Lipid nanoemulsion formulations for contrast agents and methods of using the same

Abstract

Contrast agents for blood pool and functional liver imaging are described. For example, the contrast agents can be lipid nanoemulsions that include nanoparticles containing an oil component, a surfactant component, and one or more imaging moieties. The surfactant and oil components can be derived from biocompatible molecules, such as oleic acid. The contrast agents can provide efficient contrast for x-ray computed tomography (CT) with minimal toxicity and have higher iodine payloads compared to other liposomal and micellular agents. Alternatively or additionally, the agents can be loaded with therapeutic agents, e.g., for treatment of liver-based primary and metastatic tumors.

Description

[0001] DESCRIPTION

[0002] LIPID NANOEMULSION FORMULATIONS FOR CONTRAST AGENTS AND METHODS OF USING THE SAME

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 626,884, filed January 30, 2024; the disclosure of which is incorporated herein by reference in its entirety.

[0005] TECHNICAL FIELD

[0006] The presently disclosed subject matter relates generally to contrast agent compositions and methods for using the compositions to generate images. In some embodiments, the presently disclosed subject matter relates to contrast agent compositions and methods for using the contrast agent compositions in imaging applications.

[0007] BACKGROUND

[0008] The minimally invasive treatment of lesions in liver cancer is hampered by the lack of visualization of these lesions on x-ray-based modalities without the use of iodinated contrast. Additional compositions and methods that provide for visualization of these lesions remain an ongoing need in the art.

[0009] SUMMARY

[0010] This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features. In some embodiments, the presently disclosed subject matter provides a nanoemulsion formulation comprising: (a) a surfactant component and an oil component, wherein the surfactant component and the oil component are present at a surfactant-to-oil ratio (SOR) of about 20 wt.% to about 50 wt.%; and (b) an effective amount of one or more active moieties. In some embodiments, the SOR is about 20 wt.% to about 30 wt.%.

[0011] In some embodiments, the one or more active moieties comprise a detectable moiety, a therapeutic moiety, or a combination thereof. In some embodiments, the surfactant component and / or the oil component comprise a biocompatible molecule and / or a biocompatible molecule derivatized with an active moiety. In some embodiments, the biocompatible molecule is selected from the group comprising a PEGylated molecule, oleic acid, oleylamine, ricinoleic acid, lauric acid, myristic acid, palmitic acid, and stearic acid, optionally wherein the PEGylated molecule is selected from polyethylene glycol 12-hydroxy stearate, and polyethylene glycol-substituted castor oil.

[0012] In some embodiments, the surfactant component and / or the oil component comprises a molecule that is modified covalently or non-covalently with one or more of the one or more active moieties. In some embodiments, the one or more active moieties comprise one or more detectable moieties, wherein the one or more detectable moieties comprise a detectable moiety selected from the group comprising a halogen atom-containing moiety, a fluorescent probe, Fe3O4, gadolinium, a64Cu radionucleotide, and a18F radionucleotide. In some embodiments, the one or more detectable moieties comprise an iodo-containing moiety. In some embodiments, the iodocontaining moiety comprises a triiodo-substituted phenyl group.

[0013] In some embodiments, the formulation comprises a nanoparticle having a diameter of about 30 nm to about 150 nm, wherein the nanoparticle comprises (a) and (b).

[0014] In some embodiments, the oil component comprises an iodinated oil having the structure A-X-L, where A is an iodo-substituted phenyl group, optionally a triiodo-substituted phenyl group; X is selected from -C(=O)-NH-, - C(=O)-O-, and -O-C(=O)-; and L is a saturated or unsaturated aliphatic group, wherein the saturated or unsaturated aliphatic group comprises at least 11 carbon atoms, and wherein the saturated or unsaturated aliphatic group is optionally substituted by one or more substituent having the structure -0- C(=0)-A. In some embodiments, the surfactant component comprises an iodinated surfactant having the structure [L’]m-P’, wherein L’ has a structure of the formula A’-C(=O)-X-L”-C(=O)-O-, wherein A’ is an iodo-substituted phenyl group, optionally a triiodo-substituted phenyl group, and L” is a saturated or unsaturated aliphatic chain; m is an integer that is 1 or more, optionally wherein m is 1 , 2, or 3; and P’ is a moiety comprising one or more polyethylene glycol (PEG) chains, wherein each of the one or more PEG chains is covalently attached to one of the m L’ groups.

[0015] In some embodiments, the formulation is for use as a contrast agent. In some embodiments, the formulation is for use as a contrast agent in computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound (US), and / or fluorescence imaging methods.

[0016] In some embodiments, the presently disclosed subject matter provides a method of imaging a cell and / or tissue in a subject; the method comprising administering to the subject an effective amount of a formulation comprising: (a) a surfactant component and an oil component, wherein the surfactant component and the oil component are present at a surfactant-to-oil ratio (SOR) of about 20 wt.% to about 50 wt.%, and (b) an effective amount of one or more active moieties; and detecting the formulation in the subject. In some embodiments, the subject is suffering from cancer, optionally wherein the cancer is liver cancer. In some embodiments, the imaging comprises imaging liver tissue or a blood pool in the subject.

[0017] In some embodiments, the presently disclosed subject matter provides a method of treating a disease in a subject in need of treatment, the method comprising administering to the subject an effective amount of a formulation comprising: (a) a surfactant component and an oil component, wherein the surfactant component and the oil component are present at a surfactant-to-oil ratio (SOR) of about 20 wt.% to about 50 wt.%; and (b) an effective amount of one or more active moieties. In some embodiments, the method further comprises imaging a cell or tissue in the subject, optionally wherein the imaging comprises imaging liver tissue or a blood pool in the subject. In some embodiments, the subject is suffering from cancer, optionally wherein the cancer is liver cancer.

[0018] It is thus an object of the presently disclosed subject matter to provide contrast agent compositions and related methods of imaging and treating a subject. An object of the presently disclosed subject matter having been stated herein above, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying Figures and Examples as best described herein below.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 is a schematic diagram showing an overview of lipid nanoemulsions (LNEs) including their characteristics and uses in bioimaging and drug delivery nanomedicine.

[0021] Figure 2 is a schematic diagram showing an overview of lipid nanoemulsions (LNEs) and their use as contrast agents for different imaging modalities including fluorescence, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound (US), and x-ray and computed tomography (CT) imaging.

[0022] Figure 3 is a schematic diagram showing an exemplary representation of the preparation of an iodinated lipid nanoemulsion (ILNE) contrast agent via two successive steps. On the left, frame 1 shows a chemical reaction for iodine labeling an oil compound with a carboxylic acid moiety, while frame 2, in the middle, shows a spontaneous emulsification process involving the iodinated oil prepared in frame 1 , a polyethylene glycol (PEG)ylated surfactant, and water, which provides a PEGylated nanodroplet emulsion, shown in frame 3 on the right, having an average diameter in the 30-150 nanometer (nm) size range depending on the ratio between iodinated oil and surfactant.

[0023] Figure 4 is a series of dynamic light scattering (DLS) histograms (intensity as a percentage (%) versus size as expressed as the diameter in nanometers (d, nm)) showing the size distribution of four exemplary lipid nanoemulsions (LNEs) of the presently disclosed subject matter, LNE1 (left), LNE2 (second from left), LNE3 (second from right), and LNE4 (right). Figures 5A-5D are a series of graphs showing particle size stability, polydispersity, particle concentration, and particle surface charge the exemplary liquid nanoemulsions (LNEs) described in Figure 4. Figure 5A shows the particle size (in nanometers (nm)) of LNE1 , LNE2, LNE3, and LNE4 measured by dynamic light scattering (DLS) on the day of synthesis (Day 1 ), one week after synthesis (Day 7), and one month after synthesis (Day 30), as well as after freeze drying (Freeze-dried-Day 1 ) and after freeze drying and storage for one month (Freeze-dried-Day30). Figure 5B is a graph showing the polydispersity index (PDI) of the same LNEs monitored via DLS over the same time periods and treatments. Figure 5C is a graph showing the particle concentration per one milliliter (mL) of LNE1 , LNE2, LNE3, and LNE4 as detected by nanoparticle tracking analysis (NTA). Figure 5D is a graph showing the particle surface charge (zeta potential) of LNE1 , LNE2, LNE3, and LNE4.

[0024] Figure 6 is a pair of cryo-transmission electron microscope (Cryo-TEM) images of exemplary lipid nanoemulsions (LNEs) of the presently disclosed subject matter one day (left) and 6 months (right) post-preparation. The scale bar in the lower right corner of each image represents 100 nanometers (nm).

[0025] Figures 7A-7D are a series of graphs showing the stability of the lipid nanoemulsions (LNEs) described in Figure 4 in human plasma (HP) and fetal bovine serum (FBS). Figures 7A and 7B are graphs showing mean particle size (in nanometers (nm)) and polydispersity index (PDI), respectively, of 10% and 20% LNE3 and LNE4 incubated with HP for two days. Figures 7C and 7D are graphs showing mean particle size (in nm) and PDI, respectively, of 10% and 20% LNE3 and LNE4 incubated with FBS for two days. Size (Figures 7A and 7C) and PDI (Figures 7B and 7D) were measured by dynamic light scattering (DLS).

