refer to the efficiency of delivery, retention of lipid nanoparticles, stability of lipid nanoparticles, or any combination thereof.

[0133] In some embodiments, the delivery vehicles provided herein can deliver cargo, such as nucleic acids, to target cells (RNA, DNA (e.g., minicircle DNA), etc.). In some cases, function can include the percentage of cells that received the nucleic acid from the delivery vehicle composition. In other cases, function can refer to the frequency or efficiency of protein production from the nucleic acid. For example, a delivery vehicle composition can deliver a nucleic acid encoding at least a portion of a gene, such as APC, to a cell, and the frequency of efficiency can describe the fully functional gene that is repaired or created by delivery of the cargo.

[0134] The nucleic acid cargo concentration in the delivery vehicle composition can be from 0.5 nanograms to 50 micrograms. Such concentrations can range from about 0.5 ng to 1 ng, 2 ng, 5 ng, 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1000 ng, 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, or 50 μg or more. In some cases, the amount of nucleic acid (e.g., ssDNA, dsDNA, RNA) that can be introduced into a cell by a delivery vehicle can be varied to optimize nucleic acid introduction efficiency and / or cell viability. In some cases, less than about 100 picograms of nucleic acid can be introduced into a subject.In some cases, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms , at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms of nucleic acid can be added to each cell sample (e.g., one or more cells are electroporated or otherwise targeted for cargo delivery). In some cases, the amount of nucleic acid (e.g., dsDNA, RNA) required for optimal nucleic acid transfer efficiency and / or cell viability can be cell type specific.

[0135] In some embodiments, the term "effective amount" can refer to an amount sufficient to increase the expression level of at least one gene that may be decreased in a subject prior to treatment or an amount sufficient to alleviate one or more symptoms of cancer. For example, an effective amount can be an amount sufficient to increase the expression level of at least one gene selected from the group consisting of gastrointestinal differentiation genes, cell cycle inhibitor genes, and tumor suppressor genes by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500%, or more, compared to a baseline level or the expression level in the absence of compound treatment.

[0136] In some embodiments, an effective amount can refer to an amount sufficient to reduce the expression level of at least one gene that may be elevated in a subject prior to treatment or an amount sufficient to alleviate one or more symptoms of cancer. For example, an effective amount can be an amount sufficient to reduce the expression level of a gene by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 1000%, 1500% or more compared to a baseline level or the expression level in the absence of compound treatment.

[0137] In some embodiments, treating involves at least about a 1-fold, 5-fold, 10-fold, 20-fold, 40-fold, 80-fold, 100-fold, 300-fold, 600-fold, or 1000-fold reduction in disease in a subject in need thereof compared to a comparable subject not receiving the treatment, as measured by in vitro or in vivo assays. In one aspect, the reduction in disease can be the result of an increase or decrease in the expression level of at least one gene in the subject. Various gene expression assays are available, including, but not limited to, sequencing, PCR, RT-PCR, Western blot, Northern blot, ELISA, protein quantification, mRNA quantification, FISH, RNA-Seq, SAGE, or a combination thereof. Additional assays that can be used include microscopy, histology, in vivo animal studies, human studies, or any combination thereof.

[0138] How to use The delivery vehicle compositions herein provide for delivery to epithelial cells in mucosal tissues, such as those of the digestive tract (gastrointestinal tract), as well as for use in other mucosal tissues, such as the lungs, vagina, and eye. The delivery vehicles herein provide access to epithelial cells along with penetration through the mucus layer. In some embodiments, the delivery vehicle delivers cargo to epithelial cells of the digestive tract, delivering cargo (such as those described herein) for therapeutic, diagnostic, or theranostic purposes.

[0139] Exemplary diseases treatable by the subject delivery vehicles provided herein, particularly delivery vehicles carrying therapeutic cargoes, can be cancerous or non-cancerous. Such diseases can be cardiovascular diseases, neurodegenerative diseases, eye diseases, reproductive system diseases, gastrointestinal diseases, brain diseases, skin diseases, skeletal diseases, musculoskeletal diseases, pulmonary diseases, or chest diseases, to name a few. The disease can be a genetic disease such as cystic fibrosis, Tay-Sachs disease, fragile X, Huntington's disease, neurofibromatosis, sickle cell anemia, thalassemia, Duchenne muscular dystrophy, or a combination thereof.

[0140] In some embodiments, the disease is a gastrointestinal disease. In some cases, the gastrointestinal disease is a monogenic gastrointestinal disease. In some embodiments, the gastrointestinal disease is hereditary. In some cases, the gastrointestinal disease is epithelial. Suitable gastrointestinal diseases can be familial adenomatous polyposis (FAP), attenuated FAP, microvillus inclusion body disease (MVID), chronic inflammatory bowel disease, chronic inflammatory bowel disease, ileal Crohn's disease, juvenile polyposis, hereditary diffuse gastric cancer syndrome (HDGC), Peutz-Jeghers syndrome, Lynch syndrome, gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS), Li-Fraumeni syndrome, familial gastric cancer, or a combination thereof. The gastrointestinal disease can cause polyps in the digestive tract. In some cases, the disease is FAP. FAP can progress to cancer. The gastrointestinal disease can be hereditary. For example, the genetic gastrointestinal disorder can be Gilbert syndrome, telangiectasia, mucopolysaccharidosis, Osler-Weber-Rendeus syndrome, pancreatitis, keratoacanthoma, biliary atresia, Morquio syndrome, Hurler syndrome, Hunter syndrome, Crigler-Najjar, Rotor, Peutz-Jeghers syndrome, Dubin-Johnson, osteochondrosis, osteochondrodysplasia, polyposis, or a combination thereof.

[0141] In some embodiments, subjects can be screened for the presence of a disease. Screens can be used to identify suitable subjects. In some cases, the disease can be identified by genetic, phenotypic, molecular, or chromosomal screening. In one embodiment, a suitable subject is positive for a disease provided herein. For example, a genetic screen can identify mutations in the APC gene that can result in FAP. In some cases, the screen can include analyzing genes such as CDH1, STK11, SMAD4, MLH1, MSH2, EPCAM, MSH6, PMS2, MYO5B, APC, TP53, portions thereof, promoters thereof, and combinations thereof.

[0142] In some cases, the delivery vehicles herein carry therapeutic cargo (such as a nucleic acid, protein, or drug) and are used to treat diseases affecting the gastrointestinal tract, such as familial polyposis (FAP), mild FAP, colon cancer, chronic inflammatory bowel disease, ileal Crohn's disease, microvillus inclusion body disease, and congenital diarrhea.

[0143] In other cases, the gene for delivery by liposome can be administered to the subject as a preventive measure.For example, the subject may not have been diagnosed with a disease and may appear to have a predisposition to a disease such as cancer.In some cases, the cancer may be colon cancer.

[0144] In some cases, the delivery vehicles herein carry a diagnostic cargo and are used to visualize or diagnose the state of a cell or tissue, or to diagnose or monitor a subject for a condition or disease. For example, a subject is administered an effective amount of the delivery vehicle, and a method for diagnosing FAP includes determining the level of APC integrated into the cellular genome, such that a difference in the APC level between before the start of therapy and during and / or after the therapy in the patient will provide evidence of the effectiveness of the therapy in the patient, including whether the patient has completed the therapy or whether the condition has been inhibited or eliminated.

[0145] A pharmaceutical composition comprising a delivery vehicle with a cargo can be administered chronically in some cases. Administration can include administration to a subject on an hourly, daily, monthly, or yearly basis. For example, in some cases, a subject can be administered a pharmaceutical composition daily for the entirety of the subject's life. In other cases, the pharmaceutical composition can be administered daily for the duration of the subject's disease. A subject can be administered a pharmaceutical composition, such as one comprising a delivery vehicle and a polynucleic acid cargo, to treat a disease or disorder until the disease or disorder is reduced, controlled, or eliminated. Disease management can include stabilization of the disease. For example, a cancer that is being controlled can have stopped growing or spreading, as measured by a CT scan. The cancer can be colon cancer. In other cases, the pharmaceutical composition can be administered prophylactically. In some cases, a subject can have undergone a genetic screen to identify a subject as having a predisposition to cancer, such as colon cancer. In this case, a predisposed subject can begin prophylactic treatment by receiving a pharmaceutical composition comprising a delivery vehicle and a polynucleic acid cargo. In this case, the subject contains a genetic mutation that predisposes the subject to colon cancer, and the subject can begin prophylactic treatment with such a pharmaceutical composition.

