WO2016090092A1

WO2016090092A1 - Therapeutic nanoemulsions for delivery to and imaging of the brain and methods of their preparation and use

Info

Publication number: WO2016090092A1
Application number: PCT/US2015/063634
Authority: WO
Inventors: Timothy P. Coleman; Niravkumar PATEL
Original assignee: Nemucore Medical Innovations, Inc.
Priority date: 2014-12-03
Filing date: 2015-12-03
Publication date: 2016-06-09

Abstract

Provided is a method method of targeting a nanoemulsion formulation to a specific region of the brain of a mammalian subject, comprising nanoemulsion formulations comprising a targeting agent, a drug delivery system and a therapeutic agent, an imaging or a mixture. Also provided are nanoemulsion formulation compositions and methods of preparing the same and of treating or imaging the brain, including brain cancer.

Description

THERAPEUTIC NANOEMULSIONS FOR DELIVERY TO AND IMAGING OF THE BRAIN AND METHODS OF THEIR PREPARATION AND USE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. provisional patent application number 62/086,997, filed on December 3, 2014, U.S. provisional patent application number 62/146,548, filed on April 13, 2015, and U.S. provisional patent application number 62/241,945, filed on October 15, 2015, the entire contents of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Part of the work leading to this invention was carried out with United States Government support provided under a grant from the National Institutes of Health, Grants No. U54 CA151881 and R43CA144591. Therefore, the U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present disclosure relates to medicine and pharmacology, and more particularly, to therapy, drug delivery and imaging systems for the brain, and to therapy for brain disorders such as cancer.

BACKGROUND

[0004] Chemotherapeutic agents are widely used in cancer therapy. However, in most cases these treatments do not cure the disease. Challenges for effective therapy, including therapy for brain cancers, are the serious side-effects of many cancer drugs to normal tissues, insufficient concentration and short residence time of therapeutic agents at the site of disease, multi-drug resistance (MDR), and the hydrophobicity of pharmaceutical agents. Additionally, lack of target specificity contributes to systemic toxicity as the therapeutic agent builds up in non-diseased tissues. An additional challenge for the treatment of brain tumors is the inability of many chemotherapeutic agents to cross the blood brain barrier (BBB) in sufficient quantities for effective treatment.

[0005] A number of delivery systems have been developed to address these problems. For example, nanodelivery systems with site-specific binding moieties have been developed with various levels of success to increase concentration of drug in the tumors and decrease side effects. Some delivery vehicles have been devised that improve drug delivery to tumors, for example Doxil (also known as Caelyx), comprising doxorubicin in polyethylene glycol (PEG)-coated liposomes. Another example is poly(epsilon-caprolactone) (PCL) nanoparticles. (Chawla et al. (2003) AAPS PharmSci. 5(1):28— 34). The alkyl structure of the polymer encapsulates hydrophobic compounds. Surface modification of the colloidal carrier with an agent such as a poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO-PEO) triblock copolymer can improve the solubility of the nanoparticle. However, serious side- effects of chemotherapeutic agents to normal tissue remains a challenge for effective cancer treatment.

[0006] Many first line chemotherapeutic agents elicit a response and tumors shrink, but often these tumors develop resistance to the chemotherapeutic agent before affecting a cure. For instance, platinum compounds (including cisplatin, carboplatin, and oxaliplatin), used as therapies for many types of cancers (including head and neck, testicular, ovarian, cervical, lung, colorectal, and relapsed lymphoma) have a good initial response, but later the subjects relapse because of the development of resistance. (Siddick et al. (2003) Oncogene 22:7265-7279). However the BBB remains a major obstacle impeding the delivery of effective amounts of

chemotherapeutic agents to tumors in the brain.

[0007] Effective cancer therapy also suffers from the lack of early data on the delivery of a particular pharmaceutical agent to tumors, and thus to its effectiveness. Subjects often proceed with a course of treatment for an extended period of time, while suffering associated side-effects and poor quality of life, only to find out that the particular treatment is not effective.

[0008] Hydrophobicity of pharmaceutical agents limits the range of therapies for cancer treatment. Almost one third of the drugs in the United States Pharmacopeia (http://www.usp.org/) are hydrophobic and are either insoluble or poorly soluble. (Savic et al. (2006) J. Drug Target. 14(6):343-55). As a result of poor solubility, many potential new chemical entities are being dropped in the early phases of development.

[0009] Approaches for administering hydrophobic drugs include the use of co- solvents, incorporation of complexing or solubilizing agents, chemical modification of the drug, use of micellar delivery systems such as niosomes, liposomes, and their formulation of the drug in an oily vehicle, for oral, parenteral, nasal, rectal or ophthalmic delivery. However, many of these formulations employ surfactants or co- solvents having associated toxic side-effects, and frequently, stability, sterility, and mass commercial production issues as well.

[0010] Accordingly, there exists a need for delivery systems, which can efficiently deliver therapeutic levels of drug to disease sites with fewer or no side-effects while blocking or avoiding multi-drug resistance pathways and with the ability to cross the BBB. There is also a need to expand the range of therapeutics that can be used for cancer treatment. In addition, a need also exists for imaging capabilities that will allow for quick determination as to whether a subject should proceed with a particular course of treatment.

SUMMARY

[0011] It has been discovered that certain targeted drug delivery systems

(nanoemulsion formulations) are able to cross the BBB, and that these targeted formulations accumulate in defined areas of the brain.

[0012] These discoveries have been exploited to develop the present disclosure, which, in part and in one aspect, is a nanoemulsion formulation. This nanoemulsion formulation comprises a targeting agent; a drug delivery system and a therapeutic agent, an imaging agent, or a combination thereof. The drug delivery system comprises an oil phase; an interfacial surface membrane; and an aqueous phase.

[0013] In one embodiment, the targeting ligand of the nanoemulsion formulation comprises a brain tumor targeting ligand, a brain receptor targeting ligand, or a combination thereof. In certain embodiments, the brain receptor targeting ligand comprises an μ-opioid receptor targeting ligand, an N-acetylcholine receptor targeting ligand, an integrin targeting ligand, a neurophilin targeting ligand, a bradykinin targeting ligand, or a combination thereof. In specific embodiments, the brain receptor targeting ligand comprises the μ-opioid receptor targeting ligand dermorphin, the N-acetylcholine receptor targeting ligand candoxin, the integrin and neurophilin targeting ligand cRGD, the bradykinin targeting ligand cereport, or a combination thereof. In particular embodiments, the brain tumor receptor targeting ligand comprises an EGFR-targeting ligand. In one specific embodiment, the EGFR- targeting ligand comprises peptide 4, an anti-EGFR immunoglobulin or EGFR- binding fragment thereof, EGal-PEG, or a combination thereof.

[0014] In some embodiments, the oil phase of the drug delivery system of the nanoemulsion formulation comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or a combination thereof.

[0015] In some embodiments, the interfacial surface membrane phase of the drug delivery system comprises an emulsifier and/or a stabilizer. In certain embodiments, the emulsifier comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide,

hydrogenated soy phosphatidylcholine, l,2-distearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or a combination thereof. In some embodiments, the stabilizer comprises a polyethylene glycol derivative, a phosphatide, a polyglycerol mono oleate, or a combination thereof.

[0016] The disclosure provides nanoemulsion formulations wherein the therapeutic agent is a chemotherapeutic agent comprising a platinum, a taxol, an aurora kinase inhibitor, an EGFR inhibitor, a src-c inhibitor, a PBK/mTOR inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, or a combination thereof. In certain embodiments, the therapeutic agent comprises the aurora kinase inhibitor NMI-900, the src-c inhibitor Dasatinib, the EGFR inhibitor Erlotinib, or a combination thereof.

[0017] In some embodiments, the nanoemulsion formulation further comprises a chemopotentiator. In specific embodiments, the chemopotentiator comprises ceramide or a derivative thereof, and in one embodiment, the chemopotentiator comprises C6-ceramide. [0018] In some embodiments, the imaging agent is an MRI contrasting moiety, and in certain embodiments, the MRI contrasting moiety comprises gadolinium, iron oxide, iron platinum, manganese, or a combination thereof.

[0019] The invention provides, in another aspect, a method of targeting a nanoemulsion formulation to a specific region of the brain of a mammalian subject. The method comprises administering an effective amount of the nanoemulsion formulation to the subject. The nanoemulsion formulation comprises a targeting agent; a drug delivery system comprising an oil phase; and an interfacial surface membrane; and an aqueous phase; and a therapeutic agent, an imaging agent, or a combination thereof. It is then determined if the nanoemulsion is in the targeted region of the brain.

[0020] In some embodiments, the nanoemulsion formulation is administered orally, intranasally, intraperitoneally, intraocularly, or intravenously.