[0026] Figures 8A and 8B show the X-ray attenuation and cytotoxicity of exemplary lipid nanoemulsions (LNEs). Figure 8A is a graph showing a X-ray attenuation calibration curve (in Hounsfield units (HU)) versus iodine concentration (milligrams iodine per milliliter (mg l / mL)) of iobitridol used for analysis of the phantom scan (inset, lower right) of exemplary LNEs (LNE 1 - LNE 4 from Figure 4 (which correspond to NE1 -NE4 in the phantom scan)) and reference samples with known iodine concentration (12.5, 25, 50, 100, 150, or 200 mg 1 / mL). The table in the upper left summarizes the iodine concentrations determined for the LNE samples. Figure 8B is a graph showing the results of a cytotoxicity assay of immune cells including RAW macrophages and IC21 cell line after treatment with LNE3.

[0027] Figure 9 is a graph showing ultraviolet-visible (UV-vis) absorbance measurements of iodinated lipid nanoemulsions (LNEs), i.e., LNEs 1 -4 as described in Figure 4, showing the peaks of the pegylated surfactant (pegylated castor oil) and iodinated oil (triiodophenol oleate (TIPhO)) at maximum absorbance (Amax) of about 230 nanometers (nm) and about 244 nm, respectively.

[0028] Figures 10A-10I are a series of graphs showing batch-to-batch variation in an exemplary lipid nanoemulsion (LNE). Figures 10A-10G show dynamic light scattering (DLS) histograms (intensity (%) versus diameter size in nanometers (d, nm)) of different batches of LNE3 formulations. Figure 10H is a graph showing the DLS histogram of a 35 ml batch of LNE3. Figure 10I is a pair of graphs showing the average size (in nm) and polydispersity of all batches of LNE3.

[0029] Figure 11 A-11 P are a series of graphs showing the shelf-life stability of both two lipid nanoemulsion (LNE) formulations, i.e., LNE3 and LNE4, at room temperature over one-year post-preparation. Figures 11 A-11 F show dynamic light scattering (DLS) histograms (intensity (%) versus diameter size in nanometers (d, nm)) of LNE3 after one day, one month, three months, six months, nine months and one year, respectively. Figures 11 G-11 L show DLS histograms (intensity (%) versus diameter size in nanometers (d, nm)) of LNE4 after one day, one month, three months, six months, nine months and one year, respectively. Figure 11 M is a graph comparing the size (in nanometers) of LNE3 over time, while Figure 11 N is a graph comparing the polydispersity (PDI) of LNE3 over time. Figure 110 is a graph comparing the size (in nanometers) of LNE4 over time, while Figure 11 P is a graph comparing the polydispersity (PDI) of LNE4 over time.

[0030] Figure 12 is a series of fluorescence microscopy images showing the cellular uptake of a 1 ,1 ’-dioctadecyl-3, 3, 3’, 3’ -tetramethylindocarbocyanine perchlorate (Dil) dye-loaded lipid nanoemulsions (i.e., Dil dye-loaded LNE3) in RAW 264.7 macrophage cells. Two concentrations of LNE3 (266 micrograms (pg) and 533 pg) were incubated in the cells, and the internalization of particles was observed using an optical fluorescence microscope at 4-hour (4h) and 24- hour (24h) time points. Images in each column (top to bottom) show: Hoechst dye for staining cell nucleus, Dil dye-labeled LNE3, and overlapping. The scale bar in the lower right corner of the image in the upper right indicates 150 micrometers (pm).

[0031] Figures 13A-13C are in vivo micro-computed tomography (CT) imaging images (representative coronal and transverse slices of heart and liver) in C57BL / 6 mouse animal models at different time points after intravenous (i.v.) injection of a lipid nanoemulsion (LNE) contrast agent of the presently disclosed subject matter, LNE3 as described in Figure 4. Mice identification numbers (IDs) are M1-M3 (n=3, biologically independent samples). Figure 13A shows the coronal view of mice pre- and post-injection at different time points (5 minutes, 1 , 2, 4, 24, and 72 hours). The top arrow in each image points to the heart, and the lower arrow points to the liver. Figure 13B shows a transverse view of a section of the heart. Figure 13C shows a transverse view of a section of the liver.

[0032] Figure 14 is a series of images showing hematoxylin and eosin (H&E) staining of staining histology of organs (heart, kidney, liver, lung, and spleen) harvested three days post-injection of the lipid nanoemulsion LNE3 at 2 milliliters per kilogram (mL / kg; equivalent to 800 milligrams (mg) ILNE3 / kg, n=3) compared with saline-injected mice (n=2). M1-M3 are individual mouse identification numbers.

[0033] Figure 15 is a composite of in vivo micro-computed tomography (CT) imaging images (representative transverse slices of heart, liver, and kidneys) in a porcine animal model before injection of an iodinated lipid nanoemulsion (pre-contrast, left column); arterial phase after intravenous (i.v.) injection of iohexol (middle column); and 1 hour (hr) after i.v. injection of an iodinated LNE (LNE3 CA, right column).

[0034] Figure 16 is a three-dimensional (3D) image of a porcine subject 1 -hour post-injection of an iodinated lipid nanoemulsion contrast agent (i.e. , LNE3) at a dose of 300 milligrams iodine per kilogram (mg l / kg). The image shows clear delineation of the heart chambers, arterial and venous vasculature, and hepatic vessels. Diffuse liver uptake is also observed at this time point.

[0035] Figure 17 is a schematic diagram showing conjugation reactions of exemplary molecules with triiodobenzene-based reagents for preparation of computed tomography (CT) contrast agents.

[0036] DETAILED DESCRIPTION

[0037] Currently, no clinical approval exists for blood pool CT contrast agents. All approved clinical products employ solutions of small hydrophilic iodinated molecules. Nanoparticle (NP)-based contrast agents present potential alternative candidates. However, a challenge with nanoparticle suspensions arises from the substantial amounts of excipients typically needed in the formulations, coupled with their lower stability, making it difficult to achieve widespread acceptance. As a result, none of the previously developed NPs based on polymers, micelles, liposomes, dendrimers, etc., have been successfully translated into clinical applications.

[0038] CT contrast agents that can provide for blood pool and functional liver imaging can greatly enhance the ability to follow and treat liver based primary and metastatic tumors. Blood pool agents also have functional advantages for different clinical applications. Disclosed herein, in some embodiments, are blood pool iodine-based lipid nanoemulsions (LNEs) with efficient contrast and minimal toxicity for x-ray CT imaging. In some embodiments, the presently disclosed subject matter uses biocompatible molecules, such as oleic acid, to be modified with triiodo-substituted phenyl moiety via an esterification reaction, providing high loading iodine content. This approach has an advantage of incorporating high iodine payloads as compared to other liposomal and micellar approaches, and significantly higher than commercially available pre-clinical compounds. Furthermore, this approach can limit renal toxicity yet have a straightforward scale-up in production volumes.

[0039] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0041] Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0042] I. DEFINITIONS

[0043] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0044] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0045] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0046] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0047] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March’s Advanced Organic Chemistry, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0048] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to "a surfactant" includes a plurality of such surfactants, and so forth.

[0049] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0050] As used herein, the term “about,” when referring to a value or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0051] The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

[0052] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0053] As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0054] With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0055] As used herein, the term “and / or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0056] As used herein the term “alkyl” can refer to linear ( / .e., "straight-chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated ( / .e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. In some embodiments, the term "lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms ( / .e., a C1-8 alkyl), e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In some embodiments, "higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C1-8 branched-chain alkyls. Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. In some embodiments, there can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

[0057] Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0058] The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

[0059] The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0060] Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0061] In some embodiments, the terms “phenyl group” and “benzene”, e.g., when describing iodo-substituted moieties, can be used interchangeable to refer to C6 aromatic groups, e.g., a moiety with the formula -C6H2I3.

[0062] The term “amino” refers to the group -N(R)2 wherein each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms “aminoalkyl” and “alkylamino” can refer to the group -N(R)2wherein each R is H, alkyl or substituted alkyl, and wherein at least one R is alkyl or substituted alkyl. “Arylamine” and “aminoaryl” refer to the group -N(R)2wherein each R is H, aryl, or substituted aryl, and wherein at least one R is aryl or substituted aryl, e.g., aniline (i.e. , -NHCeHs).

[0063] The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups.

[0064] The terms "hydroxyl" and “hydroxy” refer to the -OH group.

[0065] The terms “carboxylate” and “carboxylic acid” can refer to the groups - C(=O)O_and -C(=O)OH, respectively. The term “carboxyl” can also refer to the -C(=O)OH group. In some embodiments, “carboxylate” or “carboxyl” can refer to either the -C(=O)O’ or -C(=O)OH group.

[0066] The terms “bonding” or “bonded” and variations thereof can refer to either covalent or non-covalent bonding. In some cases, the term “bonding” refers to bonding via a coordinate bond. The term “conjugation” can refer to a bonding process, as well, such as the formation of a covalent linkage or a coordinate bond.

[0067] The terms “polymer” and “polymeric” refer to chemical structures that have repeating units (i.e., multiple copies of a given chemical substructure). Polymers can be formed from polymerizable monomers. A polymerizable monomer is a molecule that comprises one or more moieties that can react to form bonds (e.g., covalent or coordination bonds) with moieties on other molecules of polymerizable monomer. In some embodiments, each polymerizable monomer molecule can bond to two or more other molecules / moieties. In some cases, a polymerizable monomer will bond to only one other molecule, forming a terminus of the polymeric material.