[0146] In some cases, prophylactic treatment may prevent a disease such as cancer. Where prophylaxis may be used in connection with a condition such as local recurrence (e.g., pain), prevention of a disease such as cancer, a syndrome complex such as heart failure, or any other condition may involve administering a composition that reduces the frequency of or delays the onset of symptoms of the condition in a subject relative to subjects who do not receive the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to an untreated control population by a statistically and / or clinically significant amount, for example, relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population. Prevention of infectious diseases includes, for example, reducing the number of diagnoses of infectious diseases in a treated population relative to an untreated control population, and / or delaying the onset of symptoms of infectious diseases in a treated population relative to an untreated control population. Prevention of pain includes, for example, reducing or delaying the magnitude of pain experienced by subjects in a treated population relative to an untreated control population.

[0147] Assays can be used to determine the therapeutic efficacy of the delivery vehicles provided herein. In some cases, assays can be performed before, during, and / or after administration of the subject delivery vehicle. Assays can be performed, for example, before or after administration at -30 days, -15 days, -7 days, -3 days, 0 days, 3 days, 5 days, 7 days, 10 days, 14 days, 18 days, 20 days, 24 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 80 days, 100 days, 150 days, 250 days, 360 days, 2 years, 5 years, or 10 years. Suitable assays can be in vivo or ex vivo. In some cases, assays include scans. Suitable scans can include CT, PET, MRI, or a combination thereof. In some cases, assays include in vitro assays such as histology, serology, sequencing, ELISA, microscopy, etc.

[0148] Pharmaceutical Compositions and Formulations The compositions described throughout can be formulated into pharmaceuticals and used to treat humans or mammals in need thereof. The pharmaceuticals can be co-administered with any additional therapy.

[0149] For oral administration, excipients may include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc. If desired, the delivery vehicle composition may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

[0150] The composition may be administered orally, by subcutaneous or other injection, intravenously, intracerebrally, intramuscularly, parenterally, transdermally, intranasally, or rectally. The form in which the compound or composition is administered depends, at least in part, on the route by which the compound is administered. In some cases, the composition may be available in the form of a solid preparation for oral administration, which may be a tablet, granule, powder, capsule, or the like. In tablet formulations, the composition is typically formulated with additives, such as excipients such as sugar or cellulose preparations, binders such as starch paste or methylcellulose, fillers, disintegrants, and other additives typically used in pharmaceutical manufacturing. The composition to be administered may include a delivery vehicle in an amount that is a pharmaceutically effective amount for therapeutic use in a biological system, including a patient or subject. The pharmaceutical composition may be administered daily or as needed.

[0151] Delivery vehicles herein include those formulated as pharmaceutical compositions for administration. Suitable formulations include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the body fluids of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Suitable inert carriers may include sugars such as lactose. In some cases, the compositions may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0152] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and / or by the use of surfactants. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Solutions and dispersions of the active compound as a free acid or base or a pharmacologically acceptable salt thereof can be prepared in water or another solvent or dispersion medium, suitably mixed with one or more pharmaceutically acceptable excipients, including, but not limited to, surfactants, dispersing agents, emulsifying agents, pH-adjusting agents, and combinations thereof. Suitable surfactants can be anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include, but are not limited to, sodium, potassium, and ammonium long-chain alkyl and alkylaryl sulfonates such as sodium dodecylbenzenesulfonate, dialkyl sodium sulfosuccinates such as sodium dodecylbenzenesulfonate, dialkyl sodium sulfosuccinates such as sodium bis-(2-ethylthiol)-sulfosuccinate, and alkyl sulfonates such as sodium lauryl sulfate. Examples of cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, and stearyl dimethyl benzyl ammonium chloride, polyoxyethylene, and coconut amine.Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4 oleate, acylated sorbitan, acylated sucrose, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine. The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent. Upon reconstitution, the formulation is typically buffered to a pH of 3-8 for parenteral administration. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer. Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0153] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent or dispersion medium, optionally with one or more of the excipients listed above, followed by sterile filtration. Generally, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. For sterile powders for preparing sterile injectable solutions, the preparation method can be vacuum drying and freeze-drying techniques, which will produce a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution. Powders can be prepared in a way that the particles are porous in nature, which can increase the dissolution of the particles. Methods for producing porous particles are well known in the art.

[0154] The formulation may be an ophthalmic formulation or a topical formulation. Pharmaceutical formulations for ophthalmic administration may be in the form of a sterile aqueous solution or suspension of particles formed from one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate-buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a non-toxic, parenterally acceptable diluent or solvent, such as 1,3-butanediol. In yet other embodiments, the liposomes may be formulated for topical administration to mucosal membranes. Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, solutions, and transdermal patches. The formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The composition may include one or more chemical permeation enhancers, membrane permeabilizers, membrane transport agents, emollients, surfactants, stabilizers, and combinations thereof. In some embodiments, liposomes can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, liposomes can be formulated as a liquid, including solutions and suspensions, such as eye drops, or as a semi-solid formulation, such as an ointment or lotion for topical application to the eye or mucous membranes, such as intravaginally or rectally. The formulation can include one or more excipients, such as emollients, surfactants, emulsifiers, and penetration enhancers.

[0155] The appropriate dose ("therapeutically effective amount") of the active agent in the composition may depend, for example, on the severity and course of the condition, the mode of administration, the bioavailability of the particular agent, the age and weight of the subject, the subject's medical history and response to the active agent, the judgment of the physician, or any combination thereof. The therapeutically effective amount of the active agent in the composition to be administered to a subject may range from about 100 μg / kg body weight / day to about 1000 mg / kg body weight / day, via single or multiple administrations. In some embodiments, the range of each active agent administered daily is from about 100 μg / kg body weight / day to about 50 mg / kg body weight / day, 100 μg / kg body weight / day to about 10 mg / kg body weight / day, 100 μg / kg body weight / day to about 1 mg / kg body weight / day, 100 μg / kg body weight / day to about 10 mg / kg body weight / day, 500 μg / kg body weight / day to about 100 mg / kg body weight / day, 500 μg / kg body weight / day to about 50 mg / kg body weight / day, 500 μg / kg body weight / day to about 100 mg / kg body weight / day, The dose may be 1 mg / kg body weight / day to about 5 mg / kg body weight / day, 1 mg / kg body weight / day to about 100 mg / kg body weight / day, 1 mg / kg body weight / day to about 50 mg / kg body weight / day, 1 mg / kg body weight / day to about 10 mg / kg body weight / day, 5 mg / kg body weight / dosage to about 100 mg / kg body weight / day, 5 mg / kg body weight / dosage to about 50 mg / kg body weight / day, 10 mg / kg body weight / day to about 100 mg / kg body weight / day, and 10 mg / kg body weight / day to about 50 mg / kg body weight / day.

[0156] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweeteners, salts, buffers, etc. Pharmaceutically acceptable carriers can be prepared from a wide variety of materials, including, but not limited to, flavorings, sweeteners, and buffers and absorbents, which may be necessary to prepare a particular therapeutic composition.

[0157] In some cases, the composition containing the delivery vehicle can be formulated under sterile conditions within a reasonable time prior to administration. For example, the composition containing the delivery vehicle can be formulated about 1 month, 2 weeks, 1 week, 5 days, 3 days, 2 days, 1 day, 10 hours, 5 hours, or immediately before administration to a subject. In one embodiment, the delivery vehicle can be frozen and thawed before administration. The provided delivery vehicle can be used in combination with a secondary therapy. For example, a secondary therapy such as chemotherapy or radiation therapy can be administered before or after administration of the delivery vehicle, for example, within 12 hours to 7 days. A combination of therapies, such as both chemotherapy and radiation therapy, can be used in addition to administration of the delivery vehicle.