[0021] In specific embodiments, the nanoemulsion formulation is administered by injection into the cerebrospinal fluid.

[0022] In some embodiments, the targeting agent of the nanoemulsion formulation comprises a brain tumor targeting agent, a brain receptor targeting agent, or combinations thereof. In certain embodiments, the brain receptor targeting agent is an μ-opioid receptor targeting ligand, an N-acetylcholine receptor targeting ligand, an integrin targeting ligand, a neurophilin targeting ligand, a bradykinin targeting ligand, or a combination thereof. In particular embodiments, the brain receptor targeting agent comprises the μ-opioid receptor targeting ligand dermorphin, the N- acetylcholine receptor targeting ligand candoxin, the integrin and neurophilin targeting ligand cRGD, the bradykinin targeting ligand cereport, or combinations thereof. In certain embodiments, the brain tumor receptor targeting agent comprises an EGFR targeting ligand.

[0023] In some embodiments, the oil phase of the drug delivery system of the nanoemulsion formulation comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or a combination thereof.

[0024] In certain embodiments, the interfacial surface membrane of the drug delivery system of the nanoemulsion formulation comprises an emulsifier and/or a stabilizer. In specific embodiments, the emulsifier comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, hydrogenated soy phosphatidylcholine, 1,2-distearoyl-sn- glycero-3-phosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or a combination thereof. In particular embodiments, the nanoemulsion formulation comprises a therapeutic agent, which treats a brain disorder. In some embodiments, the therapeutic agent comprises a platinum, a taxol, an aurora kinase inhibitor, an EGFR inhibitor, a src-c inhibitor, a PI3K/mTOR inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, or a combination thereof. In certain embodiments, the therapeutic agent comprises the aurora kinase inhibitor NMI-900, the src-c inhibitor Dasatinib, the EGFR inhibitor Erlotinib, or a

combination thereof.

[0025] In some embodiments, the nanoemulsion formulation comprises an imaging agent, which images a region of the brain. In particular embodiments, the imaging agent comprises gadolinium, iron oxide, iron, platinum, manganese, or a combination thereof.

[0026] In some embodiments, the nanoemulsion formulation is targeted to a brain cancer in a region of the subject's brain. In specific embodiments, the brain cancer is a Glioblastoma or Glioma.

[0027] In one embodiment, the targeting agent of the nanoemulsion formulation comprises dermorphin, and the region of the brain to which the nanoemulsion formulation is targeted is a region which has opioid receptors.

[0028] The disclosure also provides, in another aspect, a method of inhibiting the growth of, or killing, a cancer cell, comprising contacting the cancer cell with an amount of the nanoemulsion formulation according to the disclosure which is toxic to, inhibits the growth of, or kills, the cancer cell. In one embodiment, the cancer cell is in a mammal, and the contacting step comprises administering to the mammal a therapeutically effective amount of the nanoemulsion formulation. DESCRIPTION OF THE FIGURES

[0029] The foregoing and other objects of the present disclosure, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0030] FIG. 1 is a diagrammatic representation of a generic nanoemulsion formulation of the present disclosure;

[0031] FIG. 2 is a diagrammatic representation of one non-limiting method of preparing a nanoemulsion formulation;

[0032] FIG. 3 is a schematic representation of one non-limiting method of synthesizing EGFR-MAL-PEG-DSPE;

[0033] FIG. 4 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-Dermorphin;

[0034] FIG. 5 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-Candoxin;

[0035] FIG. 6 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-cRGD;

[0036] FIG. 7 is a schematic representation of one non-limiting mode of synthesizing DSPE-PEG-Cereport;

[0037] FIG. 8 is a schematic representation of one non-limiting method of preparing Gd⁺³-DTPA-PE;

[0038] FIG. 9A is a representation of the NMR spectra of a DSPE-PEG-MAL standard;

[0039] FIG. 9B is a representation of the NMR spectra of a EGFR-binding peptide standard;

[0040] FIG. 9C is a representation of the NMR spectra of the DSPE-PEG-MAL- EGFRbp conjugate;

[0041] FIGS. 10A - 10D are a series of representations of magnetic resonance images (MRIs) of rat brain slices after treatment with μ-Opioid receptor targeted,

3+

Gd -labeled nanoemulsion formulation, or non-targeted, Gd -labeled nanoemulsion formulation; and [0042] FIG. 10E is a series of representations of the Tl weighted probability maps of magnetic resonance images (MRIs) on rat brain slices treated with a μ-Opioid receptor targeted, Gd³⁺-labeled nanoemulsion formulation as compared to a non- targeted Gd³⁺-labeled nanoemulsion formulation.

DESCRIPTION

[0043] Throughout this application, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

Definitions

[0044] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0046] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

[0047] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" or "approximately" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0048] The expression "at least one" is used herein to mean one or more and thus includes individual components as well as mixtures/combinations. [0049] "Anticancer agent" or "chemotherapeutic agent" is an agent that prevents or inhibits the development, growth or proliferation of malignant cells.

[0050] "Cancer" is the uncontrolled growth of abnormal cells.

[0051] "Stable chemotherapeutic formulation" is a formulation containing a chemotherapeutic agent or ion wherein the agent or ion is stable for transformation for a time sufficient to be therapeutically useful.

[0052] "Stabilizer" is an agent that prevents or slows the transformation or deactivation of a chemotherapeutic agent or ion in a chemotherapeutic agent formulation.

[0053] "Subject" is a human or animal in need of treatment for cancer.

[0054] A "combination index" is defined as an isobologrm equation to study combination drug effects on cells and to determine whether the drug combination produced enhanced efficacy in the form of an additive, synergistic, or antagonistic effect on cells.

[0055] "Nanoemulsion formulation" as used herein means a novel nanoemulsion (NE) comprising an oil phase; an interfacial surface membrane; an aqueous phase; and a chemotherapeutic agent dispersed in the oil phase.

[0056] "Nanoemulsion" as used herein means a colloidal dispersion comprised of omega-3, -6 or -9 fatty acid rich oils in an aqueous phase and thermo-dynamically stabilized by amphiphilic surfactants, which make up the interfacial surface membrane, produced using a high shear microfluidization process usually with droplet diameter within the range of about 80 nm to 220 nm.

[0057] "Oil phase" as used herein means the internal hydrophobic core of the nanoemulsion in which a chemotherapeutic agent or a mixture of agents is dispersed and refers either to a single pure oil or a mixture of different oils present in the core. The oil phase is comprised of generally regarded as safe grade, parenterally injectable excipients generally selected from omega-3, omega-6 or omega-9 polyunsaturated unsaturated fatty acid (PUFA) or monounsaturated fatty acid rich oils.

[0058] "Aqueous phase" is comprised of isotonicity modifiers and pH adjusting agents in sterile water for injection and forms as an external phase of the

nanoemulsion formulation in which the oil phase is dispersed.

[0059] "Amphiphilic molecule or amphiphilic compound" as used herein means any molecule of bipolar structure comprising at least one hydrophobic portion and at least one hydrophilic portion. The hydrophobic portion distributes into the oil phase and hydrophilic portion distributes into aqueous phase forming an interfacial surface membrane and has the property of reducing the surface tension of water (γ <

55 mN/m) and of reducing the interface tension between water and an oil phase. The synonyms of amphiphilic molecule are, for example, surfactant, surface-active agent and emulsifier.

[0060] "Amphiphilic or amphiphile" as used herein means a molecule with both a polar, hydrophilic portion and a non-polar, hydrophobic portion.

[0061] "Primary emulsifiers" as used herein means amphiphilic surfactants/ compounds that constitute a major percentage of amphiphilic surfactants/compounds of the nanoemulsion formulation wherein they stabilize the formulation by forming an interfacial surface membrane around oil droplets dispersed in water, and further allow for surface modification with targeting ligands and imaging agents.

[0062] "Co-emulsifiers" as used herein means amphiphilic surfactants used in conjunction with primary emulsifiers where they associate with the interfacial surface membrane, effectively lowering the interfacial tension between oil and water, and help in the formation of stable nanoemulsion formulations.

[0063] "Stabilizers" or "stealth agents" as used herein mean lipidated polyethylene glycols (PEG) where the lipid tail group distributes into the oil phase and hydrophilic PEG chains distribute into the aqueous phase of a nanoemulsion formulation, providing steric hindrance to mononuclear phagocytic system (MPS) cell uptake during the blood circulation, thus providing longer residence time in the blood and allowing for enhanced accumulation at tumor site through leaky tumor vasculature, a phenomenon termed as enhanced permeability and retention effect, largely present in wide variety of solid tumors. Other representative examples are a phosphatide, and a polyglycerol mono oleate.