[0068] Polymers can be organic, or inorganic, or a combination thereof. As used herein, the term “inorganic” refers to a compound or composition that contains at least some atoms other than carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, or one of the halides. Thus, for example, an inorganic compound or composition can contain one or more silicon atoms and / or one or more metal atoms.

[0069] As used herein “organic polymers” are those that do not include silica or metal atoms in their repeating units. Exemplary organic polymers include polyvinylpyrrolidone (PVO), polyesters, polyamides, polyethers, polydienes, and the like. Some organic polymers contain biodegradable linkages, such as esters or amides, such that they can degrade overtime under biological conditions.

[0070] The term “hydrophilic polymer” as used herein generally refers to hydrophilic organic polymers, such as but not limited to, polyvinylpyrrolidone (PVP), polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxy-propyloxazoline, polyhydroxypropylmethacrylamide, polymethy- acrylamide, polydimethylacrylamide, polyhydroxylpropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethyl-cellulose, polyethylene-imine (PEI), polyethyleneglycol (i.e., PEG) or another hydrophilic poly(alkyleneoxide), polyglycerine, and polyaspartamide.

[0071] The term “hydrophilic” refers to the ability of a molecule or chemical species to interact with water. Thus, hydrophilic polymers are typically polar or have groups that can hydrogen bond to water.

[0072] The term “hydrophobic” refers to a molecule or chemical species that does not interact with water and / or that can interact with fats and lipids. Such molecules or species are typically nonpolar. The term “amphiphilic” refers to molecules that have both hydrophilic and hydrophobic groups.

[0073] The terms “nanoscale particle,” nanomaterial,” and “nanoparticle” refer to a structure having at least one region with a dimension (e.g., length, width, diameter, etc.) of less than about 1 ,000 nm. In some embodiments, the dimension is smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm or even less than about 20 nm). In some embodiments, the dimension is between about 20 nm and about 250 nm (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).

[0074] In some embodiments, the nanoparticle is approximately spherical. When the nanoparticle is approximately spherical, the characteristic dimension can correspond to the diameter of the sphere. In addition to spherical shapes, the nanomaterial can be disc-shaped, plate-shaped (e.g., hexagonally platelike), oblong, polyhedral, rod-shaped, cubic, or irregularly-shaped.

[0075] The nanoparticle can comprise a core region (i.e., the space between the outer dimensions of the particle) and an outer surface (i.e., the surface that defines the outer dimensions of the particle). In some embodiments, the nanoparticle can have one or more coating layers surrounding or partially surrounding the nanoparticle core. Thus, for example, a spherical nanoparticle can have one or more concentric coating layers, each successive layer being dispersed over the outer surface of a smaller layer closer to the center of the particle.

[0076] The term “fluorophore” as used herein refers to a compound that can reemit light upon excitation by light. Typically, fluorophores comprise one or more aryl groups. Exemplary fluorophores include, but are not limited to xanthere derivatives, e.g., fluorescein, carboxyfluorescein, rhodamine, eosin, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine), squaraine derivatives, squaraine rotaxane derivatives, naphthalene derivatives (e.g., dansyl derivatives), coumarin and its derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives (e.g., acridine yellow, proflavine, acridine orange), arylmethine deriviatives (e.g., crystal violet, malachite green), tetrapyrrole derivatives, and dipyrrolmethene derivatives.

[0077] The term “cancer” as used herein refers to diseases caused by uncontrolled cell division and / or the ability of cells to metastasize, or to establish new growth in additional sites. The terms “malignant”, “malignancy”, “neoplasm”, “tumor,” “cancer” and variations thereof refer to cancerous cells or groups of cancerous cells.

[0078] Particular types of cancer include, but are not limited to, skin cancers (e.g., melanoma), connective tissue cancers (e.g., sarcomas), adipose cancers, breast cancers, head and neck cancers, lung cancers (e.g., mesothelioma), stomach cancers, pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers, anogenital cancers (e.g., testicular cancer), kidney cancers, bladder cancers, colon cancers, prostate cancers, central nervous system (CNS) cancers, retinal cancer, blood, neuroblastomas, multiple myeloma, and lymphoid cancers (e.g., Hodgkin’s and non-Hodgkin’s lymphomas).

[0079] The term “metastatic cancer” refers to cancer that has spread from its initial site (i.e. , the primary site) in a patient’s body.

[0080] The terms “anticancer drug”, “chemotherapeutic”, and “anticancer prodrug” refer to drugs (i.e., chemical compounds) or prodrugs known to, or suspected of being able to treat a cancer (i.e., to kill cancer cells, prohibit proliferation of cancer cells, or treat a symptom related to cancer). In some embodiments, the term “chemotherapeutic” as used herein refers to a non-PS molecule that is used to treat cancer and / or that has cytotoxic ability. Such more traditional or conventional chemotherapeutic agents can be described by mechanism of action or by chemical compound class, and can include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilones, histone deacetylase inhibitors (e.g., vorinostat), inhibitors of topoisomerase I or II (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or precursors thereof (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum based agents (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinka alkaloids (e.g., vinblastine).

[0081] II. GENERAL CONSIDERATIONS

[0082] The liver represents a common site for both primary and metastatic cancers. Hepatocellular carcinoma (HCC), is the second leading cause of cancer related mortality worldwide, the fifth most common cancer in men and the seventh most common cancer in women.1The liver is also a common site of metastases. Liver metastases occur in about 25% of patients with colorectal cancer, itself the third most common cancer.2Detecting malignant lesions in the liver using CT typically involves a three-phase CT scan in which images are acquired in the pre-contrast, arterial and portal venous phases. In MRI, a multiphase acquisition is also used, but often with a “functional” agent, such as gadoxetate disodium (sold under the tradename EOVIST® (Bayer Intellectual Property GmbH, Monheim am Rhein, Germany)), which is progressively taken up by the hepatocytes as a clearance mechanism. As a result, liver tumors typically stay hypo-enhancing in this hepatobiliary phase of imaging, which provides for functional characterization within the liver. This functional imaging significantly enhances lesion conspicuity and gives insight into liver function in patients with chronic liver diseases, themselves a risk factor for HCC. Imaging also helps define tumor morphology and potential for resection. Surgical resection remains standard of care, but both stereotactic body radiotherapy (SBRT) and embolization with chemotherapy or radio-isotopes offer minimally invasive approaches with tolerable toxicity profiles and acceptable outcomes.

[0083] However, minimally invasive treatment of liver lesions is hampered by the lack of visualization of these lesions on x-ray-based modalities without the use of iodinated contrast. For example, while cone beam CT within the angiography suite is often utilized to provide a three-dimensional image of the underlying anatomy during the transarterial procedure, long acquisition times makes it hard to perform multiphase imaging. Thus, identifying lesions at the time of procedure typically involves selective catheterization of the hepatic arteries to “localize” the lesion in the hopes of identifying all the feeding vessels. Similarly for SBRT, the lesions are not seen on radiographic views, and one must register the non-contrast pre-treatment CT with the prior MRI to localize the lesion.3To compensate for registration errors, the radiotherapy target is usually increased in size to compensate for the possibility of mis-registration. Thus, an approach that enables visualization of the lesion on a non-contrast CT, either cone-beam or immediately pre-radiotherapy, would be immensely powerful, offering the potential to improve lesion delineation, reduce planning and procedure times, and ultimately provide patients with minimally invasive treatment approaches that maximize outcomes.

[0084] In accordance with some embodiments of the presently disclosed subject matter, described herein is the successful development of a highly stable, scalable, safe, and injectable blood pool CT contrast agent suitable for vascular and hepatic imaging, with dose volumes comparable to clinically relevant hydrophilic CT contrast agents (~2 ml / kg). The presently disclosed subject matter provides the following representative aspects of CT contrast agents: (1 ) manufacturing long shelf-life stable blood pool X-ray CT contrast agents, ensuring extended shelf life for enhanced stability; (2) low-priced, safe, injectable, and efficient CT Agents, providing cost-effective, secure, injectable, and efficient solutions; (3) high reproducibility and scalability, ensuring high batch-to-batch consistency and scalability for widespread production; (4) matching clinical dose volumes of standard CT contrast agents, aligning with the established dose volumes used in clinical settings; (5) clinical applications, CT Imaging of vasculature (heart angiography) and liver, enabling precise imaging of both vasculature, particularly in heart angiography, and the liver; and (6) selective targeting of hepatocytes for functional and anatomical imaging, facilitating targeted imaging for functional and anatomical purposes, supporting the staging and monitoring of fatty liver disease, quantifying liver tumor burdens for improved diagnostic accuracy. In short, the presently disclosed subject matter provides in some embodiments blood pool contrast agents that offer a range of benefits for clinical applications in CT imaging.

[0085] III. REPRESENTATIVE COMPOSITIONS AND METHODS

[0086] In accordance with aspects of the presently disclosed subject matter, lipid nanoemulsions (LNEs) are utilized as soft nanoparticles with maximized contrast and minimal toxicity for clinical CT and other forms of imaging and / or therapy. Some production methods for lipid nanoemulsions involve significant energy depositions and strong shear forces that can overcome interfacial tension during the formation of finely dispersed droplets. Although methods that do not involve high shear forces exist, they are not typically applicable to industrial production because they generally use elevated surfactant concentrations and involve complex preparation procedures.