[0158] In some cases, the provided delivery vehicle may include a coating. The coating may be an enteric coating. An enteric coating may be utilized to prevent or minimize dissolution in the stomach while allowing dissolution in the small intestine. In some embodiments, the coating may include an enteric coating. An enteric coating may be a barrier applied to oral pharmaceuticals that prevents release of the pharmaceutical before reaching the small intestine. Delayed-release formulations such as enteric coatings may have an irritating effect on the stomach from dissolution in the stomach after administration of the pharmaceutical. Such coatings are used to protect acid-labile drugs from acidic exposure in the stomach and deliver them to a basic pH environment (intestinal pH 5.5 or higher) where they may otherwise not be degraded.

[0159] Dissolution can occur in any organ. For example, dissolution can occur in the duodenum, jejunum, ilium, and / or colon, or any combination thereof. In some cases, dissolution can occur near the duodenum, jejunum, ilium, and / or colon. Some enteric coatings work by providing a surface that is stable at the highly acidic pH present in the stomach but rapidly degrades at less acidic (relatively more basic) pH. Thus, an enteric-coated pill may not dissolve in the acidic environment of the stomach, but can dissolve in the alkaline environment present in the small intestine. Examples of enteric coating materials include, but are not limited to, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, sodium alginate, and stearic acid.

[0160] The enteric coating can be applied at a functional concentration. The enteric coating can be cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose acetate succinate, poly(methacrylic acid-co-ethyl acrylate) 1:1, poly(methacrylic acid-co-ethyl acrylate) 1:1, poly(methacrylic acid-co-methyl methacrylate) 1:1, poly(methacrylic acid-co-methyl methacrylate) 1:1, poly(methacrylic acid-co-methyl methacrylate) 1:2, poly(methacrylic acid-co-methyl methacrylate) 1:2, poly(methyl acrylate-co-methyl methacrylate-co-methacrylic acid) 7:3:1, or any combination thereof. The enteric coating can be applied at a concentration of about 6 mg / (cm 2 ) ~ approx. 12mg / (cm 2 The enteric coating can be applied to the structure at a concentration of about 1 mg / (cm 2 ) to 2mg / (cm 2 ), 3mg / (cm 2 ), 4mg / (cm 2 ), 5mg / (cm 2 ), 6mg / (cm 2), 7mg / (cm 2 ), 8mg / (cm 2 ), 9mg / (cm 2 ), 10mg / (cm 2 ), 11mg / (cm 2 ), 12mg / (cm 2 ), 13mg / (cm 2 ), 14mg / (cm 2 ), 15mg / (cm 2 ), 16mg / (cm 2 ), 17mg / (cm 2 ), 18mg / (cm 2 ), 19mg / (cm 2 ), approx. 20mg / (cm 2 ) can be applied.

[0161] In some embodiments, pharmaceutical compositions containing the subject delivery vehicles can be orally administered from various drug formulations designed to provide delayed release. Delayed oral dosage forms include, for example, tablets, capsules, and caplets, and may also include multiple granules, beads, powders, or pellets, which may or may not be encapsulated. Tablets and capsules may represent oral dosage forms, in which case solid pharmaceutical carriers may be utilized. In delayed-release formulations, one or more barrier coatings may be applied to the pellets, tablets, or capsules to promote slow dissolution and simultaneous release of the drug into the intestine. Typically, the barrier coating may comprise one or more polymers that encase, surround, or form a layer or membrane around the therapeutic composition or active core. In some embodiments, an active agent, such as a polynucleic acid, may be delivered in a formulation that provides delayed release at a predetermined time after administration. The delay may be up to about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or up to one week in length. In some cases, an enteric coating may not be used to coat the particles.

[0162] Polymers or coatings that can be used to achieve enteric release can in some cases be anionic polymethacrylates (copolymers of methacrylic acid with either methyl methacrylate or ethyl acrylate (Eudragit®), cellulosic polymers such as cellulose acetate phthalate (Aquateric®), or polyvinyl derivatives such as polyvinyl acetate phthalate (Coateric®).

[0163] In some cases, the formulations may be presented in single-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored frozen or freeze-dried (lyophilized), requiring only the addition of a sterile liquid carrier immediately prior to use. For oral administration, the compositions may take the form of tablets or capsules prepared by conventional techniques with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets may in some cases be coated. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or gum arabic), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). Preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents as needed. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration, the compositions can take the form of tablets or lozenges formulated in a conventional manner. In some cases, the compositions can also be formulated as preparations for implantation or injection. Thus, for example, the structures can be formulated with suitable polymeric, aqueous, and / or hydrophilic materials or resins, or as sparingly soluble derivatives (e.g., as sparingly soluble salts). The compounds may also be formulated in rectal compositions, creams or lotions, or as a transdermal patch.

[0164] In some cases, pharmaceutical compositions may contain salts. Salts may be relatively non-toxic. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitable inorganic bases for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and sufficiently strong to form such salts. To illustrate, types of such organic bases may include mono-, di-, and trialkylamines such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids such as arginine and lysine; guanidine, N-methylglucosamine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, (trihydroxymethyl)aminoethane, and the like.

[0165] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be utilized. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0166] Example 1: Preparation of an exemplary delivery vehicle of the present disclosure This example provides an exemplary method for preparing a delivery vehicle of the present disclosure. The lipid components of the delivery vehicle, DODMA (Sigma Aldrich), deoxycholate (Sigma Aldrich), MVL5 (Avanti Polar Lipids), DSPC (Avanti Polar Lipids), DMG-PEG2000 (Avanti Polar Lipids), DOPC (Avanti Polar Lipids), DiI (ThermoFisher Scientific), and DiO (ThermoFisher Scientific), were dissolved in ethanol and heated above their phase transition temperatures, for example, if the phase transition temperature was higher than 37°C. For example, when using DSPC, the lipid and aqueous phases were heated to 70°C. When DOPC was used, the lipid and aqueous phases were not heated and were used at room temperature. The nucleic acid was dissolved in an aqueous buffer heated above the phase transition temperature of the lipid.

[0167] The aqueous buffer pH was set below the pKa of the bile salts and cationic lipids. In this way, the lipids were strongly cationic when formulated with nucleic acids. To form cargo-bearing delivery vehicles, lipids and nucleic acids were mixed using a microfluidic channel, followed by dialysis to remove ethanol. Other suitable methods can also be used for this process. For example, lipid structures such as liposomes can be formed by thin film hydration, in which lipids are dissolved in an organic phase and dried using a rotating rotovap. The formed thin film can be hydrated in water. The hydrated lipids can be heated to 70°C, for example, for DSP, or at room temperature, for example, for DOPC, and extruded through an appropriate extruder pore size. Nucleic acid cargo can be mixed with lipids to form lipoplexes.

[0168] Another suitable alternative method for preparing exemplary delivery vehicles is to use thin film hydration. The lipids are dissolved and mixed in an organic solvent. The solvent is removed, and the formed thin film is hydrated in an aqueous solution. Sonication or extrusion is used to size the lipids appropriately. The nucleic acid can be complexed by mixing the lipid mixture and the nucleic acid together.

[0169] Exemplary Delivery Vehicle Formulations To prepare an exemplary delivery vehicle containing encapsulated nucleic acid, 300 μg of plasmid DNA encoding Gaussia luciferase under the cytomegalovirus (CMV) promoter was dissolved in a final volume of 3 mL of 50 mM sodium acetate buffer (pH 4.8). Appropriate molar amounts of MVL5, DODMA, deoxycholate, MVL5, DSPC, DMG-PEG2000, and / or DOPC were mixed in ethanol according to their molar amounts and cationic lipid:nucleic acid ratio (see Table 2 for the molar percentages of lipids in the various formulations prepared). The cationic lipid:nucleotide molar ratio was maintained at approximately 16. Fluorescently labeled lipids, such as DiI and DiO, were added to the mix at 0.5% of the total lipid molar ratio, if used. The ethanol volume was increased to 1 mL.