[0064] "Targeting agents" as used herein are molecules, which direct a

nanoemulsion particle towards a particular cell in the body, on example of which is a tumor/cancer cell. Such targeting agents allow for interaction with cells, such as tumor cells in vivo, forming a ligand-receptor complex, which is taken up by the cells.

[0065] "Isotonicity modifiers" as used herein means agents that provide an osmolality (285-310 mOsm/kg) to the nanoemulsion formulation, thus maintaining isotonicity for parenteral injection. [0066] " H modifiers" as used herein means buffering agents that adjust the pH of nanoemulsion formulation to a value of about pH 6-7.4, thus preventing the hydrolysis of phospholipids upon storage.

[0067] "Preservatives" as used herein means antimicrobial agents that when added to the nanoemulsion formulation at about 0.001-0.005% w/v prevent bacterial growth during the storage of nanoemulsion formulation.

[0068] "Antioxidants" as used herein means agents that stop oxidation of oils comprised of fatty acids, thus preventing rancidification of oil phase and

destabilization of the nanoemulsion formulation.

[0069] "Chemopotentiator" as used herein means a drug or chemotherapeutic agent used in combination with other drugs or chemotherapeutic agents to enhance, increase or strengthen the effect, for instance decreases the IC5₀, and thus increasing the efficacy, of the drug or chemotherapeutic agent.

1. Nanoemulsion Formulations

[0070] The present disclosure provides novel methods to target new nanoemulsion formulations to a specific region of the brain. On administering an effective amount of the nanoemulsion formulation the formulation crosses the blood brain barrier and are useful, among others, for treatment of tumors and cancer cells in the brain. The nanoemulsion formulation comprises a targeting agent, a drug delivery system, and a therapeutic agent and/or an imaging agent. The drug delivery system comprises an oil phase, an interfacial surface membrane, and an aqueous phase. After administering the nanoemulsion formulation a determination is made if the nanoemulsion is in the targeted region of the brain.

[0071] FIG. 1 is a non-limiting schematic representation of a nanoemulsion formulation of the present disclosure. In this figure, 4 represents a chemotherapeutic agent dispersed in the oil phase 8 of the nanoemulsion formulation. 8 is encapsulated within the interfacial membrane 7 which comprise emulsifiers 5 and stabilizers 3. The polar, hydrophilic portions of the amphiphiles of the interfacial surface membrane project into the aqueous phase 9, and the non-polar, hydrophobic portions of these amphiphiles project into the oil phase 8. 1 represents a targeting ligand linked to stabilizers 3 in the interfacial surface membrane, 2 represents an imaging agent attached to an emulsifier 5 in the interfacial surface membrane 7 and 6 represents a chemopotentiator or second chemotherapeutic agent dispersed in the oil phase 8 of the nanoemulsion formulation.

[0072] The nanoemulsion formulation of the present disclosure may also comprise a co-agent, a co-emulsifier, a preservative, an antioxidant, a pH adjusting agent, an isotonicity modifier, or any combination thereof.

[0073] Various non-limiting examples of the drug delivery system components of the nanoemulsion formulations and their corresponding proportions are provided in Table I and discussed below.

Table I

A. Therapeutic Agents

[0074] The novel nanoemulsion formulations according to the disclosure contain certain therapeutic agents which can be chemotherapeutic agents or combinations of chemotherapeutic agents. Useful, non-limiting examples of such agents are platinums, taxols, Aurora kinase inhibitors, EGFR inhibitors, Src-c inhibitors, PI3K/mTOR inhibitors, cytoskeletal inhibitors, kinase inhibitors, or Inflammatory Signal Inhibitors. These are listed in Table II.

Table II

Chemotherapeutic Agents and Co-Agents for Nanoemulsion Formulations

NA = Not available

[0075] Non-limiting examples of platinums are Dimyrisplatin, Dipalmiplatin and Distearysplatin. Without being limited to any mechanism of action platinum complexes may react with nucleophilic centers on purine bases of DNA and cross- linking of adjacent guanine bases. To a lesser extent, the platinum center coordinates to guanine bases from different DNA strands to form interstrand cross-links. The major intrastrand dGpG cross-link induces a significant distortion in the DNA double helix. The DNA lesion is then recognized by cellular machinery that either repairs the lesion, bypasses it, or initiates apoptosis. The most significant mechanism by which platinum complexes are believed to induce apoptotic cell death is inhibition of transcription. When RNA polymerases transcribe DNA, they stall at the platinum cross-link and recruit the transcription-coupled repair machinery. If this machinery is unable to repair the lesion, then the cell evokes a programmed cell death pathway.

[0076] Non-limiting examples of Taxols are Docetaxel and Paclitaxel. Docetaxel acts by disrupting the microtubular network in cells that is essential for mitotic and interphase cellular functions and binds to free tubulin and promotes the assembly of tubulin into stable microtubules while simultaneously inhibiting their disassembly leading to the production of microtubule bundles without normal function and to the stabilization of microtubules, which results in the inhibition of mitosis in cells.

Paclitaxel stabilizes the microtubular polymer and protects it from disassembly. Chromosomes are then unable to achieve a metaphase spindle configuration. This stabilization blocks progression of mitosis, and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the G-phase of the cell cycle without cell division.

[0077] A non-limiting example of an aurora kinase inhibitor is NMI-900 {3-(4-(4-(2- (3 -((dimethylamino)-methyl)phenyl)- 1 H-pyrrolo[2,3 -b]pyridin-4-yl)- 1 -ethyl- 1 H- pyrazol-3-yl)phenyl)-l, l-dimethylurea} . NMI-900 is an ATP-competitive inhibitor of the serine/threonine kinases Aurora B and C and binds to and inhibits the activity of Aurora kinases B and C, which may result in inhibition of cellular division and a decrease in the proliferation of tumor cells that overexpress the Aurora kinases B and C. Aurora kinases play essential roles in mitotic checkpoint control during mitosis, and are overexpressed by a wide variety of cancer cell types.

[0078] Non-limiting examples of EGFR inhibitors are Erlotinib, Gefitinib,

Rociletinib, and AZD9291. Erlotinib is designed to block tumor cell growth by targeting a protein EGFR (epidermal growth factor) that is present on the surface of some cancer cells and some normal cells. Erlotinib inhibits an enzyme within the cell (tyrosine kinase) that is associated with EGFR. Gefitinib works by binding to the intracellular enzyme (tyrosine kinase) of the EGFR to directly block signals turned on by triggers outside or inside the cell and inhibits the intracellular phosphorylation of numerous tyrosine kinases associated with transmembrane cell surface receptors, including the tyrosine kinases associated with the epidermal growth factor receptor (EGFR-TK). Rociletinib was designed to form a covalent bond in a highly directed and controlled manner to potently inhibit the mutant forms of the EGFR, while sparing normal EGFR, providing efficacy without "off-target" side effects or side effects due to inhibition of normal receptor functions. AZD9291 is a potent and selective mutated forms of the EGFR inhibitor.

[0079] Non-limiting examples of Src-c inhibitors are Dasatinib and AZD0530.

Dasatinib at nanomolar concentrations, inhibits the following kinases: BCR-ABL, SRC family (SRC, LCK, YES, FYN), c-KIT, EPHA2, and PDGFR , and inhibits cell growth. AZD0530 is a highly selective, dual Src/Abl kinase inhibitor and reverses ABCB 1 -mediated resistance in vitro and in vivo by directly inhibiting ABCB 1 transport function, without altering ABCB 1 expression or AKT phosphorylation.

[00801 Non-limiting examples of PBK/mTOR inhibitors are XL765 and NVP- BEZ235. XL765 s an orally available small molecule that has been shown in preclinical studies to selectively inhibit the activity of phosphoinositide-3 kinase (PI3K) and mammalian target of rapamycin (mTOR). NVP-BEZ235 seems to inhibit effectively both wild-type and mutant form of PIK3CA and in vivo models have confirmed these potent antineoplastic effects of dual mTOR/PI3K inhibitors.

[0081] A non-limiting example of a cytoskeletal inhibitor is noscapine, which binds to tubulin and alters its conformation, resulting in a disruption of the dynamics of microtubule assembly, thereby arresting cell growth and inducing apoptosis.

[0082] Non-limiting examples of kinase inhibitors are UCN-01, staurosporine, and/or SP600125. UCN-01 is a potent Chkl inhibitor that binds to the ATP-binding pocket of Chkl,which abrogates the G2/M checkpoint, resulting in cell cycle arrest.