[0087] In contrast, the presently disclosed LNEs can be produced via a spontaneous emulsification process that does not involve high pressure or energy, resulting in saving time, money, and risk of damaging the quality of the materials. It also makes it possible to scale up from research and development (R&D) to pilot scale to production scale with repeatable, linearly scalable results. Spontaneous emulsification has advantages including efficient size reduction with uniform size distribution. This plays a role in producing a stable nanoemulsion with better bioavailability and longer shelf-life. Additional benefits can include the ability to sterile filter (with little clog in) and / or produce transparent emulsions.

[0088] The chemical structure and composition ratio between the surfactant component and oil component (e.g., iodinated oil component) (i.e., surfactant- to-oil ratio (SOR)) in the nanoformulations can impact the size, surface charge, blood circulation time, active agent content (e.g., iodine content for contrast enhancement), and biodistribution (nanoparticle uptake). A smaller size can lead to a more rapid uptake by the kidneys, potentially causing kidney dysfunction, as in clinical CT agents. Conversely, nanosized formulations (e.g., about 20 nm to about 200 nm) tend to preferentially accumulate in the liver due to the enhanced permeability and retention (EPR) phenomenon.

[0089] It is noted that formulating efficient nanodroplet emulsions with compounds, such as lipidic compounds, to be employed as surfactant and / or oil components and containing free reactive functional groups can present a significant challenge in achieving desirable physicochemical properties, injectability, and bioavailability. While it is not desired to be bound by any particular theory of operation, difficulty is likely attributed to either physical features or chemical interactions, such as hydrogen bonding, Van der Waals forces, and ionic forces, between these active functional groups on oil components and other functional groups found in surfactants (excipients). However, in accordance with an aspect of the presently disclosed subject matter, when these groups are blocked, such by the binding of an active moiety or other moiety, the modified compounds (e.g., oil component and / or surfactant component) undergo changes in their physical properties, resulting in variations in homogeneity with surfactants and consequently better affecting the overall formulation efficiency. For example, as shown in Figure 17, carboxylic acid, amino, and hydroxyl functional groups can be blocked with active agents (e.g., iodinated moieties, such as a triiodophenol or triiodobenzoic acid) before implementation in a nanoformulation in accordance with the presently disclosed subject matter.

[0090] In some embodiments, the mixture of oil and surfactant (e.g., active- agent-modified oil and surfactant) can be tailored to enhance the stability and uniformity of nanodroplet emulsions prepared from the mixture. For example, in some embodiments, the choice of oil and surfactant components (e.g., active agent-modified oil and surfactant) and their ratios can be varied to provide a more homogenous mixture, which can result in more stable and uniform nanodroplet emulsions.

[0091] In some embodiments, an aspect of the presently disclosed subject matter is the significantly reduction of excipients while maximizing the presence of active agents (e.g., iodinated molecules), which can enhance safety and efficiency. In some embodiments, opting for a SOR of about 20 wt.% to about 50 wt.% (e.g., about 20 wt.%, about 25 wt.%, about 30 wt.%, about 35 wt.%, about 40 wt.%, about 45 wt.% or about 50 wt.%), results in enhanced stability, reduced toxicity, and improved efficiency, aligning with a desired clinical dose. In some embodiments, the SOR is about 20 wt.% to about 30 wt.% (e.g., about 20 wt.%, about 21 wt.%, about 22 wt.%, about 23 wt.%, about 24 wt.%, about 25 wt.%, about 26 wt.%, about 27 wt.%, about 28 wt.%, about 29 wt.%, or about 30 wt.%). In some embodiments, when the nanoemulsion comprises an iodinated contrast agent, the clinical dose is approximately 2 ml / kg. In some embodiments, a desired dose of iodine ranges from about 100 milligrams iodine per milliliter (mg l / mL) to about 200 mg l / mL, including about 125 mg l / mL, 150 mg 1 / mL, and 175 mg l / mL. See also the dosage information provided in the Figures. These formulations exhibited optimal characteristics for use as a contrast agent, as they accumulated in the liver for about three days before being entirely cleared from the body.

[0092] Thus, an aspect of the presently disclosed subject matter pertains to the role the SOR ratio of a nanoemulsion plays in varying physicochemical properties, toxicity, contrast enhancement, and suitability for clinical applications.

[0093] Accordingly, in some embodiments, the presently disclosed subject matter provides a nanoemulsion formulation. In some embodiments, the nanoemulsion formulation comprises (a) a surfactant component and an oil component, wherein the surfactant component and the oil component are present at a surfactant-to-oil ratio (SOR) of about 20 wt.% to about 50 wt.%; and (b) an effective amount of one or more active moieties. In some embodiments, the SOR is about 20wt.%, about 22 wt.%, about 24 wt.%, about 26 wt.%, about 28 wt.%, about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, or about 50wt.%. In some embodiments, the surfactant component and the oil component are present at a SOR of about 20wt.% to about 30 wt.%. In some embodiments, the SOR is about 30 wt.% or less.

[0094] In some embodiments, an amount of an aqueous phase of the nanoemulsion formulation is kept constant. In some embodiments, the nanoemulsion has a surfactant-to-oil-to water ratio (SOWR) of about 40 wt.%. Thus, in some embodiments, the nanoemulsion formulation comprises a phase comprising the surfactant component and the oil component and an aqueous phase wherein the aqueous phase comprises about 60% by weight of the formulation and the phase comprising the surfactant and oil components comprises about 40% by weight of the formulation.

[0095] In some embodiments, the one or more active moieties comprise one or more detectable moieties and / or one or more therapeutic moieties. Thus, in some embodiments, the nanoemulsion comprises one or more moieties suitable for detection (e.g., via CT, MRI, US, etc.), one or more moieties that provide a therapeutic effect in treating a disease or a symptom thereof in a subject (e.g., a mammalian subject), or both one or more detectable moieties and one or more therapeutic moieties.

[0096] In some embodiments, the surfactant component and / or the oil component comprises a biocompatible molecule or a biocompatible molecule derivatized with one or the one or more active moieties. Thus, in some embodiments, the surfactant component and / or the oil component comprise a biocompatible molecule that has been covalently modified with one or more active moieties (i.e. , a detectable or therapeutic moiety). In some embodiments, the surfactant component and / or the oil component comprise two or more biocompatible molecules or biocompatible molecules derivatized with an active moiety.

[0097] In some embodiments, the biocompatible molecule is selected from a PEGylated molecule, a fatty acid, a fatty alcohol, and a fatty amine. In some embodiments, the fatty acid, fatty alcohol, or fatty amine comprises an aliphatic chain comprising at least 11 carbon atoms. In some embodiments, the term “aliphatic” as used herein refers to a saturated or unsaturated alkyl group where the unsaturated aliphatic group is an aliphatic group that contains one or more carbon-carbon double bonds. In some embodiments, the fatty acid, fatty alcohol or fatty amine contains a single reactive functional group, e.g., carboxylic acid, amine, or hydroxy group, e.g., available to be conjugated to an active moiety. In some embodiments, the single reactive functional group can be at one of the ends of the aliphatic chain (e.g., take the place of a -CH3 group at the end of an alkyl chain). In some embodiments, the fatty acid, fatty alcohol, or fatty amine can further contain one or more additional functional group (e.g., carboxylic acid, alcohol, or amine) attached to a carbon atom that is not at the end of the aliphatic chain. In some embodiments, the fatty acid, fatty alcohol, or fatty amine contains 11 to 26 carbon atoms or 11 to 20 carbon atoms (e.g., 12, 14, 16, or 18 carbon atoms). In some embodiments, the biocompatible molecule is a PEGylated molecule (i.e., a molecule comprising one or more polyethylene glycol (PEG) groups), oleic acid, oleylamine, ricinoleic acid, lauric acid, myristic acid, palmitic acid, and stearic acid. In some embodiments, the PEGylated molecule is polyethylene glycol 12-hydroxy stearate (commercially available under the tradename SOLUTOL® HS15 (BASF Akteingeselllschaft, Ludwigshafen, Germany) or PEG-substituted castor oil (e.g., PEG35 castor oil, sold under the tradename CREMOPHORE® ELP (BASF SE Ludwigshafen am Rhein, Germany). See Figure 17.

[0098] In some embodiments, the surfactant component comprises a PEGylated molecule. In some embodiments, the surfactant component comprises a single surfactant. In some embodiments, the surfactant component comprises a combination of two or more surfactants. For example, in some embodiments, the surfactant comprises polyethylene glycol 12- hydroxy stearate, polyethylene glycol castor oil, or a both polyethylene glycol 12-hydroxy stearate and polyethylene glycol castor oil.

[0099] In some embodiments, the surfactant component and / or the oil component comprise a molecule that is covalently or non-covalently modified with one or more of the one or more active moieties. In accordance with some embodiments of the presently disclosed subject matter, surfactants and / or oils containing free reactive functional groups are employed, wherein these groups are blocked, such by the binding of an active moiety or other moiety. The resulting modified oil or surfactant compounds undergo changes in their physical properties, resulting in variations in homogeneity with other surfactants and / or oils and consequently are better at affecting overall formulation efficiency. For example, as shown in Figure 17, carboxylic acid, amino, and hydroxyl functional groups are blocked with active agents (e.g., iodinated moieties, such as triiodophenol and triiodobenzoic acid) before implementation in a nanoformulation in accordance with the presently disclosed subject matter.