[0170] The nucleic acid is in an aqueous sodium acetate buffer phase in a 3 mL syringe. The lipid is in ethanol in a 1 mL syringe. The two syringes are attached to a NanoAssemblr (Precision Nanosystems), and the two samples are then mixed using a microfluidic chip on the NanoAssemblr.

[0171] For this study, samples were placed in syringes (nucleic acid in a 3 mL syringe and lipid in a 1 mL syringe, as described above) on the NanoAssemblr Benchtop and preheated to 65°C for DSPC formulations or at room temperature (approximately 25°C) for DOPC formulations. Samples were mixed using the NanoAssemblr Benchtop microfluidic chip system at a flow rate of 6 mL / min. The pH was neutralized with 300 mM HEPES buffer at pH 7.5. Ethanol was removed using overnight dialysis. Samples were concentrated using an Amicon Ultra-4 with a 100 kDa molecular weight cutoff.

[0172] [Table 2]

[0173] Example 2: Nucleic acid transfection of exemplary delivery vehicles of the present disclosure In this study, the transfection efficiency of an exemplary delivery vehicle (prepared using the process described in Example 1 above) was evaluated. HEK cells cultured to 50-80% confluency were used for transfection. 1 μg of Gaussia luciferase-expressing plasmid DNA encapsulated in lipid nanoparticles (listed in Table 2 above) was used per well in a 24-well plate. Transfection efficiency was assessed by removing 30 μl of medium after 24 hours and performing a flash luciferase assay (Pierce Gaussia Luciferase Assay Kit). An increase in relative light units (RLU) corresponded to higher transfection efficiency.

[0174] It was observed that the presence of the polyvalent cationic lipid MVL5 significantly increased nucleic acid transfer, likely by exerting positive or neutral properties on the bile salt-stabilized system. This was likely due to increased endosomal escape. MVL5 and other polyvalent lipids may be optimal for this system due to their multivalency (+3 at physiological pH and +5 at lysosomal pH) and the high molar ratio of negatively charged bile salts required for stability. The data are shown in Figure 1.

[0175] Example 3: Stability of exemplary delivery vehicles of the present disclosure In this study, the stability of exemplary delivery vehicles in a high bile salt environment was evaluated. To determine delivery vehicle stability, 0.5 mol % DiI and DiO were incorporated into the delivery vehicles used in this assay. DiI and DiO are fluorescent dyes that are a FRET pair. Bile salts were simulated by using an equal mixture of cholate and deoxycholate at the concentrations indicated (Figures 2-4). It was expected that a decrease in FRET intensity would occur if the delivery vehicle was susceptible to bile salt degradation. Relative fluorescence units (RFU) were determined by excitation at 465 nm and emission readings at 501 nm and 570 nm. The RFU reading at 570 nm was divided by the reading at 501 nm. Readings were normalized to the FRET intensity of the system without any treatment. The data are shown in Figures 2, 3, and 4.

[0176] This study demonstrated that DSPC / deoxycholate (Formulation No. 10) was stable to bile salts, whereas DOPC / deoxycholate (Formulation No. 11) was not. It should be noted that DOPC / deoxycholate resembles elastic liposomes, which were found to be highly susceptible to bile salts. In contrast, DSPC / deoxycholate was found to be highly resistant to bile salt attack. Furthermore, DSPC / cholesterol (Formulation No. 13) was also found to be inresistant to bile salts. This indicated that the presence of saturated lipid tails was not sufficient to confer stability against bile salts, and that bile salts (e.g., deoxycholate) must be incorporated into the lipid nanoparticles to confer stability.

[0177] Furthermore, as can be seen in Figure 4, it was observed that PEGylation (Formulation No. 16) was not required for stability, and omission of the high phase transition temperature lipid (Formulation No. 15) or bile salts (Formulation No. 14) resulted in a loss of bile salt stability of the delivery vehicle.

[0178] Example 4: Encapsulation of nucleic acids in exemplary delivery vehicles of the present disclosure For this study, delivery vehicles containing 1 μg of DNA encapsulated in lipid nanoparticles (formulation number 5 in Table 2) were loaded into lanes of an agarose gel either untreated (lane 2 in Figure 5), (ii) treated with 7% Triton-X100 (lane 3 in Figure 5), or (iii) treated with 7% Triton-X100 plus 70°C for 30 minutes (lane 4 in Figure 5), followed by electrophoresis. DNA was detected using UV light with SYBR Safe. No DNA bands were observed with any of the cationic lipids containing the bile salt-stabilized system (lane 2, untreated), indicating that encapsulation and DNA were not released from the delivery vehicle. However, DNA bands were observed when the system was perturbed using detergent and heat (lanes 3 and 4), indicating that the vehicle was unstable in this environment and that DNA was released upon treatment. The data are shown in Figure 5. This demonstrated the advantage of having cargo (such as DNA) encapsulated within a delivery vehicle that is stable in bile salt environments, especially for efficient protection in high bile salt environments such as the gastrointestinal tract.

[0179] Example 5: Preparation of cargo-bearing delivery vehicles Encapsulation of nucleic acid cargo was carried out as follows: Lipids were dissolved in ethanol and heated above their phase transition temperature. Nucleic acid was dissolved in an aqueous buffer heated above the lipid's phase transition temperature. The aqueous buffer pH was set below the pKa of the bile salts and cationic lipids. In this way, the lipids were strongly cationic when formulated with nucleic acid. Lipids and nucleic acid were mixed using a microfluidic channel. The pH was raised to neutral, the sample was concentrated, and ethanol was removed using dialysis.

[0180] Materials: DODMA (Sigma Aldrich), deoxycholate (Sigma Aldrich), MVL5 (Avanti Polar Lipids), DSPC (Avanti Polar Lipids), DMG-PEG2000 (Avanti Polar Lipids), DOPC (Avanti Polar Lipids), DiI (ThermoFisher Scientific), DiO (ThermoFisher Scientific), and GMO (MP Biomedicals). formulation 375 μg of plasmid DNA encoding Gaussia luciferase under the CMV promoter was dissolved in a final volume of 3 mL of 50 mM sodium acetate buffer (pH 4.8). Appropriate molar amounts of MVL5, DODMA, deoxycholate, MVL5, DSPC, GMO, DMG-PEG2000, and / or DOPC were mixed in ethanol according to their molar amounts and cationic lipid:nucleic acid ratio. The cationic lipid:nucleotide molar ratio was kept constant at 16. When lipids were fluorescently labeled with DiI and DiO, each DiI and DiO was added to the mix at 0.5% mol of the total lipid moles. The ethanol volume was increased to 1 mL. Samples were placed in syringes on a NanoAssemblr Benchtop (Precision Nanosystems) and preheated to 65°C for DSPC formulations or at room temperature for DOPC formulations. The samples were mixed at a flow rate of 6 mL / min using a NanoAssemblr Benchtop microfluidic chip system. The pH was neutralized, and then ethanol was removed using overnight dialysis. The samples were concentrated using an Amicon Ultra-4 (Merck Millipore Ltd, Ireland) with a 100 kDa molecular weight cutoff.

[0181] The following formulations were prepared as shown in Table 3.

[0182] [Table 3]

[0183] In summary, particles with DMG-PEG were stable and did not form aggregates, even at 1% DMG-PEG. DSG has a stearic acid lipid tail that exists in the gel phase at 37°C. DMG has a myristyl phosphate lipid tail that exists in the liquid phase at 37°C. DMG-PEG was present in the liquid phase of the vehicle, thereby stabilizing the cationic lipid and preventing aggregation, while DSG-PEG was present in the gel phase and did not have the same stabilizing effect.