Staurosporine is a Chkl inhibitor that binds to the ATP-binding pocket of Chkl abrogating the G2/M checkpoint, resulting in cell cycle arrest. SP600125 inhibits Jun N-terminal kinase and mediates downstream effects resulting in inhibition of cell proliferation. It is also a mullerian-inhibiting substance (MIS) agonist and activates the MIS transduction pathway by binding to MIS type II receptors, resulting in cell growth inhibition. [0083] A non-limiting example of inflammatory signal inhibitors is BAY 1 1-7082, which inhibits transcription factor NFkB that controls cell growth, apoptosis and differentiation, resulting in CSCs cell death.

[0084] These chemotherapeutic agents are lipophilic, forming stable nanoemulsions either alone or in combination with chemopotentiators or co-agents, and thus are available for use as anticancer agents having a high specificity and selectivity to cancer cells. Moreover, their liposolubility makes them useful as slowly and steadily released and sustained nanomedicines. The encapsulation of chemotherapeutic agents, including those in combination with chemopotentiators and/or co-agents in the nanoemulsion formulations of the present disclosure, aid in mitigating undesirable side-effects known to sometimes accompany their use and allow them to cross the

BBB.

[0085] The nanoemulsion formulations of the present disclosure may comprise a chemopotentiator, such as an apoptosis enhancer. Non-limiting examples for apoptosis enhancers are ceramide (CER), cyclopamine, sulforaphane, curcumin, or ceramide or curcumin derivatives. These chemopotentiators enhance apoptosis and the increase the ability of chemotherapeutic agents to kill cancer cells.

[0086] C-6 CER is one such chemopotentiator, whose structure is:

[0087] Over-expression of Glucosylceramide synthase is associated with decreased rates of apoptosis in many cancer types. Endogenous addition of CER can

significantly enhance the apoptotic potential of chemotherapies, thus improving efficacy (Shabbits et al. (2003) Biochim..Biophvs. Acta, 1612:98-106). Replacement of CER mediates induction of apoptosis via the inhibition of Akt pro-survival pathways, mitochondrial dysfunction, and stimulation of caspase activity, ultimately leading to DNA fragmentation. [0088] Cyclopamine inhibits the Hedgehog signaling pathway by directly binding to a membrane receptor smoothened, resulting in apoptosis and supression of renewal of CSCs. Sulforaphane inhibits Akt pro-survival pathways and down regulates the Wnt/ -catenin pathway that is critical to the self-renewal of CSCs and differentiation. These events lead to cell growth arrest, overcoming drug resistance, and apoptosis.

[0089] Curcumin interferes with the NFkB, Akt/mTOR/p70S6K molecular signaling pathways and drug efflux pumps, resulting in apoptosis and sensitization of cells to chemotherapy. It also nhibits Wnt patway invovled in CSCs growth and self-renewal.

[0090] While chemopotentiators seem to enhance the efficacy of chemotherapeutic agents, there are obstacles to the delivery of these compounds. First, their effectiveness is limited due to their hydrophobicity and possible precipitation when administered in aqueous solutions. In addition, the structures of the

chemopotentiators, such as the existence of a second aliphatic chain of CER, can hinder cellular permeability. Also, some of the free chemopotentiators are susceptible to metabolic inactivation by specific enzymes in the systemic circulation. The present nanoemulsion formulations exploit the benefits of the chemopotentiators/apoptosis enhancers by providing increased solubility, intracellular permeability, and protection from systemic enzymatic degradation.

B. Drug Delivery System (1) Oil Phase

[0091] The oil phase of the nanoemulsion formulation according to the present disclosure comprises individual oil droplets. The average diameter of the oil droplets in the oil phase ranges from about 5 nm to 500 nm. This component is the internal hydrophobic or oil core and may be a single entity or a mixture.

[0092] A wide variety of oils and methods for forming nanoemulsion formulations therefrom are known in the art of drug delivery. The oil phase of the disclosed nanoemulsion formulations may include at least one polyunsaturated fatty acid (PUFA)-rich oil, for example, a first oil that may contain a polyunsaturated oil, for example linolenic acid, and optionally an oil that may be for example a saturated fatty acid, for example icosanaic acid. [0093] Any oil can be used in accordance with the present invention. Oils can be natural or unnatural (synthetic) oils. Oils can be homogeneous or oils comprising two or more monounsaturated fatty acid or PUFA-rich oils. Contemplated oils may be biocompatible and/or biodegradable.

[0094] Biocompatible oils do not typically induce an adverse response (such as, but not limited to, an immune response with significant inflammation and/or acute rejection) when inserted or injected into a living subject. Accordingly, the therapeutic nanoemulsion formulations contemplated herein can be non-immunogenic.

[0095] One simple test to determine biocompatibility is to expose a nanoemulsion formulation to cells in vitro. Useful biocompatible oils in the nanoemulsion formulation do not result in significant cell death at moderate concentrations, e.g., 50 μg/10⁶ cells. For example, these biocompatible oils can cause less than about 20% cell death when exposed to or taken up by, fibroblasts or epithelial cells. Non-limiting examples of biocompatible oil useful in nanoemulsion formulations of the present disclosure include alpha linolenic acid, pinolenic acid, gamma linolenic, linoleic acid, oleic acid, icosenoic acid, palmitic acid, stearic acid, icosanaic acid, and derivatives thereof.

[0096] The biocompatible oils may be biodegradable, i.e., able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. As used herein, "biodegradable" oils are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells. Both the biodegradable oils and their degradation byproducts can be biocompatible.

[0097] Another useful oil is flaxseed oil which is a biocompatible and

biodegradable oil of alpha linolenic, linoleic, and oleic. Useful forms of this oil can be characterized by the ratio of alpha linolenic: linoleic: oleic. The degradation rate of flaxseed oil can be adjusted by altering the alpha linolenic:linoleic:oleic ratio, e.g., having a molar ratio of about 65:5:30, about 65:20: 15, about 55: 15:30, or about 55:20:25.

[0098] The nanoemulsion formulations may include an oil phase of saturated fatty acid, monounsaturated fatty acid or PUFA rich oils that are biocompatible and/or biodegradable. [0099] Oil compositions suitable for use as the oil phase of the nanoemulsion formulations according to the present disclosure can be from any source rich in mono- saturated or PUFAs, such as plant or animal sources. Chemically or enzymatically derivatized, or completely synthetic, monounsaturated or PUFAs are included within the scope of suitable components for the oil phase of the nanoemulsion formulations of the present disclosure. The concentration of the mono-unsaturated or PUFA in the oil phase can range from about 2% to about 100% (w/w), from about 5% to about 100% (w/w), or greater than 10% (w/w), and from about 20% to about 80% (w/w). The concentration of the oil phase, in the nanoemulsion formulation can vary from about 5% to about 40% (w/w), or from about 5% to about 30% (w/w). The concentration of the chemotherapeutic agent soluble in the oil phase can range from about 0.01% to about 90% (w/w), from about 0.1% to about 45% (w/w), or greater than 0.5%, or from about 1% to about 30% (w/w). For example, the oils may contain high concentrations of mono-saturated or PUFAs such as a concentration of greater than or equal to 10% (w/w) of at least one mono-unsaturated or PUFA of the omega- 3, omega-6, or omega-9 family. A useful oil is one that can solubilize high concentrations of a chemotherapeutic agent, such as those containing high concentrations of linolenic or linoleic acid (e.g., oils of flax seed oil, black currant oil, pine nut oil or borage oil), and fungal oils such as spirulina and the like, alone or in combination.

(2) Aqueous Phase

[00100] The aqueous phase of the drug delivery system of the nanoemulsion formulations according to the disclosure is purified and/or ultrapure water. This aqueous phase can also contain isotonicity modifiers such as, but not limited to, glycerine, low molecular weight polyethylene glycol (PEG), sorbitol, xylitol, or dextrose. The aqueous phase can alternatively or also contain pH adjusting agents such as, but not limited to, sodium hydroxide, hydrochloric acid, free fatty acids (oleic acid, linoleic acid, stearic acid, palmitic acid) and their sodium and potassium salts, preservative parabens, such as, but not limited to, methyl paraben or propyl paraben; antioxidants such as, but not limited to, ascorbic acid, a-tocopherol, and/or butylated hydroxy anisole. The concentration of the aqueous phase in the present nanoemulsion formulations can vary from about 30% to about 95% (w/w), from about 30% to about 75% (w/w), or from about 50% to about 80% (w/w), or from about 40% to about 70% (w/w), or from about 60% to about 90% (w/w).

(3) Interfacial Surface Membrane

[00101] The term "interfacial surface membrane" as used herein applies to the interface of the oil and aqueous phase and may refer either to a single pure emulsifier or a mixture of different emulsifiers and/or a mixture of emulsifiers and other components, such as stealth agents (stabilizers) present in the interfacial surface membrane of the nanoemulsion formulation. The interfacial surface membrane or corona can comprise degradable lipids or emulsifiers bearing neutral, cationic and/or anionic side chains. The average surface area of the interfacial surface membrane corona on the nanoemulsion formulations described herein from may range from 100 nm² to 750,000 nm².