[0100] In some embodiments, the one or more detectable moieties are selected from the group consisting of a halogen atom-containing moiety (optionally an iodo group), a fluorescent probe, Fe3O4, gadolinium, a superparamagnetic iron oxide nanoparticle (SPION), a64Cu radionucleotide, and a18F radionucleotide. In some embodiments, the one or more detectable moieties comprise an iodo-containing moiety. In some embodiments, the iodocontaining moiety is an iodo-substituted aryl group, e.g., an iodo-substituted phenyl group. The iodo-containing moiety can comprise more than one iodo group (e.g., can be a monoiodo-, diiodo-, or triiodo-substituted group). In some embodiments, the iodo-substituted phenyl group is a mono-iodophenyl group, a di-iodophenyl group, or a triiodophenyl group. In some embodiments, the iodo-substituted phenyl group is a derivative of 1 ,3,5-triiodobenzene or 1 ,2,4- triiodobenzene.

[0101] In some embodiments, the one or more active moieties comprise a therapeutic agent, e.g., an anticancer agent. In some embodiments, the anticancer agent is an anticancer agent that comprises a hydroxy or carboxylic acid moiety that can form an ester or amide linkage with a hydroxyl, amino, or carboxylic acid reactive functional group present of an oil or surfactant molecule.

[0102] In some embodiments, the formulation comprises a nanoparticle having a diameter ranging from about 30 nm to about 150 nm (e.g., about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, or about 150 nm). In some embodiments, the nanoparticle comprises the oil component, the surfactant component and the one or more active moieties. In some embodiments, the formulation comprises a population of nanoparticles having a mean diameter of about 30 nm to about 150 nm. In some embodiments, the nanoparticles have a polydispersity index of about 0.2 or less, about 0.19 or less, about 0.18 or less, about 0.17 or less, about 0.16 or less, about 0.15 or less, about 0.14 or less, about 0.13 or less, about 0.12 or less, about 0.11 or less, about 0.10 or less, about 0.09 or less, or about 0.08 or less. In some embodiments, the nanoparticle diameter and / or PDI is stable for at least 30 days, at least 3 months, at least 6 months, or at least 12 months.

[0103] In some embodiments, the oil component comprises an iodinated oil having the structure A-X-L, where A is an iodo-substituted phenyl group, X is an amide linkage or an ester linkage (e.g., -C(=O)-NH- (amide linkage), -O- C(=O)- (ester linkage) or -C-(=O)-O- (ester linkage); and L is saturated or unsaturated aliphatic chain, wherein the saturated or unsaturated aliphatic group comprises at least 11 carbon atoms. In some embodiments, A is a triiodosubstituted phenyl group. In some embodiments, L is an unsaturated aliphatic chain comprising one or more double bonds. In some embodiments, the saturated or unsaturated aliphatic chain is optionally substituted by a one or more substituent having the structure -O-C(=O)-A. Thus, in some embodiments, the iodinated oil can comprise more than one iodo-substituted group. In some embodiments, a range for the number of carbon atoms in the L group that would provide oils suitable for forming emulsions with SORs between 20 wt% and 50 wt.% is 11-17. In some embodiments, the oil component comprises 2,4,6-triiodophenyl oleate (TIPhO).

[0104] In some embodiments, the oil component comprises one or more of the group comprising oleoyl polyoxyl-6 glycerides (sold under the tradename LABRAFIL®, Gattefosse SA, Saint Priest, France), vitamin E (a-tocopherol), vitamin D3 (cholecalciferol), oleic acid, castor oil, capric acid mono- and digycerides (sold under the tradename CAPMUL® MCM C8, ABITEC Corporation, Columbus, Ohio, United States of America), and the like. In some embodiments, the performance of these formulations significantly improves when using their derivatives (e.g., active agent-modified compounds, such as iodine-modified compounds).

[0105] In some embodiments, the surfactant component comprises an iodinated surfactant having the structure [L’]m-P’, wherein L’ has a structure of the formula A’-C(=O)-X-L”-C(=O)-O-, wherein A’ is an iodo-substituted phenyl group (e.g., triiodophenyl) and L” is a saturated or unsaturated aliphatic chain; m is an integer that is 1 or more (e.g., 1 , 2, or 3), and P’ is a moiety comprising one or more polyethylene glycol (PEG) chains, wherein each of the one or more PEG chains is covalently attached to one of the m L’ groups. In some embodiments, m is 1 and P’ is a single PEG chain. In some embodiments, m is 3 and P’ is a PEGylated glycerol.

[0106] In some embodiments, the oil component comprises TIPhO and the surfactant component comprises polyethylene glycol 12-hydroxy stearate, polyethylene glycol castor oil, or a both polyethylene glycol 12-hydroxy stearate and polyethylene glycol castor oil. In some embodiments, the formulation is LNE1 , LNE2, LNE3, or LNE4. In some embodiments, the formulation is LNE3.

[0107] In some embodiments, the formulation is suitable for use as a contrast agent (CA). In some embodiments, the formulation is suitable for use as a contrast agent in CT, PET, MRI, US, and / or fluorescence imaging methods. In some embodiments, the formulation is suitable for use in CT imaging. In some embodiments, a method of imaging a cell and / or tissue in a subject is provided. In some embodiments, the method comprises administering to the subject an effective amount of a formulation as disclosed herein (e.g., a formulation wherein the one or more active moieties comprise one or more detectable moieties); and detecting the formulation in the subject. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the imaging comprises imaging a blood pool in the subject. In some embodiments, the imaging comprises imaging liver tissue (e.g., the entire liver or a portion thereof). In some embodiments, the one or more active moieties comprise an iodo-containing moiety (e.g., a triiodo-substituted phenyl group) and the imaging comprises CT imaging.

[0108] In some embodiments, a method of treating a subject in need of treatment is disclosed. In some embodiments, the method comprises administering to the subject an effective amount of a formulation as disclosed herein (e.g., wherein the one or more detectable moieties comprise at least one therapeutic moiety). In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is liver cancer.

[0109] In some embodiments, a formulation can be provided in a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the carrier is pharmaceutically acceptable for use in humans. The carrier or adjuvant desirably should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.

[0110] Pharmaceutically acceptable carriers in imaging compositions and therapeutic compositions can additionally contain liquids such as water and saline, glycerol and ethanol. Additionally, auxiliary substances, such as pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated for administration to the patient.

[0111] Pharmaceutical compositions (e.g., imaging and / or therapeutic compositions) of the presently disclosed subject matter can include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze- dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Any other agents conventional in the art having regard to the type of formulation in question can be used. In some embodiments, the carrier is pharmaceutically acceptable. In some embodiments the carrier is pharmaceutically acceptable for use in humans.

[0112] Pharmaceutical compositions (e.g., imaging and / or therapeutic compositions) of the presently disclosed subject matter can have a pH between 5.5 and 8.5, preferably between 6 and 8, and more preferably about 7. The pH can be maintained by the use of a buffer. The composition can be sterile and / or pyrogen free. The composition can be isotonic with respect to humans. Pharmaceutical compositions of the presently disclosed subject matter can be supplied in hermetically-sealed containers.

[0113] An effective amount or dose of a pharmaceutical composition of the presently disclosed subject matter is administered to a subject in need thereof. The terms “therapeutically effective amount,” “therapeutically effective dose,” “effective amount,” “effective dose,” and variations thereof are used interchangeably herein and refer to an amount of a therapeutic composition or imaging composition of the presently disclosed subject matter sufficient to produce a measurable response. Actual dosage levels can be varied so as to administer an amount that is effective to achieve the desired therapeutic response for a particular subject.

[0114] In some embodiments, the quantity of an imaging and / or therapeutic composition of the presently disclosed subject matter administered to a subject will depend on a number of factors including but not limited to the subject’s size, weight, age, the target tissue or organ, the route of administration, the condition to be treated, and the seventy of the condition to be treated.

[0115] The potency of an imaging and / or therapeutic composition can vary, and therefore an “effective” amount can vary. However, using the assay methods described herein below, one skilled in the art can readily assess the potency and efficacy of the pharmaceutical compositions of the presently disclosed subject matter and adjust the regimen accordingly.

[0116] The subject of the presently disclosed subject matter is desirably a human subject, although it is to be understood that the principles of the disclosed subject matter indicate that the compositions and methods are effective with respect to invertebrate and to all vertebrate species, including mammals, which are intended to be included in the term “subject.” Moreover, a mammal is understood to include any mammalian species in which treatment and / or imaging is desirable, particularly agricultural and domestic mammalian species.

[0117] The methods of the presently disclosed subject matter are particularly useful in warm-blooded vertebrates. Thus, the presently disclosed subject matter concerns mammals and birds.