[0184] Example 6: In vivo administration of delivery vehicles Mice were intrarectally administered approximately 30 micrograms of DNA encapsulated in DiI- and DiO-labeled nanoparticles. Four hours after dosing, mice were sacrificed, and the intestines were embedded in OCT, frozen on dry ice, and stored at -80°C. Tissues were cryosectioned at 30 micrometer slices and imaged using BioTek Citation. DiI fluorescence was measured in the RFP channel.

[0185] PEGylated particles cannot reach intestinal epithelial cells MVL5 / DODMA / DSPC / deoxycholate / DMG-PEG (Particles 5–9) particles were formed with increasing amounts of DMG-PEG, and particle behavior was investigated in vivo. Increasing the amount of DMG-PEG decreased intestinal tissue distribution. This contradicts the current theory that increasing PEGylation increases intestinal epithelial delivery. We believe that increased PEGylation reduces the exposure of positive charges at the surface due to its shielding properties. This reduces the dual nature of the particles, as shown in Figure 6 (Particle 5), Figure 7 (Particle 6), Figure 8 (Particle 7), Figure 9 (Particle 8), and Figure 10 (Particle 9).

[0186] Example 7: Delivery vehicle in vivo testing As shown in Figures 11A, 11B, 12A, 12B, 13A, 13B, 14A, and 14B, the effect of increasing the positive charge was investigated by varying the ratio of MVL5 / DODMA in DSPC / deoxycholate / DMG-PEG with DiI and DiO. The following ratios of MVL5 / DODMA were formed in particles: (0% / 25%), (6.25% / 18.75%), (12.5% / 12.5%), (18.75%, 6.25%), and (25% / 0%). Because DODMA is nearly neutral at neutral pH and monovalent, the negative charge of deoxycholate and the multivalent charge of MVL5 governed the particle behavior. Therefore, increasing the MVL5 content increased the charge.

[0187] The data indicate that a 12.5% / 12.5% ​​MVL5 / DODMA ratio is optimal for intestinal epithelial distribution of particles in vivo. Too much MVL5 resulted in too strong a cationic character, leading to adhesion to negatively charged mucus. Too little MVL5 resulted in negatively charged particles that may have been repelled by mucus or did not interact at all. Furthermore, MVL5 / DODMA / DSPC / cholesterol / DMG-PEG particles were produced but found to fail to reach intestinal epithelial cells. In summary, as shown in Figures 11A, 11B, 12A, 12B, 13A, 13B, 14A, and 14B, a double charge is required to reach intestinal epithelial cells with careful balancing of charges.

[0188] Example 8: Dual Phase Delivery Vehicles vs. Zwitterionic Delivery Vehicles Delivery vehicles were generated as described in Example 1 and tested in vivo as described in Example 7. Zwitterionicity has previously been shown to increase mucus penetration without the presence of PEG. To investigate whether zwitterionicity, rather than biphasicity, is sufficient, particles designed to be single-phase were formulated. To produce single-phase particles, low-phase transition temperature lipids (i.e., containing DOPC or GMO) were substituted for DSPC. Charge was kept the same across particles. Particles that were only in the liquid phase (containing DOPC or GMO instead of DSPC) were found to have significantly reduced or no intestinal epithelial cell reach.

[0189] In summary, the data show that the presence of zwitterions alone is insufficient to allow access to intestinal epithelial cells, as shown in Figures 16A, 16B, 16C, and 16D.

[0190] Example 9: Stability of delivery vehicles with bile salts Using the method described above in Example 1, the following formulations were prepared: MVL5:MC2 (Biofine International LLC, Vancouver, BC, Canada):bile salts:DSPC:DMG-PEG2000:DiI:DiO in the molar ratios of 0.96:0.96:2.592:3.168:0.0768:0.0384:0.0384, where the bile salt component was either ursodiol, deoxycholate, lithocholate, isolithocholate, alloisolithocholate, dehydrolithocholate, or 5β-cholanic acid. Nucleic acid was not incorporated into the lipid nanoparticles. Alternative formulations, such as those provided in Table 4, can also be generated.

[0191] [Table 4]