[00102] The interfacial surface membrane component of the drug delivery system of the present nanoemulsion formulations comprises an emulsifier and may also comprise a stabilizer (stealth agent).

(a) Emulsifiers

[00103] At least one emulsifier forms part of the interface between the hydrophobic or oil phase and the aqueous phase. They comprise individual amphiphilic lipids and/or amphiphilic polymers. The emulsifier can be an amphiphilic molecule such as a nonionic and ionic amphiphilic molecule. For example, the emulsifier can consist of neutral, positively-charged, or negatively-charged, natural or synthetic phospholipids molecules such as, but not limited to, natural phospholipids including soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol,

phosphatidylethanolamine, phosphatidic acid, sphingomyelin, diphosphatidyl- glycerol, phosphatidylserine, phosphatidylcholine and cardiolipin; synthetic phospholipids including 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine, dimyristoylphosphatidylcholine, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, dimyristoylphosphatidylglycerol, distearoylphos- phatidylglycerol and dipalmitoylphosphatidylcholine; and hydrogenated or partially hydrogenated lecithins and phospholipids, e.g., from a natural source are used. The concentration of amphiphilic lipid in the nanoemulsion formulations can vary from about 0.5% to about 15% (w/v), or from about 1% to about 10% (w/v).

[00104] One non-limiting example of a nanoemulsion formulation of the present disclosure comprises oil and amphiphilic compounds of the interfacial surface membrane which surround or are dispersed within the oil and which form a continuous or discontinuous monomolecular layer. The interfacial surface membrane lowers the interfacial tension between the oil and aqueous phases, thereby enhancing the stability of the dispersed oil droplets in the surrounding aqueous phase. Further, the interfacial surface membrane of the nanoemulsion formulation localizes drugs, thereby providing therapeutic advantages by releasing the encapsulated

chemotherapeutic drug at predetermined, appropriate times.

[00105] An amphiphilic compound may have a polar head attached to a long hydrophobic tail. The polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. Exemplary amphiphilic compounds include, for example, one or a plurality of the following: naturally derived lipids, surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.

[00106] Non-limiting examples of amphiphilic compounds making up a

representative emulsifier include phospholipids, such as 1,2 distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of about 0.5% to about 2.5% (weight lipid/w oil), about between 1.0% to about 1.5% (weight lipid/w oil). Useful phosphatidic acids, phosphatidyl cholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines, phosphatidylglycerols,

phosphatidylserines, phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, and β-acyl-y-alkyl phospholipids. Examples of phospholipids include, but are not limited to, phosphatidylcholines such as l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine, dioleoylphosphatidylcholine, dimyristoylphos-phatidylcholine, dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine, and

phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or

1 -hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another acyl chain of 12 carbons) may also be used.

[00107] An amphiphilic compound of the interfacial membrane may include lecithin or phosphatidylcholine.

(b) Stabilizers

[00108] When the interfacial surface membrane comprises a stabilizer or stealth agent, it can be added with the emulsifier when preparing a nanoemulsion formulation of the present disclosure. The stabilizer may be an amphiphilic molecule.

[00109] One non-limiting representative stabilizer is a PEGylated lipid. Some useful phospholipid molecules are natural phospholipids including polyethylene glycol (PEG) repeat units, which can also be referred to as a "PEGylated" lipid or lipidated PEG. Such PEGylated lipids can control inflammation and/or immunogenicity (i.e., the ability to provoke an immune response) and/or lower the rate of clearance from the circulatory system via the reticuloendothelial system (RES) and/or the mononuclear phagocyte system (MPS), due to the presence of the poly(ethylene glycol) groups, PEGylated soybean lecithin, PEGylated egg lecithin, PEGylated phosphati-dylglycerol, PEGylated phosphatidylinositol, PEGylated

phosphatidylethanolamine, PEGylated phosphatidic acid, PEGylated sphingomyelin, PEGylated diphosphatidylglycerol, PEGylated phosphatidylserine, PEGylated phosphatidylcholine and PEGylated cardiolipin; synthetic phospholipids including PEGylated dimyristoylphosphatidylcholine, PEGylated dimyristoylphosphatidyl- glycerol, PEGylated distearoylphosphatidylglycerol and PEGylated

dipalmitoylphosphatidylcholine; and hydrogenated or partially hydrogenated

PEGylated lecithins and PEGylated phospholipids. Such amphiphilic PEGylated lipids can be used alone or in combination. The concentration of amphiphilic

PEGylated lipid in the nanoemulsions can vary from about 0.01% to 15% (w/v), or from about 0.05% to 10% (w/v). [00110] Exemplary lipids that can be part of the PEGylated lipid include, but are not limited to, fatty acids such as long chain (e.g., C8-C50), substituted, or unsubstituted hydrocarbons. A fatty acid group can be a C10-C20 fatty acid or salt thereof, a C15- C20 fatty acid or salt thereof, or a fatty acid can be unsaturated, monounsaturated, or polyunsaturated. For example, a fatty acid group can be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.

[00111] Other exemplary stabilizers are phosphatide, a polyglycerol mono oleate, PEGioooDSPE, PEG₂₀ooDSPE, PEG₃₄₀₀DSPE, PEG₅₀₀₀DSPE, or any combination thereof. Additional useful stabilizers are PEG derivatives, a phosphatide, and/or polyglycerol mono oleate and useful non-limiting PEG derivatives are PEGioooDSPE, PEG₂₀ooDSPE, PEG₃₄₀oDSPE, PEG₅₀ooDSPE.

[00112] The PEGylation density may be varied as necessary to facilitate long- circulation in the blood (Perry et al. (2012) Nano. Lett. 12:5304-5310). In some cases, the addition of PEG repeat units may increase plasma half-life of the nanoemulsion formulation, for instance, by decreasing the uptake of the nanoemulsion formulation by the MPS, while decreasing transfection/uptake efficiency by cells. Those of ordinary skill in the art will know of methods and techniques for PEGylating a lipid, for example, by using EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxy-succinimide) to react a lipid that will be in the corona of the nanoemulsion formulation to a PEG group terminating in an amine, by ring opening polymerization techniques (ROMP), and the like.

[00113] PEG may include a terminal end group, for example, when PEG is not conjugated to a ligand. For example, PEG may terminate in a hydroxyl, a methoxy or other alkoxyl group, a methyl or other alkyl group, an aryl group, a carboxylic acid, an amine, an amide, an acetyl group, a guanidino group, or an imidazole. Other contemplated end groups include azide, alkyne, maleimide, aldehyde, hydrazide, hydroxylamine, alkoxyamine, or thiol moieties.

[00114] The molecular weight of the PEG on interfacial membrane surface of the nanoemulsion formulation can be optimized for effective treatment as disclosed herein. For example, the molecular weight of a PEG may influence particle degradation rate (such as adjusting the molecular weight of a biodegradable PEG), solubility, water uptake, and drug release kinetics. For example, the molecular weight of the PEG can be adjusted such that the particle biodegrades in the subject being treated within a period of time ranging from a few hours, to 1 week to 2 weeks, 3 weeks to 4 weeks, 5 weeks to 6 weeks, 7 weeks to 8 weeks, etc. One exemplary useful nanoemulsion formulation comprises a copolymer PEG conjugated to a lipid, the PEG having a molecular weight of about 1 kDa to about 20 kDa, about 5 kDa to about 20 kDa, or about 10 kDa to about 20 kDa, and the lipid can have a molecular weight of about 200 D to about 3 kDa, about 500 D to about 2.5 kDa, or about 700 D to about 1.5 kDa. An exemplary nanoemulsion formulation includes about 5 weight percent (wt %) to about 30 wt % monounsaturated or polyunsaturated fatty acid rich oil, or about 0.5 wt % to about 5 wt % primary emulsifier, or about 0.1 wt % to about 1.0 wt % co-emulsifiers, or about 0.1 wt % to about 0.75 wt %, PEG-derivatives. Exemplary lipid-PEG copolymers can include a number average molecular weight of about 1.5 kDa to about 25 kDa, or of about

2 kDa to about 20 kDa.

[00115] The ratio of oil to emulsifier to stabilizer in the nanoemulsion formulation for example, flax seed oil to emulsifier to PEGylated lipid stabilizer, may be selected to optimize certain parameters such as size, chemotherapeutic agent release, and/or nanoemulsion formulation degradation kinetics.