[0118] More particularly, provided herein is the imaging and / or treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and / or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, provided herein is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

[0119] EXAMPLES

[0120] The following Examples provide further illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. EXAMPLE 1

[0121] PREPARATION AND CHARACTERIZATION OF LNES

[0122] 2,4,6-Triiodophenyl oleate (TIPhO) was synthesized on gram scale using an esterification reaction between 2,4,6-triiodophenol and oleic acid in presence of 4-dimethylaminopyridine (DMAP) and N,N'- dicyclohexylcarbodiimide (DCC) in a nonpolar aprotic solvent, such as dichloromethane (DCM). See Figure 2, left-hand panel. Other iodinated oils and / or surfactants of the presently disclosed subject matter can be prepared by the same method or via other esterification or amide formation reactions known in the field. See Figure 17. Purification was simple using column chromatography, yielding high-purity compounds with 89% yield. 2,4,6- Triiodophenyl oleate was characterized by proton and carbon-13 nuclear magnetic resonance spectroscopy (1HNMR and13CNMR).

[0123] The formulation of nanoemulsion contrast agents (CAs) was performed by a spontaneous emulsification process in which different ratios of iodinated lipophilic molecules were mixed with surfactant (e.g., different ratios of nonionic PEGylated solubilizers such as PEG35 castor oil, a nonionic surfactant sold under the tradename KOLLIPHOR ELP® (formerly CREMOPHOR ELP® (CrEL), BASF SE Ludwigshafen am Rhein, Germany) or polyethylene glycol 12-hydroxy stearate (a nonionic surfactant sold under the tradename SOLUTOL® HS 15 (BASF Aktiengesellschaft, Ludwigshafen, Germany)) and an aqueous phase (e.g., saline) to produce uniform, homogeneous and opaque nanosuspension in the 30-150 nm size range, which varied based on the ratio between iodinated compounds and surfactants. See Figure 3, middle and righthand panels and Figure 4. See also Table 1 , below. The LNE CAs were then filtered through a 0.22 pm syringe filter and sterilized for further in vitro and in vivo characterizations. This was performed as previously published.6-8 Table 1. Characterization of LNE suspensions prepared with PEG castor oil (CrEL) as the surfactant component. As summarized in Table 1 , above, the iodinated LNE CAs were prepared at a surfactant-to-oil (SOR) ranging from about 20 wt.% to about 50 wt.% and at a constant surfactant-to-oil-to-water (SOWR) of 40 wt.%, producing stable NPs with a size range of ~30-150 nm and low PDI (< 0.2). potentials were -12 to -27 mV. Size, PDI, and zeta potential were determined by DLS. Stability of the LNEs was studied in serum and human plasma, and in vitro iodine quantification in function of X-ray attenuation. Excellent stability was observed after recording the size over a year.

[0124] More particularly, Figures 5A and 5B show particle size stability and polydispersity over time for up to 30 days monitored by DLS. Figure 5C shows particle concentration per one milliliter of all LNEs detected by nanoparticle tracking analysis (NTA) using an instrument sold under the tradename ZETAVIEW® (Particle Metrix GmbH, Inning, Germany). Figure 5D shows particle surface charge measured by DLS using an instrument sold under the tradename ZETASIZER® (Malvern Panalytical Limited, Malvern, United Kingdom). Figure 6 shows cryo-transmission electron microscope (Cryo-TEM) images of LNEs one day (left) and 6 months (right) post-preparation.

[0125] Figures 7A and 7B show particle stability of 10% and 20% LNE3 and 10% and 20% LNE4 upon incubation in human plasma for up to 2 days. Figure 7A shows change in particle size and Figure 7B shows change in PDI over time as measured via DLS. Figures 7C and 7D show particle stability of the same compositions upon incubation in fetal bovine serum (FBS) for up to two days, where Figure 7C follows particle size and Figure 7D follows PDI over time. Again, particle size and PDI are measured via DLS.

[0126] Figure 8A shows an in vitro evaluation of the X-ray attenuation properties exemplary LNEs of the presently disclosed subject matter, i.e., LNE1 , LNE2, LNE3, and LNE4 using a calibration curve (X-ray attenuation (in Hounsfield units (HU)) versus iodine concentration (milligrams iodine per milliliter (mg l / mL))) made using different concentrations of the clinical CT contrast agent lodixanol (sold under the tradename VISIPAQUE® (GE Healthcare AS, Oslo, Norway). The inset shows a phantom scan of the different LNEs and of reference samples with various iodine concentrations, plus water and air for normalization. The table in the upper left summarizes the values obtained for the NLEs. Figure 8B, shows the results of a Cell Counting Kit-8 (CCK-8) cytotoxicity assay of immune cells, including RAW macrophages and IC21 cell line after treatment with LNE3 for 48 hours. Based on the cell viability assay, 50% inhibitory concentrations (ICsos) of 0.91 mg l / mL and 0.41 mg l / mL were calculated for LNE3 in RAW cells and IC21 cells, respectively.

[0127] To further characterize the LNEs, UV-vis absorbance analysis was conducted on LNE1 -LNE4 at a 3200 times dilution of the stock solution. The analysis revealed two distinct peaks of maximum wavelength (Xmax) at approximately 230 nm and 244 nm (see Figure 9), associated with the surfactant (CrEL) and the TIPhO moiety, respectively, confirming the composition of the LNEs.

[0128] As viscosity is important for injectable products, viscosity measurements were performed on LNE1 -LNE4 formulations, as well as of a formulation referred to as LNE5 (a 2x dilution of LNE3). For comparison, the viscosities of the presently disclosed LNEs were compared to that of iodixanol stock solution (300 mg 1 / mL) at 60% wt / v. See Table 2 provides the viscosities of the LNE formulations (in centipoise (cP)) at room temperature (RT, 25°C) and at 37°C. Diluting ILNE3 2x reduced its viscosity to 5.9 ± 0.2 and 4.1 ± 0.1 cP at 25°C and 37°C, respectively. ILNE3 had a lower viscosity than the clinical X-ray imaging agent iohexol (300 mg / mL), as well as a lower viscosity than a commercially available pre-clinical liver functional imaging agent, a-tocopheryl 2,3,5-triiodobenzoate (sold under the tradename FENESTRA™ (Medilumine, Montreal, Canada).

[0129] Table 2. Viscosity of LNE Formulations.

[0130] Figures 10A-10I shows batch-to-batch variation of LNE3, indicating that the LNEs can be prepared with consistent physical properties. Figures 11 A-11 P show that LNE3 and LNE4 formulations are stable at room temperature for at least one-year post-preparation.

[0131] Understanding nanoparticle uptake by cells can be of use in evaluating toxicity and biocompatibility. Inadequate internalization or cytotoxic effects can compromise cellular function. NPs should be able to overcome biological barriers like the cell membrane and endosomal / lysosomal compartments to deliver therapeutic or contrast agents effectively. To study the in vitro cellular uptake of the LNEs, two concentrations of LNE3 (266 pg / mL and 533 pg / mL) were prepared, encapsulating 1 ,1 ’-dioctadecyl-3,3,3’,3’- tetramethylindocarbocyanine perchlorate Dil lipophilic dye within their lipidic core to monitor their internalization into RAW and 264.7 macrophages. See Figure 12. The samples were incubated with the cells for 4 and 24 hours. After incubation, the cells were washed, and the nuclei were stained with Hoechst dye before imaging. The images show that the higher concentration of particles exhibited slightly greater uptake after 4 hours. However, at both concentrations, LNE3 displayed similar uptake intensity at 24 hours, significantly higher than at 4 hours. Overall, LNE3, with a size of about 72.9 ± 5.1 nm and a ^-potential of -12.8 mV (measured by NTA) demonstrated remarkable internalization within the cytoplasm of macrophage cells, indicating their suitability for targeting and biocompatibility.

[0132] Tables 3 and 4, below, summarize the characteristics of different formulations of LNEs at varying SOR ratios and the same surfactant-to-oil-to- water (SOWR) ratio (40 wt.%). Table 3 shows the characteristics of LNE formulations prepared using the PEGylated non-ionic surfactant polyethylene glycol 12-hydroxy stearate (surfactant sold under the tradename SOLUTOL™ HS15 (BASF Aktiengesellschaft, Ludwigshafen, Germany). Table 4 shows the characteristics of LNE formulations prepared using a 50:50 wt.% mixture of polyethylene glycol 12-hydroxy stearate and PEGylated castor oil (surfactant sold under the tradename KOLLIPHOR® EL, formerly known as CREMOPHOR® EL, BASF SE, Ludwigshafen Am Rhein, Germany).

[0133] Table 3. LNE Formulations with single PEGylated Surfactant. Table 4. LNE Formulations with Surfactant Mixture.

[0134] EXAMPLE 2 MURINE STUDIES

[0135] In vivo micro-computed tomography (micro-CT, microCT, or p-CT) imaging was performed in small and large animal models. For imaging in a small animal model, murine studies were performed to compare LNE uptake, clearance and toxicity of the LNE against commercially available pre-clinical liver directed contrast agents. Select iodinated LNE sizes are compared against a commercially available pre-clinical liver functional imaging agent, a- tocopheryl 2,3,5-triiodobenzoate (sold under the tradename FENESTRA™ LC, Medilumine, Montreal, Canada) using micro-CT imaging in normal mice. The degree of enhancement, clearance rate and toxicity are evaluated. The agents are formulated to match the iodine dose of the commercial compound at 100 mg / mL.