[0192] The stability of lipid nanoparticles in bile salts was measured up to 10 g / L as previously discussed. FRET signals from DiI and DiO were normalized to untreated. The stability levels of the salt form vehicles are shown in Figure 20. (Appendix 1) A delivery vehicle comprising (i) a cargo and (ii) a lipid nanoparticle, the lipid nanoparticles comprise at least one saturated lipid and a bile salt; A delivery vehicle wherein the at least one saturated lipid is a saturated cationic lipid or the lipid nanoparticle further comprises at least one cationic lipid. (Appendix 2) 2. The delivery vehicle of claim 1, wherein the lipid nanoparticle further comprises at least one unsaturated cationic lipid or unsaturated non-cationic lipid, and optionally, the concentration of the at least one unsaturated cationic lipid or unsaturated non-cationic lipid in the lipid nanoparticle is less than 50 molar % of the total lipid concentration of the lipid nanoparticle. (Appendix 3) 3. The delivery vehicle of claim 1 or 2, wherein the saturated cationic lipid has a phase transition temperature of at least about 37° C. (Appendix 4) 3. The delivery vehicle of claim 1 or 2, wherein the saturated lipid comprises a saturated non-cationic lipid having a phase transition temperature of at least about 37° C. (Appendix 5) 5. The delivery vehicle of any one of claims 1 to 4, wherein the lipid nanoparticles further comprise at least one of a non-cationic lipid, a multivalent cationic lipid, a permanently charged cationic lipid, or any combination thereof. (Appendix 6) 5. The delivery vehicle of claim 4, wherein the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, GL67, or any combination thereof. (Appendix 7) 7. The delivery vehicle of claim 6, wherein the polyvalent cationic lipid comprises MVL5. (Appendix 8) 8. The delivery vehicle of claim 6 or 7, wherein the polyvalent cationic lipid is about 25 mol % or less of the total lipid concentration. (Appendix 9) 6. The delivery vehicle of claim 5, wherein the permanently charged cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol·HCl), or any combination thereof. (Appendix 10) The saturated cationic lipids include 1,2-stearoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-distearoyl-3-dimethylammonium-propane, dimethyldioctadecylammonium, 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammoniumpropane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(2-methyl- ... 10. The delivery vehicle of any one of claims 1 to 9, comprising at least one of (alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-distearoyl-3-dimethylammonium-propane (DSDAP), or any combination thereof. (Appendix 11) The saturated non-cationic lipids include 1,2-dialkyl-sn-glycero-3-phosphocholines, 1,2-dialkyl-sn-glycero-3-phosphoethanolamines, 1,2-diacyl-sn-glycero-3-phosphorylglycerols, 1,2-dialkyl-sn-glycero-3-phosphatidylserines, 1,2-dialkyl-sn-glycero-3-phosphates, monoglycerol alkylates, glyceryl hydroxyalkylates, sorbitan monoalkylates, 1,2-dialkyl-sn- 10. The delivery vehicle of claim 4, comprising at least one of glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof. (Appendix 12) The unsaturated cationic lipid may be dimethyldioctadecylammonium, 1,2-dialkyl-sn-glycero-3-ethylphosphocholine, 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammonium propane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N1-[2-((1S)-1-[(3-amino 12. The delivery vehicle of any one of claims 2-11, comprising at least one of: N,N-dimethylamino]-4-[di(3-amino-propyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, 1,2-dialkyloxy-N,N-dimethylaminopropane, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkylethylphosphocholine, MC3, MC2, MC4, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, N4-cholesteryl-spermine, 7-(4-(dimethylamino)butyl)-7-hydroxytridecane-1,13-diyldioleate (CL1H6), or any combination thereof. (Appendix 13) 13. The delivery vehicle of claim 12, wherein the unsaturated cationic lipid comprises at least MC2 or CL1H6. (Appendix 14) The unsaturated non-cationic lipids include 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-diacyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, 1,2-dialkyl-sn-glycero 14. The delivery vehicle of any one of claims 2 to 13, comprising at least one of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof. (Appendix 15) 2. The delivery vehicle of claim 1, wherein the at least one saturated lipid or cationic lipid is a multivalent cationic lipid. (Appendix 16) 16. The delivery vehicle of claim 15, further comprising a non-cationic lipid. (Appendix 17) 17. The delivery vehicle of claim 16, wherein the polyvalent cationic lipid, the non-cationic lipid, or the polyvalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37°C. (Appendix 18) 18. The delivery vehicle of claim 17, wherein the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, and GL67, or any combination thereof. (Appendix 19) 19. The delivery vehicle of claim 17 or claim 18, wherein the non-cationic lipid comprises a saturated non-cationic lipid. (Appendix 20) The saturated non-cationic lipids include 1,2-dialkyl-sn-glycero-3-phosphocholines, 1,2-dialkyl-sn-glycero-3-phosphoethanolamines, 1,2-diacyl-sn-glycero-3-phosphorylglycerols, 1,2-dialkyl-sn-glycero-3-phosphatidylserines, 1,2-dialkyl-sn-glycero-3-phosphates, monoglycerol alkylates, glyceryl hydroxyalkylates, sorbitan monoalkylates, 1,2-dialkyl-sn- 20. The delivery vehicle of claim 19, comprising at least one of glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof. (Appendix 21) 21. The delivery vehicle of any one of claims 1 to 20, wherein the delivery vehicle is stable in a high bile salt environment compared to an otherwise identical delivery vehicle that does not contain bile salts. (Appendix 22) 22. The delivery vehicle of claim 21, wherein the high bile salt environment comprises a gastrointestinal environment. (Appendix 23) 23. The delivery vehicle of claim 21 or 22, wherein the delivery vehicle (i) exhibits increased stability in a solution containing at least about 5 g / L of bile salts compared to an otherwise identical delivery vehicle that does not contain bile salts, the stability being measured by the relative fluorescence intensity of a fluorescent lipid incorporated into the lipid nanoparticle in a Förster resonance energy transfer (FRET) assay. (Appendix 24) 24. The delivery vehicle of claim 23, wherein the delivery vehicle (i) exhibits increased stability in a solution containing at least about 5 g / L of a mixture of about 50% cholic acid and about 50% deoxycholate compared to an otherwise identical delivery vehicle that does not contain bile salts, wherein the stability is measured by the relative fluorescence intensity of a fluorescent lipid incorporated into the lipid nanoparticle in a Forster resonance energy transfer (FRET) assay. (Appendix 25) N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DODAP), N-(1,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), N-(1-(2,3-dioleyloxy)propyl)N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), diocmdecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethyl Ammonium-propane (DODAP), DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLin-K-C2-DMA, DLin-M-C3-DMA, 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z 25. The delivery vehicle of any one of claims 1-24, comprising at least one of: N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), N-(4-octadeca-9,12-dienyloxyl)propan-1-amine (CLinDMA), MC4, O-alkylethylphosphocholine, didodecyldimethylammonium bromide (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), or any combination thereof. (Appendix 26) 26. The delivery vehicle of any one of claims 1 to 25, comprising at least one of diacylphosphatidylcholine, diacylphosphatidylevanolamine, ceramide, sphingomyelin, cephalin, cerebroside, diacylglycerol, or any combination thereof. (Appendix 27) 27. The delivery vehicle of any one of claims 1 to 26, comprising at least one of phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), or any combination thereof. (Appendix 28) Distearoylphosphatidylcholine (DSPC), phosphatidylcholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (OPEC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylolmyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine (DOPE). 28. The delivery vehicle of any one of claims 1 to 27, comprising at least one of phosphatidylethanolamine 4-(4-maleimidomethyl)coriolehexane-1-carboxylate (DOPE-teal), dipafnitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoevanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPS), 1,2-dielideyl-sn-glycero-3-phosphatidyethanolamine (transDOPE), or any combination thereof. (Appendix 29) 29. The delivery vehicle of claim 28, comprising at least DSPC or DMPC. (Appendix 30) 30. The delivery vehicle of any one of claims 1-29, further comprising a conjugated lipid, wherein the conjugated lipid comprises a lipid conjugated to a stabilizing component. (Appendix 31) 31. The delivery vehicle of claim 30, wherein the stabilizing component comprises a hydrophilic polymer. (Appendix 32) 32. The delivery vehicle of claim 31, wherein the hydrophilic polymer comprises polyethylene glycol, poly(2-alkyl-2-oxazoline), polyvinyl alcohol, or any combination thereof. (Appendix 33) 33. The delivery vehicle of claim 32, wherein the hydrophilic polymer has a molecular weight of about 50 kDa to about 500 kDa. (Appendix 34) 34. The delivery vehicle of claim 32 or 33, wherein the hydrophilic polymer comprises polyethylene glycol (PEG) and the conjugated lipid comprises a pegylated lipid. (Appendix 35) 35. The delivery vehicle of claim 34, wherein the pegylated lipid comprises DSPE-PEG, DSG-PEG, DMG-PEG, or DPPE-PEG. (Appendix 36) 36. The delivery vehicle of claim 35, wherein the pegylated lipid comprises DSPE-PEG or DMG-PEG. (Appendix 37) 37. The delivery vehicle of any one of claims 30 to 36, wherein the concentration of the conjugated lipid is less than 25 mol %. (Appendix 38) 37. The delivery vehicle of any one of claims 30 to 36, wherein the concentration of the conjugated lipid is less than 5 mol%. (Appendix 39) 37. The delivery vehicle of any one of claims 30 to 36, wherein the concentration of the conjugated lipid is about 0.5 mol % to about 20 mol %. (Appendix 40) 40. The delivery vehicle of any one of claims 5 to 39, comprising the non-cationic lipid, wherein the concentration