[00116] An alternative stabilizer may contain poly(ester-ether)s. For example, the interfacial membrane surfaces of the nanoemulsion formulation can have repeat units joined by ester bonds (e.g., R— C(O)— O— R' bonds) and ether bonds (e.g., R— O— R' bonds). A biodegradable component of the interfacial membrane surface of the nanoemulsion formulation, such as a hydrolyzable biopolymer containing carboxylic acid groups, may be conjugated with poly(ethylene glycol) repeat units to form a poly(ester-ether) coating on the interfacial membrane surface of the nanoemulsion formulation.

(c) Targeting Ligands

[00117] The novel nanoemulsion formulations according to the disclosure further comprise certain targeting ligands which guide the nanoemulsion to a particular region of the brain. Representative useful targeting ligands are, e.g., a low-molecular weight ligand, protein, carbohydrate, or nucleic acid. Use of a targeting ligand enables more accurate delivery of the nanoemulsion formulation to specific region of the brain, such as to the cancer cells in the brain.

[00118] One such useful target is epidermal growth factor receptor (EGFR) (see, e.g., Magadala et al. (2008) AAPS. J. 10:565-576). EGFR is a member of the human epidermal growth factor receptor HER/erb family of receptor tyrosine kinases, which plays important roles in both cell growth and differentiation. Overexpression of EGFR is associated negatively with progression-free and overall survival in a wide variety of human cancers, including brain cancer. Its positive signaling causes increased proliferation, decreased apoptosis, and enhanced tumor cell motility and angiogenesis.

[00119] Useful EGFR-targeting ligands include, but are not limited to, peptide 4: Y-H-W-Y-G-Y-T-P-Q-N-V-I (SEQ ID. NO: l) or an anti-EGFR immunoglobulin, e.g., a nanobody such as EGal-PEG.

[00120] Other useful targets for the brain are Penetratin, the μ-Opioid Receptor, the N-Acetylcholine Receptor, Integrin, Neuropilin, and Bradykinin in the brain. These targets and useful non-limiting examples of targeting ligands are listed in Table III (on following page).

Table III

Brain Receptor Targeting Ligands

[00121] In some nanoemulsion formulations, the targeting moieties are attached, e.g., covalently bonded, to a lipid component of the nanoemulsion formulation. One exemplary nanoemulsion formulation comprises a chemotherapeutic agent, an oil core comprising functionalized and non-functionalized oils, an interfacial surface membrane or corona, and a low-molecular weight targeting ligand, wherein the targeting ligand is covalently bonded, to the lipid component of the nanoemulsion formulation's interfacial surface membrane.

2. Preparation of Nanoemulsion Formulations

[00122] The nanoemulsion formulations of the present disclosure can be prepared from various intermediates and component constituents, e.g., as described in Examples 1-4 below, and can be made using a microfluidizer (Microfluidics Corp., Newton, MA).

[00123] FIG. 2 shows a representative synthesis scheme for one non-limiting, μ- opioid receptor-targeted, Gd-labeled nanoemulsion formulation of the present disclosure. In this figure, 1 is a chemotherapeutic agent. 2 is a second

chemotherapeutic agent, or a proapoptotic agent. 3 represents the compounds 1 and 2 being dissolved in chloroform and added to flaxseed oil. Chloroform is removed using nitrogen, and mixture is then heated at 60°C for 2 min resulting in oil phase formation. 4 is the imaging moiety Gd-DTPA-PE. 5 is the targeting ligand Dermorphin-PEG- DSPE. 6 represents the compounds of 4 and 5 being added to egg lecithin and

PEG₂₀₀₀DSPE in glycerol water solution, and mixture is then heated at 60°C for 2 min resulting in aqueous phase formation 6. 7 represents the oil phase of 3 and aqueous phase of 6 being combined, and mixed for 30 seconds to form the coarse emulsion. 8 represents the coarse emulsion of 7 being emulsified using a high pressure homogenizer (LVl Microfluidizer) at 25,000 psi for 10 cycles to obtain nanoemulsion formulation droplets of a size below 150 nm. 9 is the resulting nanoemulsion formulation of one embodiment of the present disclosure with a size below 150 nm. 10 is a representative drawing of an individual resulting nanoemulsion formulation.

[00124] An initial screening step for chemotherapeutic agents that may be suitable candidates for the present nanoemulsion formulations is to test their solubility in various oils that can be used to form the oil phase of the nanoemulsion formulation. The solubility of representative chemotherapeutic agents in an oil phase is shown in Table IV. The solubility was estimated by visual observation. The nanoemulsion formulations of the present disclosure were prepared with a concentration of chemotherapeutic agent from about 0.5 mg/ml to about 20 mg/ml.

Table IV

Drug Solubility in

3. Characterization of Nanoemulsion Formulations

[00125] The nanoemulsion formulations of the present disclosure may have a substantially spherical or non-spherical shape. For instance, the nanoemulsion formulations initially may appear to be spherical, but upon shrinkage, may adopt a non-spherical configuration. These nanoemulsion formulations may have a characteristic dimension of less than about 1 μιη, where the characteristic dimension of a nanoemulsion formulation is the diameter of a perfect sphere having the same volume as the nanoemulsion formulation. For example, the characteristic dimensions of the nanoemulsion formulation can be less than about 300 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, or less than about 50 nm. Some disclosed nanoemulsion formulations may have a diameter of about 50 nm to 200 nm, about 50 nm to about 180 nm, about 80 nm to about 160 nm, or about 80 nm to about 150 nm. The size of the nanoemulsion formulation particles can be determined by dynamic light scattering (DLS) (Zetasizer ZS, Malvern Instruments Ltd.,

Worcestershire, United Kingdom). The size distribution and zeta potential values of specific exemplary nanoemulsion formulations are shown in Table V. Table V

Size and Charge of Demorphin, Candoxin and Cereport-targeted NE

[00126] These nanoemulsion formulations were prepared using Microfluidics LVI (10 cycles of 25,000 PSI). Nanoemulsion samples were diluted 1 : 1000 in distilled water for size and charge analysis. Size and charge were measured using Malvern Zetasizer ZS. The average particle size of the control nanoemulsion formulation containing no chemotherapeutic agent was below 200 nm in diameter. The incorporation of chemotherapeutic agents alone or with chemopotentiators and/or co- agents in the nanoemulsion formulations did not significantly change the

hydrodynamic particle size and size remained below 200 nm. The average surface charges of the nanoemulsion formulations were in the range of about -38 mV to -56 mV.

[00127] Nanoemulsion formulation size distribution of control blank nanoemulsion formulation, and nanoemulsion formulations with chemotherapeutic agents alone or with chemopotentiators and/or co-agents were determined using Zetasizer ZS (Malvern Instruments, Worcestershire, United Kingdom) at 4°C for up to one month. The results are shown in below Tables VI and VII.

Table VI

Physical Stability of Candoxin and Dermorphin-Targeted NE Formulations

* Size in nm; * * Polydispersity Index Table VII

Physical Stability of Candoxin and Dermorphin-Targeted NE Formulations

* Zeta Potential in mV; * * Standard Deviation

[00128] The average particle size of the targeted nanoemulsion formulation containing an agents remained below 200 nm in diameter for up to about 1 month and the Polydispersity index and zeta potentials also did not change significantly indicating that the nanoemulsion formulations were stable at 4°C for up to 1 month.

[00129] The nanoemulsion formulations of the present disclosure may have an interior and a surface, where the surface has a composition different from the interior, i.e., there may be at least one compound present in the interior but not present on the surface (or vice versa), and/or at least one compound is present in the interior and on the surface at differing concentrations. For example, a compound, such as a targeting moiety ligand, may be present in both the interior and the surface of the nanoemulsion formulation, but at a higher concentration on the surface than in the interior of the nanoemulsion formulation, although in some cases, the concentration in the interior of the nanoemulsion formulation may be essentially nonzero, i.e., there is a detectable amount of the compound present in the interior of the nanoemulsion.

[00130] To determine what effect targeted nanoemulsion formulations have on cellular uptake, the uptake of the formulation into cancer cells can be measured, e.g., using fluorescence. For example, cancer cells growing on cover slips in 6-well plate at 3000 cells/well are incubated with NBD-CER fluorescently labeled nanoemulsion formulations for varying times, e.g., 5 minutes, 15 minutes, or 30 minutes. At the end of incubation period, cells are washed thrice with phosphate buffered saline (PBS) and incubated with Lyso Tracker and DAPI for 10 minutes, which stains lysosomes and nucleus of the cells, respectively. Cells are further washed with PBS, inverted, and mounted on glass slides using Flouromount G mounting media. [00131] DIC/Fluorescent images of fluorescently labeled cells treated with nanoemulsion formulation according to the present disclosure are acquired, e.g., using a Confocal Zeiss LSM 700 microscope with an object 63 x oil immersion over a 30 minute period.