[0136] More particularly, three C57BL / 6 mice were injected with LNE3 contrast agent (CA) (which has an average particle size of 75 nm) and two mice were injected with saline at a dose of 2 mL / kg for comparison. Imaging is performed via micro-CT using a standard abdominal imaging protocol under isoflurane anesthesia. Sequential imaging is performed on the Quantum GX2 microCT scanner (PerkinElmer Inc., Waltham, Massachusetts, United States of America) which provides for rapid, repeated scans of the same region. This approach is used to replicate the four-phase liver CT imaging approach on clinical scanners. Images are acquired during the administration of the contrast agent at total iodine doses matched to that of the commercial iodinated contrast agent a-tocopheryl 2,3,5-triiodobenzoate (sold under the tradename FENESTRA™ LC, Medilumine, Montreal, Canada) at 0.005 mL / g. Dynamic images are acquired every 5 seconds for 60 seconds during which a tail vein injection of the contrast agent is administered. Repeat single-phase scans are subsequently conducted at 5 min, 1 , 2, 4, 24, and 72 hours post-injection. For this Example, non-tumor bearing mice were utilized. A reference sample of the contrast agent is scanned within the field-of-view of the liver to provide an external reference for the degree of iodine attenuation.

[0137] In Vivo CT Contrast and Pharmacokinetics in Mice:

[0138] Inspired by the remarkable x-ray attenuation ability of the LNEs, LNE3 was administered intravenously at 2 mL / kg to C57BL / 6 mice. Enhanced contrast was observed in the heart and liver compared to pre-injection images, evident in both coronal and transverse views. See Figure 13A. This confirms the rapid distribution of LNE3 throughout the vasculature, highlighting the major veins of the heart ventricles, aorta, and liver. Over 72 hours, no clinical signs of disorder or toxicity were observed in mice. LNE3 consistently exhibited enhanced contrast, distribution and clearance behavior. The images revealed a gradual increase in contrast in the heart and liver up to 4 hours post-injection. See Figures 13B and 13C. By 24 hours, LNE3 had been entirely cleared from the bloodstream, with no detectable signals, while notably accumulating in the liver, resulting in pronounced contrast. By 72 hours, CT signals in the liver were significantly reduced, confirming elimination from the body. Collectively, LNE3 functions as a blood pool CT contrast agent (BPCA), boasting a half-life of at least 4 hours, prolonging the scan window, and subsequently undergoing hepatic metabolism, circumventing renal clearance. It is effectively eliminated from the body within approximately 3 days post-injection. These attributes match the criteria for optimal BPCAs in clinical settings, featuring a kidney-safe formulation with improved contrast enhancement, followed by rapid clearance from the body post-scanning.

[0139] Toxicity analysis:

[0140] To further ensure the safety profile of LNE3 as a CT contrast agent, two studies were performed in C57BL / 6 mice, varying time points and escalating doses. In an initial study, a control group of two mice received saline (2 mL / kg) while a group of three mice each received LNE3 at a dose of 300 mg l / kg. Three days post-injection and CT scanning, both groups were sacrificed for blood analysis and histological examination of major organs (heart, liver, kidney, lung, and spleen). Analysis showed no significant differences in liver / kidney functions or complete blood count (CBC) between groups. See Tables 5A, below which provides CBC and blood chemistry data as the mean ± SD. Macroscopic examination and H&E stains indicated no visible damage, toxicity, necrosis, or pathological alterations in tissue architectures. See Figure 14. These findings, showcasing normal histological features with no deviations, provide compelling evidence of the nontoxic nature of LNE3. In a second study, three groups were compared, saline-injected mice (n=5), LNE3-injected mice at 300 mg l / kg (n=5), and LNE3-injected mice at 750 mg l / kg (n=3). After 10 days, CBC, blood chemistry and thyroid-stimulating hormone (TSH) levels were analyzed. Data showed now significant weight changes in the mice or differences in blood analysis and liver / kidney / function tests. See Table 5B. AST levels were slightly elevated three days post-injection but normalized after 10 days. The higher dose of 750 mg l / kg (a 2.5-fold increase of clinical dose) showed only a minor AST increase, suggesting LNEs accumulate in the liver before dissipating. These findings support LNE3’s exemplary biosafety for translational studies.

[0141] Table 5A. Blood Analysis in Mice Three Days after LNE3 Injection.

[0142]

[0143] Table 5B. Blood Analysis in Mice Ten Days after LNE3 Injection.

[0144]

[0145] Abbreviations: RBC (red blood cells), HGB (hemoglobin), HCT

[0146] (hematocrit), MCV (mean corpuscular volume), MCHC (mean corpuscular hemoglobin concentration), RET (reticulocytes), PLT (platelets), MPV (mean platelet volume), WBC (white blood cells), NELIT (neutrophils), LYMPH (lymphocytes), MONO (monocytes), EO (eosinophils), ALP (alkaline phosphatase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), CERA (creatinine).

[0147] EXAMPLE 3

[0148] PORCINE STUDIES

[0149] A porcine animal model (16.6 kg) was injected with the LNE3 CA at a dose of 300 mg l / kg and CT imaging of heart, liver, kidneys levels was performed at one hour post-injection of the LNEs. See Figure 15. A reference clinical agent, iohexol, was also injected and the porcine subject was scanned at arterial phase within the field-of-view of the liver, kidneys, and heart to provide an external reference for the degree of iodine attenuation. The porcine subject was anesthetized and intubated prior to the procedure. Intravenous access was obtained in an ear, or other appropriate vein. Intubation was required for breath holding during the CT acquisition.

[0150] Image analysis. Contrast-to-noise ratios were performed comparing the HU units of the liver parenchyma as compared to adipose tissue and muscle during the phases of CT between the different contrast agents. The half-life of the contrast agent was evaluated by evaluating the HU differences relative to time and fitting the exponential curve. Reader study: A reader study aided by board certified radiologists with abdominal imaging experience was performed. Using a Likert scale, they evaluated image quality, confidence of assessment of the liver parenchyma against background, and identification of the hepatic arterial and portal venous structures and their branches. Radiologists were asked to comment on the presence of artifacts.

[0151] Analysis: The relative contrast to noise ratios at each time point were evaluated relative to conventional iohexol as a standard. The radiologist confidence was scored in interpretation each of the categories listed above.

[0152] More particularly, imaging of a 16.6 kg porcine was performed on a clinical CT scanner (sold under the tradename FORCE™; Siemens Medical Solutions USA, Inc., Malvern, Pennsylvania, United States of America) using an abdominal protocol at 120kVp and mean x-ray tube current of 32mA. Images were reconstructed with a standard soft tissue kernel. Imaging was acquired at 2 hours after the i.v. administration of the ILNE3 contrast agent (300 mg l / mL). Iohexol (300 mg l / mL) based imaging was performed in a different animal for reference prior to and after the i.v. administration of contrast agent at 1 .5 mL / kg. Imaging was performed during the arterial phase of contrast administration. Vital signs were continuously monitored. No acute changes in the animal’s vital signs were observed during the administration of contrast or during the scan.

[0153] A 3D rendering of the porcine subject 1 -hour post-injection with LNE3 at a dose of 300 mg l / kg is shown in Figure 16. The image shows clear delineation of the heart chambers, arterial and venous vasculature, and hepatic vessels. Diffuse liver uptake is also observed at this time point.

[0154] EXAMPLE 4

[0155] DISCUSSION OF EXAMPLES 1 -3

[0156] The Examples above pertain, in some embodiments, to blood pool iodine-based LNEs with efficient contrast enhancement and minimal toxicity for X-ray CT imaging. The Examples use biocompatible molecules such as unsaturated fatty acids (e.g., oleic acid) to be modified with a triiodobenzene moiety via esterification reaction providing high loading iodine content in their final chemical structures, such as 51.7 wt.%. The synthesized iodinated lipophilic molecules were then incorporated with PEGylated solubilizer by low energy emulsification method producing stable radiopaque nano contrast agents containing about 120 to about 177 mg l / mL in the final suspension in the 30-150 nm size range. See Figure 4 and Table 1 , below. The iodine payloads are relatively higher than iodinated liposomes and micelles in the literature and largely higher than commercial products (e.g., 55 mg l / ml for iodinated contrast agent sold under the tradename FENESTRA™ VC (Medilumine, Montreal, Canada). Aspects of the presently disclosed subject matter rest on the facile formulation process of CAs, safety, improved contrast enhancement and thus reduction of the injected dose, stability of the particles extended to more than a year, prolonged blood circulation, limitation of renal toxicity, and easy scale-up and commercialization.

[0157] LNEs are remarkably stable particles with a high payload capacity to encapsulate high amounts of lipophilic compounds in their cores. See Figures 1 and 2. Injectable nanoemulsions, formulated to include iodinated agents, are designed for medical imaging applications in both animals and humans. These nanoemulsions are prepared using a low-energy process. In some embodiments, the iodinated agents comprise iodine atoms directly attached to a phenyl group bound to lipophilic compounds, such as oleic acid or other vitamins, and are prepared using simple chemical reactions. See Figures 3 and 17. These iodinated compounds can be incorporated into or can constitute the internal phase of oil-in-water nano-emulsions. Further, lipophilic fluorescent probes and / or FesCM or gadolinium chelates can be easily loaded in the internal phase of the iodinated LNEs for multimodal imaging modalities such as fluorescence, MRI, and X-ray CT imaging. See Figure 2. In addition, these formulations can be conjugated with64Cu or18F radionucleotides, generating nanoparticles-based radiotracer for PET imaging with blood half-life time greater than the current clinical tracers that exhibit 2-100 min for better investigation of biological processes. See Figure 2. In some embodiments, the presently disclosed iodinated nanoparticles can be utilized for radiotherapy enhancement of glioblastoma and other cancers.