of the non-cationic lipid is about 5 mol % to about 75 mol %. (Appendix 41) 41. The delivery vehicle of any one of claims 1 to 40, wherein the lipid nanoparticles have a net positive or near-neutral charge. (Appendix 42) 42. The delivery vehicle of any one of claims 1-41, further comprising cholesterol. (Appendix 43) 1. A delivery vehicle comprising a cargo and a nanoparticle, wherein the nanoparticle comprises a first portion that is positively charged at a pH of about 5.5 to 8.0 and a second portion that is negatively charged at a pH of about 5.5 to 8.0, the first and second portions being separated such that the positive and negative charges are not dispersed, and the nanoparticle is capable of passing through a mucus barrier to reach an epithelial cell. (Appendix 44) 44. The delivery vehicle of claim 43, wherein reaching an epithelial cell comprises the delivery vehicle approaching within 20 microns of the cell surface, binding to the epithelial cell surface, or being absorbed by the epithelial cell. (Appendix 45) 45. The delivery vehicle of claim 43 or 44, wherein the nanoparticle comprises a lipid, a polymer, or a combination thereof. (Appendix 46) 46. ​​The delivery vehicle of any one of claims 43 to 45, wherein the first portion is included in a first phase and the second portion is included in a second phase, and the first phase and the second phase are physically separated from each other. (Appendix 47) 47. The delivery vehicle of claim 46, wherein the first phase is a liquid. (Appendix 48) 48. The delivery vehicle of claim 47, wherein the second phase is a gel. (Appendix 49) 47. The delivery vehicle of claim 46, wherein the first phase is a gel. (Appendix 50) 50. The delivery vehicle of claim 49, wherein the second phase is a liquid. (Appendix 51) 51. The delivery vehicle of any one of claims 43 to 50, further comprising a stability component. (Appendix 52) 52. The delivery vehicle of claim 51, wherein the stability component is polyethylene glycol (PEG). (Appendix 53) 48. The delivery vehicle of claim 47, wherein the first moiety comprises an unsaturated lipid or a short tail lipid. (Appendix 54) 54. The delivery vehicle of claim 53, wherein the unsaturated lipid comprises a cationic lipid or an ionizable cationic lipid. (Appendix 55) 55. The delivery vehicle of claim 54, wherein the cationic lipid comprises a polyvalent cationic lipid or a monovalent cationic lipid. (Appendix 56) 56. The delivery vehicle of claim 55, wherein the cationic lipid is selected from the group consisting of N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N4-cholesteryl-spermine HCl (GL67), a salt of any of these, and any combination thereof. (Appendix 57) 57. The delivery vehicle of claim 56, wherein one or more lipids in the first phase are PEGylated. (Appendix 58) 58. The delivery vehicle of any one of claims 43 to 57, wherein the first moiety further comprises at least one of 1,2-dioleyloxy-3-(dimethylamino)propane (DODMA), 6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propanoate (MC2), or any combination thereof. (Appendix 59) 59. The delivery vehicle of any one of claims 43 to 58, wherein the second moiety comprises at least one of 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), depot medroxyprogesterone acetate (DMPA), diphenylphosphoryl azide (DPPA), 1,2-distearoyl-sn-glycero-3-sodium phosphatidate (DSPA), 1,2-dipalmitoylphosphatidylglycerol (DPPG), or 2,4-diacetylphloroglucinol (DAPG). (Appendix 60) 60. The delivery vehicle of claim 59, wherein the second moiety further comprises at least one of 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis(dimethylphosphino)ethane (DMPE), 1,2-bis(diphenylphosphino)ethane (DPPE), 1,2-distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine 20:0 PC (DAPC), or 1,2-diradyl-3-phosphatidylethanolamine 20:0 PE (DAPE). (Appendix 61) 59. The delivery vehicle of any one of claims 43 to 58, wherein the second moiety comprises deoxycholate and at least one of 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis(dimethylphosphino)ethane (DMPE), 1,2-bis(diphenylphosphino)ethane (DPPE), 1,2-distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine 20:0 PC (DAPC), or 1,2-diradyl-3-phosphatidylethanolamine 20:0 PE (DAPE). (Appendix 62) 48. The delivery vehicle of claim 47, wherein the first phase has a transition temperature less than 37°C and the second phase has a transition temperature greater than 37°C. (Appendix 63) 50. The delivery vehicle of claim 49, wherein the first phase has a transition temperature greater than 37°C and the second phase has a transition temperature less than 37°C. (Appendix 64) 64. The delivery vehicle of claim 62 or 63, wherein the phase having a transition temperature less than 37° C. comprises DODMA, MVL5, MC2, a cationic lipid, or an ionizable cationic lipid. (Appendix 65) 64. The delivery vehicle of claim 62 or 63, wherein the phase having a transition temperature greater than 37° C. comprises DSPC. (Appendix 66) 66. The delivery vehicle of any one of claims 43 to 65, wherein the ratio of cationic charge in the first portion to anionic charge in the second portion at pH 7.4 is about 0.25 to about 3.0. (Appendix 67) 67. The delivery vehicle of claim 66, wherein the ratio is about 0.75 to about 1.25. (Appendix 68) 68. The delivery vehicle of claim 66 or 67, wherein the first phase comprises MVL5 and an ionizable cationic lipid. (Appendix 69) 69. The delivery vehicle of claim 68, wherein the ionizable cationic lipid is selected from the group consisting of DODMA, MC2, MC3, and KC2. (Appendix 70) 70. The delivery vehicle of claim 69, wherein the ionizable cationic lipid is DODMA or MC2, and the molar percentage ratio of MVL5:ionizable cationic lipid in the delivery vehicle is about 6.25%:18.75%, 12.5%:12.5%, or 18.75%:6.25%. (Appendix 71) 71. The delivery vehicle of claim 70, wherein the ratio of MVL5:ionizable cationic lipid in the delivery vehicle is about 12.5%:12.5%. (Appendix 72) 70. The delivery vehicle of any one of claims 61 to 69, wherein the second phase comprises deoxycholate. (Appendix 73) 73. The delivery vehicle of claim 72, further comprising 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), or a salt thereof. (Appendix 74) 73. The delivery vehicle of claim 72, further comprising DMPE-PEG or a salt thereof. (Appendix 75) 44. The delivery vehicle of claim 43, wherein the first portion comprises a cationic lipid and the second portion comprises an anionic compound. (Appendix 76) 76. The delivery vehicle of claim 75, wherein the cationic lipid is MVL5. (Appendix 77) 77. The delivery vehicle of claim 75 or 76, wherein the anionic compound comprises a bile salt. (Appendix 78) 80. The delivery vehicle of any one of claims 1 to 32 or 77, wherein the bile salt is selected from the group consisting of cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, isolithocholic acid, isolithocholate, lithocholic acid, and lithocholate. (Appendix 79) 79. The delivery vehicle of claim 78, wherein the bile salt is selected from the group consisting of lithocholate, deoxycholate, and isolithocholate. (Appendix 80) 79. The delivery vehicle of claim 78, wherein the bile salt is deoxycholate. (Appendix 81) 79. The delivery vehicle of claim 78, wherein the bile salt is isolithocholate. (Appendix 82) 82. The delivery vehicle of any one of claims 1 to 42 or 78 to 81, wherein the bile salt is at a concentration of about 10 mol % to about 80 mol %. (Appendix 83) 83. The delivery vehicle of any one of claims 1 to 82, wherein the cargo is at least partially surrounded by the lipid nanoparticle. (Appendix 84) 84. The delivery vehicle of any one of claims 1 to 83, wherein the cargo comprises a therapeutic agent. (Appendix 85) 85. The delivery vehicle of any one of claims 1 to 84, wherein the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, a biologic, or any combination thereof. (Appendix 86) 86. The delivery vehicle of claim 85, wherein the cargo is a nucleic acid, and the nucleic acid comprises DNA, modified DNA, RNA, modified RNA, miRNA, siRNA, antisense RNA, or any combination thereof. (Appendix 87) 87. A delivery vehicle according to any one of claims 1 to 86, further comprising a component for cellular internalization. (Appendix 88) 88. The delivery vehicle of claim 87, wherein the component is a peptide, carbohydrate, or ligand. (Appendix 89) 89. The delivery vehicle of any one of claims 1-88, further comprising a cell-penetrating peptide, a ligand, a mucus-penetrating polymer, a mucus-penetrating peptide, a non-mucus-adherent cell-penetrating peptide, or any combination thereof. (Appendix 90) A pharmaceutical composition comprising a delivery vehicle according to any one of claims 1 to 89. (Appendix 91) 91. A method of delivering a cargo to the gastrointestinal tract, comprising administering a delivery vehicle according to any one of claims 1 to 89 or a pharmaceutical composition according to claim 90, wherein the delivery vehicle reaches the gastrointestinal tract and wherein the delivery vehicle protects the cargo from bile salts present in the gastrointestinal tract. (Appendix 92) 92. The method of claim 91, wherein the delivery vehicle readily crosses the mucus barrier. (Appendix 93) 93. The method of claim 91 or 92, wherein the delivery vehicle is capable of reaching epithelial cells in the gastrointestinal tract. (Appendix 94) 94. The method of claim 93, wherein reaching the epithelial cell comprises the delivery vehicle approaching within 20 microns of the cell surface. (Appendix 95) 94. The method of claim 93, wherein the delivery vehicle contacts the surface of an epithelial cell. (Appendix 96) 96. The method of claim 95, wherein the cargo is absorbed by epithelial cells after the delivery vehicle contacts the epithelial cells. (Appendix 97) 97. The method of any one of claims 91 to 96, wherein the delivery vehicle or pharmaceutical composition is administered orally or parenterally to a subject in need thereof. (Appendix 98) 98. The method of any one of claims 91 to 97, wherein the cargo comprises a nucleic acid, a protein, an antibody, a peptide, a small molecule, or a biologic. (Appendix 99) 99. The method of claim 98, wherein the nucleic acid encodes a therapeutic agent and the epithelial cells express the therapeutic agent after absorbing the cargo. (Appendix 100) 99. The method of claim 99, wherein the therapeutic agent is secreted by the epithelial cells.