[00132] A fluorescently labeled agent, such as 0.01% NBD-Ceramide at 0.01% w/v is incorporated in all formulations. Lyso Tracker and DAPI were used to monitor the co-localization of the nanoemulsion formulations in the cells.

[00133] These images show that the fluorescently labeled agent co-localizes with Lyso Tracker, indicating entry of the agent into lysosomes. The study demonstrates that the nanoemulsion formulations of the present disclosure are able to evade the drug degradative lysosomal pathway, thereby enhancing drug concentrations in cells in vitro.

[00134] In some cases, the interior of the nanoemulsion formulation is more hydrophobic than the surface of the nanoemulsion formulation. For instance, the interior of the nanoemulsion formulation may be relatively hydrophobic with respect to the surface of the nanoemulsion formulation, and a drug or other payload may be hydrophobic, and readily associates with the relatively hydrophobic center of the nanoemulsion formulation. The drug or other payload can thus be contained within the interior of the nanoemulsion formulation, which can shelter it from the external environment surrounding the nanoemulsion formulation (or vice versa). For example, a chemotherapeutic drug or other payload contained within the delivery system of the nanoemulsion formulation administered to a subject will be protected from a subject's body, and the body may also be substantially isolated from the drug for at least a period of time.

[00135] An exemplary nanoemulsion formulation may have a PEG derivative corona with a density of about 1.065 g/cm³, or about 1.01 g/cm³ to about 1.10 g/cm³.

[00136] The nanoemulsion formulations of the present disclosure may have controlled release properties, e.g., may be capable of delivering an amount of active agent to a subject, for example to a specific site in a subject, over an extended period of time, for example over 1 day, 1 week, or more. Some disclosed nanoemulsion formulations substantially immediately release (for example over about 1 minute to about 30 minutes), less than about 2% in 6 hours, less than about 4% in 24 hours, less than about 7% in 48 hours, or less than about 10% of a chemotherapeutic agent in 72 hours, for example when placed in a phosphate buffer saline solution at room temperature and/or at 37°C.

4. Method of Treatment

[00137] The nanoemulsion formulation in accordance with the present disclosure may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a brain disorder such as a cancer or tumor.

[00138] The term "cancer" includes pre-malignant as well as malignant cancers. Cancer cells can be in the form of a tumor or exist alone within a subject (e.g., leukemia cells). The term "tumor" includes nonmalignant and malignant tumors.

[00139] In certain cases, targeted nanoemulsion formulation may be used to treat any cancer where EGFR is expressed on the surface of cancer cells or in the tumor neovasculature, including the neovasculature of brain or other solid tumors. Examples of the EGFR-receptor-related indications include, but are not limited to, brain cancers.

[00140] When treating cancer, a therapeutically-effective amount of the

nanoemulsion formulation of the present disclosure is administered and is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of the cancer.

[00141] As will be appreciated by those of ordinary skill in this art, the effective amount of the nanoemulsion formulation may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of the nanoemulsion formulation is the amount that results in a reduction in tumor size by a desired amount over a desired period of time. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the subject being treated; diet, time and frequency of administration; reaction sensitivities; and tolerance/response to therapy.

[00142] The nanoemulsion formulations of the present disclosure can be used to inhibit the growth of, or kill, cancer cells. As used herein, the term "inhibits growth of cancer cells" or "inhibiting growth of cancer cells" refers to any slowing of the rate of cancer cell proliferation and/or migration, arrest of cancer cell proliferation and/or migration, or killing of cancer cells, such that the rate of cancer cell growth is reduced in comparison with the observed or predicted rate of growth of an untreated control cancer cell. The term "inhibits growth" can also refer to a reduction in size or disappearance of a cancer cell or tumor, as well as to a reduction in its metastatic potential. Such an inhibition at the cellular level may reduce the size, deter the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a cancer in a subject. Those skilled in the art can readily determine, by any of a variety of suitable indicia, whether cancer cell growth is inhibited.

[00143] Inhibition of cancer cell growth may be evidenced, for example, by arrest of cancer cells in a particular phase of the cell cycle, e.g., arrest at the G2/M phase of the cell cycle. Inhibition of cancer cell growth can also be evidenced by direct or indirect measurement of cancer cell or tumor size. In human cancer subjects, such

measurements generally are made using well known imaging methods such as magnetic resonance imaging, computerized axial tomography and X-rays. Cancer cell growth can also be determined indirectly, such as by determining the levels of circulating carcinoembryonic antigen, prostate specific antigen or other cancer- specific antigens that are correlated with cancer cell growth. Inhibition of cancer growth is also generally correlated with prolonged survival and/or increased health and well-being of the subject.

[00144] Also provided herein are prophylactic therapeutic protocols that include administering a therapeutically effective amount of a disclosed therapeutic nanoemulsion formulation to a healthy individual (i.e., a subject who does not display any symptoms of cancer and/or who has not been diagnosed with cancer). For example, healthy individuals may be "immunized" with an inventive targeted or non- targeted particle, such as a nanoemulsion formulation, prior to development of cancer and/or onset of symptoms of cancer; at risk individuals (for example, subjects who have a family history of cancer; subjects carrying one or more genetic mutations associated with development of cancer; subjects having a genetic polymorphism associated with development of cancer; subjects infected by a virus associated with development of cancer; subjects with habits and/or lifestyles associated with development of cancer; etc.) can be treated substantially contemporaneously with (for example, within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of cancer. Individuals known to have cancer may receive inventive treatment at any time.

[00145] Nanoemulsion formulations disclosed herein may be combined with pharmaceutical acceptable carriers that do not change the therapeutic and other characteristics of the formulation to form the pharmaceutical formulation. As would be appreciated by one of skill in this art, the carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.

5. Methods of Administration

[00146] The nanoemulsion formulations of this disclosure can be administered to a subject by any means known in the art including oral and parenteral routes. The term "subject" as used herein, refers to mammals such as humans and non-humans, and non-mammals including, for example, mammals, birds, reptiles, amphibians, and fish. Sometimes parenteral routes are chosen since they avoid contact with the digestive enzymes that are found in the alimentary canal. These compositions may be administered by injection (e.g., intravenous, intraocular, subcutaneous or

intramuscular, intraperitoneal, cerebrospinal fluid), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).

[00147] For example, the nanoemulsion formulations of the present disclosure are administered to a subject in need thereof systemically, e.g., by intravenous infusion or injection. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parentally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. [00148] The nanoemulsion formulations may also be administered orally. Solid dosage forms for oral administration include liquids, capsules, tablets, pills, and granules.

[00149] It will be appreciated that the exact dosage of the nanoemulsion formulation of the present disclosure is chosen by the individual physician in view of the subject to be treated. In general, dosage and administration are adjusted to provide an effective amount of the nanoemulsion formulation to the subject being treated. As used herein, the "effective amount" of a nanoemulsion formulation refers to the amount that elicits the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of the nanoemulsion formulations may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of the nanoemulsion formulation is the amount that results in a reduction in tumor size by a desired amount over a desired period of time. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the subject being treated; diet, time and frequency of administration; reaction sensitivities; and tolerance/response to therapy.

[00150] The nanoemulsion formulation of the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of the nanoemulsion formulation appropriate for the subject to be treated. It will be understood, however, that the total daily usage of the nanoemulsion formulations of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. For any nanoemulsion formulation, the therapeutically- effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of the nanoemulsion formulations can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD5₀/ED5₀. Nano- emulsion formulations, which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

[00151] For example, the nanoemulsion formulation may contain a chemotherapeutic agent at a concentration of about 0.001% to about 2% (about 0.01 mg/ml to about 20 mg/ml). The dosage administered by injection may contain chemotherapeutic agents in the range of about 5 mg to about 1000 mg in the first day of every 1 week to 4 weeks depending upon the subject. One might administer a dosage of about

O.Olmg/kg of subject body weight to about 500 mg/kg subject body weight in the first day of every 1 week to 4 weeks. Such dosages may prove useful for subjects having a body weight outside this range. The nanoemulsion formulation may also contain a proapoptotic agent, such as ceramide, sulforaphane, curcumin or cyclopanine, that act to enhance the cytotoxicity of other chemotherapeutic agents in the cancer cells. The concentration of proapoptotic agent in the composition is about 0.001% to about 2% (about 0.01 mg/ml to about 20 mg/ml).

[00152] The nanoemulsion for oral administration are of about the same volume as those used for injection. However, when administering the drug orally, higher doses may be used when administering by injection. For example, a dosage containing about 10 mg to about 1500 mg chemotherapeutic agent in the first day of every 1 week to 4 weeks may be used. In preparing such liquid dosage form, standard-making techniques may be employed.