[0158] The presently disclosed class of LNEs showed high integrity profile in vivo and greatly passively accumulated in tumor tissues through the EPR effect as reported by Redouane et al.5The presently disclosed preliminary data on a triiodophenyl oleate LNE contrast agent revealed that the blood circulation exceeds 4 hours, indicating a utility of this approach as a blood pool agent. Subsequently, the imaging demonstrates substantial liver uptake, enabling them to serve as functional liver imaging agent. Overall, the simple formulation, high CT contrast, lower toxicity, and ability to scale up production support the use of the presently disclosed LNEs as contrast agents.

[0159] Aspects of this study include evaluating the triiodophenyl oleate LNEs against commercially available liver CT imaging agents, and then demonstrating scaling to imaging in a large animal model.

[0160] Existing, traditional X-ray CT contrast agents are hydrophilic-based iodinated molecules associated with multiple clinical problems including short blood circulation times and rapid renal clearance, which can cause severe nephrotoxicity and other unwanted side effects. The presently disclosed subject matter pertains to the design of long-circulating iodinated lipid nanoemulsion contrast agents with high biocompatibility and significant contrast enhancement. Simple formulation and stability over time are key points for clinical translation. The 30-150 nm size range of LNEs facilitates tumor perfusion, giving rise to a clear mapping of tumor vasculature. Further embodiments can translate these formulations to PET imaging through conjugation with64Cu or18F radionucleotides, producing efficient nano tracers. Ligand-functionalized iodinated LNE CAs can also help in reducing toxicity and off-target distribution. Those CAs are also promising in enhancing radiotherapy in cancer in mice. Access to this kind of CAs could drastically improve diagnosis of diseases, treatment assessment, and prediction of therapy.

[0161] The facile conjugation of iodo-containing compounds is a simple chemical reaction for yielding iodinated lipophilic compounds at the gram scale and beyond. Formulation of iodinated lipid nanoemulsion contrast agents is also easily achieved by scale-up using low energy spontaneous emulsification technique, which will be potential for industrial scale-up. REFERENCES

[0162] All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and / or compositions employed herein.

[0163] 1 . Mittal S, El-Serag HB. Epidemiology of HCC: Consider the Population. J Clin Gastroenterol. 2013 Jul;47(0):S2-S6. PMCID: PMC3683119.

[0164] 2. Griscom JT, Wolf PS. Liver Metastasis. StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 [cited 2023 Apr 18], Available from: www.ncbi.nlm.nih.gov / books / NBK553118 / PMID: 31971757.

[0165] 3. Current practices and perspectives on the integration of contrast agents in MRI-guided radiation therapy clinical practice: A worldwide survey - Clinical and Translational Radiation Oncology [Internet], [cited 2023 Apr 18], Available from: www.ctro.science / article / S2405-6308(23)00040-X / fulltext.

[0166] 4. Tan MJ, Fernandes N, Williams KC, Ford NL. In vivo micro-computed tomography imaging in liver tumor study of mice using Fenestra VC and Fenestra HDVC. Sci Rep. Nature Publishing Group; 2022 Dec 27;12(1 ):22399.

[0167] 5. Bouchaala R, Mercier L, Andreiuk B, Mely Y, Vandamme T, Anton N, Goetz JG, Klymchenko AS. Integrity of lipid nanocamers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice. J Control Release Off J Control Release Soc. 2016 Aug 28;236:57-67. PMCID: PMC4968657.

[0168] 6. Attia MF, Anton N, Chiper M, Akasov R, Anton H, Messaddeq N, Fournel S, Klymchenko AS, Mely Y, Vandamme TF. Biodistribution of X-ray iodinated contrast agent in nano-emulsions is controlled by the chemical nature of the oily core. ACS Nano. 2014 Oct 28;8(10): 10537-10550. PMID: 25284066.

[0169] 7. Attia MF, Anton N, Akasov R, Chiper M, Markvicheva E, Vandamme TF. Biodistribution and Toxicity of X-Ray Iodinated Contrast Agent in Nanoemulsions in Function of Their Size. Pharm Res. 2016 Mar;33(3):603-614. PMID: 26511860. 8. Attia MF, Akasov R, Elbaz NM, Owens TC, Curtis EC, Panda S, Santos-Oliveira R, Alexis F, Kievit FM, Whitehead DC. Radiopaque lodosilane- Coated Lipid Hybrid Nanoparticle Contrast Agent for Dual-Modality Ultrasound and X-ray Bioimaging. ACS Appl Mater Interfaces. 2022 Dec 14; 14(49):54389- 54400. PMID: 36449986.

[0170] 9. Schachtschneider KM, Schwind RM, Darfour-Oduro KA, De AK, Rund LA, Singh K, Principe DR, Guzman G, Ray CE, Ozer H, Gaba RC, Schook LB. A validated, transitional and translational porcine model of hepatocellular carcinoma. Oncotarget. 2017 Sep 8;8(38):63620-63634. PMCID: PMC5609948.

[0171] It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A nanoemulsion formulation comprising: (a) a surfactant component and an oil component, wherein the surfactant component and the oil component are present at a surfactant-to-oil ratio (SOR) of about 20 wt.% to about 50 wt.%; and (b) an effective amount of one or more active moieties.

2. The formulation of claim 1 , wherein the SOR is about 20 wt.% to about 30 wt.%.

3. The formulation of claim 1 or claim 2, wherein the one or more active moieties comprise a detectable moiety, a therapeutic moiety, or a combination thereof.

4. The formulation of any one of claims 1-3, wherein the surfactant component and / or the oil component comprise a biocompatible molecule and / or a biocompatible molecule derivatized with an active moiety.

5. The formulation of claim 4, wherein the biocompatible molecule is selected from the group consisting of a PEGylated molecule, oleic acid, oleylamine, ricinoleic acid, lauric acid, myristic acid, palmitic acid, and stearic acid, optionally wherein the PEGylated molecule is selected from polyethylene glycol 12-hydroxy stearate, and polyethylene glycol-substituted castor oil.

6. The formulation of any one of claims 1-5, wherein the surfactant component and / or the oil component comprises a molecule that is modified covalently or non-covalently with one or more of the one or more active moieties.

7. The formulation of any one of claims 1 -6, wherein the one or more active moieties comprise one or more detectable moieties, wherein the one ormore detectable moieties comprise a detectable moiety selected from the group consisting of a halogen atom-containing moiety, a fluorescent probe, FesCM, gadolinium, a64Cu radionucleotide, and a18F radionucleotide.

8. The formulation of claim 7, wherein the one or more detectable moieties comprise an iodo-containing moiety.

9. The formulation of claim 8, wherein the iodo-containing moiety comprises a triiodo-substituted phenyl group.

10. The formulation of any one of claims 1 -9, wherein the formulation comprises a nanoparticle having a diameter of about 30 nm to about 150 nm, wherein the nanoparticle comprises (a) and (b).

11. The formulation of any one of claims 1 -10, wherein the oil component comprises an iodinated oil having the structure A-X-L, where A is an iodo-substituted phenyl group, optionally a triiodo-substituted phenyl group; X is selected from -C(=O)-NH-, -C(=O)-O-, and -O-C(=O)-; and L is a saturated or unsaturated aliphatic group, wherein the saturated or unsaturated aliphatic group comprises at least 11 carbon atoms, and wherein the saturated or unsaturated aliphatic group is optionally substituted by one or more substituent having the structure -O-C(=O)-A.

12. The formulation of any one of claims 1 -10, wherein the surfactant component comprises an iodinated surfactant having the structure [L’]m-P’, wherein L’ has a structure of the formula A’-C(=O)-X-L”-C(=O)-O-, wherein A’ is an iodo-substituted phenyl group, optionally a triiodo-substituted phenyl group, and L” is a saturated or unsaturated aliphatic chain; m is an integer that is 1 or more, optionally wherein m is 1 , 2, or 3; and P’ is a moiety comprising one or more polyethylene glycol (PEG) chains, wherein each of the one or more PEG chains is covalently attached to one of the m L’ groups.

13. The formulation of any one of claims 1 -12, for use as a contrast agent.

14. The formulation of claim 13, for use as a contrast agent in computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound (US), and / or fluorescence imaging methods.

15. A method of imaging a cell and / or tissue in a subject; the method comprising administering to the subject an effective amount of a formulation according to any one of claims 1-14; and detecting the formulation in the subject.

16. The method of claim 15, wherein the subject is suffering from cancer, optionally wherein the cancer is liver cancer.

17. The method of claim 15, wherein the imaging comprises imaging liver tissue or a blood pool in the subject.

18. A method of treating a disease in a subject in need of treatment, the method comprising administering to the subject an effective amount of a formulation according to any one of claims 1 -14.

19. The method of claim 18, wherein the method further comprises imaging a cell or tissue in the subject, optionally wherein the imaging comprises imaging liver tissue or a blood pool in the subject.

20. The method of claim 18 or 19, wherein the subject is suffering from cancer, optionally wherein the cancer is liver cancer.

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