Claims

1. A delivery vehicle comprising a cargo comprising a nucleic acid and a lipid nanoparticle, wherein the lipid nanoparticle comprises a saturated lipid, a cationic lipid, and a bile salt; the saturated lipid has a phase transition temperature of at least about 37°C; A delivery vehicle wherein said saturated lipid is different from said cationic lipid, or alternatively, said saturated lipid and said cationic lipid are the same.

2. A delivery vehicle as described in claim 1, wherein the bile salt is at a concentration of about 10 mol% to about 80 mol%.

3. 3. The delivery vehicle of claim 1 or 2, wherein the saturated lipid comprises a saturated non-cationic lipid.

4. The saturated noncationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-diacyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, 1,2-dialkyl-sn- 4. The delivery vehicle of claim 3, comprising at least one of glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof.

5. A delivery vehicle described in any one of claims 1 to 4, wherein the lipid nanoparticles further comprise at least one unsaturated cationic lipid or unsaturated non-cationic lipid, and optionally, the concentration of the at least one unsaturated cationic lipid or unsaturated non-cationic lipid in the lipid nanoparticles is less than 50 mol% of the total lipid concentration of the lipid nanoparticles.

6. (a) The unsaturated cationic lipid is selected from the group consisting of 1,2-dialkyl-3-dimethylammonium-propane, 1,2-dialkyl-3-trimethylammonium-propane, 1,2-di-O-alkyl-3-trimethylammonium propane, 1,2-dialkyloxy-3-dimethylaminopropane, N,N-dialkyl-N,N-dimethylammonium, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(alkyloxy)propan-1-aminium, 1,2-dialkyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[alkyl]-benzamide, 1,2-dialkyloxy-N,N-dimethyl and / or at least one of methylaminopropane, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, O-alkylethylphosphocholine, MC3, 1,2-dioleyloxy-3-(dimethylamino)propane (DODMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propanoate (MC2), MC4, 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol, N4-cholesteryl-spermine, 7-(4-(dimethylamino)butyl)-7-hydroxytridecane-1,13-diyldioleate (CL1H6), or any combination thereof, optionally wherein the unsaturated cationic lipid comprises at least MC2 or CL1H6; and / or (b) The unsaturated non-cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-diacyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan monoalkylate, 1,2-dialkyl-sn-glycero-3-phosphoethanoate, 6. The delivery vehicle of claim 5, comprising at least one of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof.

7. A delivery vehicle described in any one of claims 1 to 6, wherein the delivery vehicle has a size of 20 to 200 nm.

8. The lipid nanoparticles further comprise at least one of a non-cationic lipid, a multivalent cationic lipid, a permanently charged cationic lipid, or any combination thereof, and optionally: (a) the concentration of the non-cationic lipid is from about 5 mol % to about 75 mol %; (b) the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, GL67, or any combination thereof, and optionally the polyvalent cationic lipid is less than or equal to about 25 mol % of the total lipid concentration; (c) the delivery vehicle of any one of claims 1 to 7, wherein the permanently charged cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol.HCl), or any combination thereof.

9. The saturated lipid or the cationic lipid is a polyvalent cationic lipid, Optionally, the delivery vehicle further comprises a non-cationic lipid, and optionally the multivalent cationic lipid, the non-cationic lipid, or the multivalent cationic lipid and the non-cationic lipid have a phase transition temperature of at least about 37° C., and further optionally, (i) the polyvalent cationic lipid comprises at least one of MVL5, TMVLBG2, TMVLG3, TMVLBG1, GL67, or any combination thereof; and / or (ii) the non-cationic lipid comprises a saturated non-cationic lipid, and optionally the saturated non-cationic lipid is selected from the group consisting of 1,2-dialkyl-sn-glycero-3-phosphocholine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphorylglycerol, 1,2-dialkyl-sn-glycero-3-phosphatidylserine, 1,2-dialkyl-sn-glycero-3-phosphate, monoglycerol alkylate, glyceryl hydroxyalkylate, sorbitan mono ...

10. The delivery vehicle of claim 1, comprising at least one of 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N-methyl, 1,2-dialkyl-sn-glycero-3-phosphomethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanol, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl, 1,2-dialkyl-sn-glycero-3-phosphopropanol, 1,2-dialkyl-sn-glycero-3-phosphobutanol, or any combination thereof.

10. A delivery vehicle described in any one of claims 1 to 9, wherein the delivery vehicle is stable in a high bile salt environment compared to an otherwise identical delivery vehicle that does not contain bile salts, and optionally the high bile salt environment includes a gastrointestinal environment.

11. The delivery vehicle may be N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), diocmdecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), DMDMA, 1,2-dilinoleyl 11. The delivery vehicle of any one of claims 1 to 10, comprising at least one of: oxy-N,N-dimethylaminopropane (DLinDMA), 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLin-K-C2-DMA, DLin-M-C3-DMA, 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienyloxyl]propan-1-amine (CLinDMA), MC4, O-alkylethylphosphocholine, didodecyldimethylammonium bromide (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), or any combination thereof.

12. A delivery vehicle described in any one of claims 1 to 11, wherein the delivery vehicle comprises at least one of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cerebroside, diacylglycerol, or any combination thereof.

13. The delivery vehicle is selected from the group consisting of distearoylphosphatidylcholine (DSPC), phosphatidylcholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (OPEC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylolmyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine (DOPE).

13. The delivery vehicle of any one of claims 1 to 12, comprising at least one of dimethicone 4-(4-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-teal), diphosphatidyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoevanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPS), 1,2-dielideyl-sn-glycero-3-phosphatidyethanolamine (transDOPE), or any combination thereof, optionally wherein the delivery vehicle comprises at least DSPC or DMPC.

14. The delivery vehicle further comprises a conjugated lipid, optionally at a concentration of less than 25 mol%, or less than 5 mol%, or from about 0.5 mol% to about 20 mol%, wherein the conjugated lipid comprises a lipid conjugated to a stabilizing component; Optionally, the stabilizing component comprises a hydrophilic polymer; Optionally, the hydrophilic polymer comprises polyethylene glycol (PEG), poly(2-alkyl-2-oxazoline), polyvinyl alcohol, or any combination thereof; Optionally, the hydrophilic polymer comprises PEG and the conjugated lipid comprises a PEGylated lipid; 14. The delivery vehicle of any one of claims 1 to 13, wherein optionally the PEGylated lipid comprises DSPE-PEG, DSG-PEG, DMG-PEG, or DPPE-PEG. (a) the lipid nanoparticles have a positive or near-neutral net charge, and / or (b) the delivery vehicle of any one of claims 1 to 14, further comprising cholesterol.

16. The bile salt is selected from the group consisting of cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, isolithocholic acid, isolithocholate, lithocholic acid, and lithocholate; 16. The delivery vehicle of any one of claims 1 to 15, wherein optionally the bile salt is selected from the group consisting of lithocholate, deoxycholate, and isolithocholate.

17. (a) the delivery vehicle further comprises a component for cellular internalization, optionally the component being a peptide, carbohydrate, or ligand; and / or (b) the delivery vehicle of any one of claims 1 to 16, wherein the delivery vehicle further comprises a cell-penetrating peptide, a ligand, a mucus-penetrating polymer, a mucus-penetrating peptide, a non-mucus-adherent cell-penetrating peptide, or any combination thereof.

18. The delivery vehicle of claim 1 or 2, wherein the saturated lipid is different from the cationic lipid.

19. A delivery vehicle described in claim 1 or 2, wherein the saturated lipid and the cationic lipid are the same.

20. A pharmaceutical composition comprising a delivery vehicle described in any one of claims 1 to 19.

21. A composition comprising the delivery vehicle of any one of claims 1 to 19 for use in a method of treating a subject in need thereof, the method comprising administering said composition to said subject; the delivery vehicle reaches the digestive tract, the delivery vehicle protects the cargo from bile salts present in the digestive tract; Optionally, the delivery vehicle is administered orally or rectally to a subject in need thereof.

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