6. Imaging Moieties

[00153] Nanoemulsion formulations of the present disclosure can include imaging or contrast agents. The use of imaging agents on the nanoemulsion formulation of the present disclosure enables tracking in real time of the chemotherapeutic agent as it moves to the site of disease and also allows a determination of the amount which reaches its destination. This enables physicians to quickly decide whether a particular subject should continue with treatment. Exemplary, useful imaging agents include paramagnetic agents such as gadolinium (Gd), iron oxide, iron platinum, and manganese. Exemplary, useful gadolinium derivatives include 1 ,2-dimyristoyl-sn- glycero-3 -phosphoethanolamine-N-diethylene-triaminepentaacetic acid (Gd-DTPA- PE), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- 1,4,7, 10-tetraazacyclo- dodecane-l,4,7,10-tetraacetic acid (Gd-DOTA-PE), and l,2-dimyristoyl-sn-glycero-3- paraazoxyphenetole-N- 1 ,4,7, 10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (Gd- PAP-DOTA) (Avanti Polar Lipids, Inc. Alabaster, Alabama). These gadolinium- based MRI contrast moieties can be prepared or obtained and incorporated into a nanoemulsion formulation as described herein. Useful imaging agents are gadolinium, iron oxide, iron, platinum, and manganese. Examples of suitable gadolinium imaging agents are Gd-DTPA-PE, Gd-DOTA-PE, Gd-PAP-DOTA.

[00154] Accordingly, a representative nanoemulsion formulation comprises an imaging moiety attached, e.g., covalently bonded, to a lipid component of the nanoemulsion formulation. One exemplary nanoemulsion formulation comprises a chemotherapeutic agent, an oil phase comprising functionalized and non- functionalized oils, an interfacial surface membrane or corona, an EGFR targeting ligand, and an imaging agent, wherein the imaging agent is covalently bonded to the lipid component of the nanoemulsion formulation's interfacial surface membrane.

[00155] In another exemplary nanoemulsion formulation, imaging moieties are solubilized in the oil phase. For example, a nanoemulsion formulation comprises a chemotherapeutic agent, an oil phase comprising functionalized and non- functionalized oils, an interfacial surface membrane, an EGFR targeting ligand, and an imaging agent, wherein the imaging agent is soluble in the oil phase.

7. Imaging Methods

[00156] The nanoemulsion formulations in accordance with the present disclosure may be used to image regions of the body, such as regions of the brain, and including images of tumors or cancer cells therein. These nanoemulsion formulations are small enough to travel into minute body regions and crossing the BBB and, when coupled with paramagnetic elements, such as gadolinium ions (Gd³⁺), iron oxide, iron, platinum, or manganese, can enhance tissue contrast in an MRI. Once the

nanoemulsion formulation has reached the region of interest, such as the site of a tumor, its efficacy is determined, which can be done using an in vivo imaging modality such as MRI. Image-guided therapy using nanoemulsion formulations couples drug delivery with tissue imaging to allow clinicians to efficiently deliver chemotherapeutic agents, while simultaneously localizing the drugs and visualizing their physiological effects.

[00157] The nanoemulsion formulations combined with an appropriate imaging agent can act as MRI contrast agents to enhance tissue image resolution. Contrast agents such as Gd³⁺ have unpaired electrons that interact with surrounding water molecules to decrease their proton spin time, also referred to as Ti. Relaxation time is defined as the period it takes for a proton to return to its equilibrium position following a magnetization pulse. MRI can measure Ti by creating a magnetic field that reverses the sample's magnetization, and then recording the time required for the spin directions to realign in their equilibrium positions again. The decreased Ti relaxation time of the target tissue allows an MRI machine to better distinguish between it and its surrounding aqueous environment.

[00158] The nanoemulsion formulations according to the disclosure can serve as a new Gd³⁺ chelated, EGFR- or brain receptor-targeted nanoemulsion formulation that not only exhibits MRI contrast but can also carry an encapsulated chemotherapeutic agent, a chemotherapeutic agent and a chemopotentiator or a chemotherapeutic agent and co-agent to the target tissue for successful image-guided therapy. To examine the MRI contrast potential of these nanoemulsions, in vivo studies were conducted using MRI, while efficacy studies are conducted to examine the drug delivery potential of the nanoemulsion formulation.

[00159] The method of imaging includes administering to a subject a diagnostically effective amount of a nanoemulsion formulation according to the disclosure. The nanoemulsion formulation can be administered by a variety of techniques including orally, intranasally, intraperitoneally, intraocularly, subcutaneously and intravenously and injection into the cerebrospinal fluid, as described above. The method is effective for imaging cancers, such as those accessible by the lymphatic) system.

[00160] The following examples provide specific exemplary methods of the invention, and are not to be construed as limiting the invention to their content. EXAMPLES

EXAMPLE 1

Synthesis of EGFR_RP-PEG-DSPE

[00161] EGFRBP-PEG-DSPE was prepared according to the scheme shown in FIG. 3. Briefly, the synthetic EGFR-targeting peptide Y-H-W-Y-G-Y-T-P-Q-N-V-I (SEQ ID NO: 1) with a linker sequence G-G-G-G-C (SEQ ID NO:2) was synthesized by standard peptide organic synthesis methods. The carboxyl group of terminal cysteine of the peptide was reacted with the maleimide of the PEG2000-DSPE construct. To accomplish this reaction, 9.4 mg of EGFR-binding peptide Y-H-W-Y-G-Y-T-P-Q-N- V-I G-G-G-G-C (SEQ ID NO:3) was added to 14.7 mg MAL-PEG2000-DSPE (2.942 kDa mol. wt.) dissolved in HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid ) buffer (pH 7.4) solution at 1 : 1 molar ratio while mixing at 400 rpm) under nitrogen at 4°C for 24 hr.

[00162] The EGFR_Bp conjugate was then purified by dialysis against deionized distilled water at RT using a 3500 molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories, Rancho Dominguez, CA). The purified sample was then transferred into tubes and freeze-dried for 24 hr. The sample was stored at -20°C until use.

[00163] EGFRBP-PEG2000DSPE conjugate formation was confirmed by nuclear magnetic resonance spectroscopy (NMR) analysis (FIG. 9A-C). A 2 mg sample was dissolved in 1 ml DMSO (dimethyl sulfoxide) and the NMR spectra was recorded using a Varian 400AS Spectrometer (400 MHz, Varian Inc., Palo Alto, CA).

EXAMPLE 2

Synthesis of Brain Receptor Targeting Ligands DSPE-PEG-Dermorphin. DSPE-PEG-Candoxin. DSPE-PEG-cRGD and DSPE-PEG-Cereport

[00164] The lipidated brain receptor targeting ligands were prepared according to the schemes in FIGS. 4 - 7. Generally, DSPE-PEG2000-MAL was dissolved in 50 mM HEPES buffer. Then peptide was added to the DSPE-PEG2000-MAL solution. This reaction mixture was stirred for 4 hr at room temperature and then for 20 hr at 4°C. Next, the mixture was dialyzed at 25°C against deionized water using MWCO 6- 8K dialysis membrane for 72 hr with frequent changes of water. The dialyzed solution was frozen and then freeze-dried for 72 hr. The final product was a white powder.

[00165] The quantities of individual reaction components and the product yield for each brain receptor targeting ligand can be found in Table VIII.

Table VIII

Reaction Component Quantities and Product Yields for Lapidated Brain

Receptor-Targeting Ligands

EXAMPLE 3

Synthesis of Gd⁺³-DTPA-PE

[00166] Gd⁺³-DTPA-PE chelate was prepared according to the scheme shown in FIG. 8. 30 μΐ of triethylamine (Sigma) was added to 100 mg of L-a- phosphatidylethanolamine, transphosphatidylated (egg chicken) (841 118C, Avanti Polar Lipids, Birmingham, AL) dissolved in 4 ml of chloroform (extra dried). This solution was then added drop-wise to 400 mg (1 mM) of diethylene

triaminepentacetic dianhydride (DTPA anhydride) (Sigma) in 20 ml of

dimethylsulfoxide and the mixture was stirred for 3 hr under nitrogen atmosphere at RT. Nitrogen was then blown on to a sample to remove the chloroform.

[00167] The DTPA-PE conjugate was then purified by dialysis against deionized distilled water at RT using a 3 kDa molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories). The purified sample was then transferred into tubes and freeze-dried for 48 hr. The DTPA-PE complex formation and purity of the complex were monitored by thin layer chromatography (TLC) using a mobile phase of chloroform: methanol: water at a 3.25: 1.25:0.5 (v/v) ratio and using ninhydrin as a visualizing reagent. For this, reactants (DTPA, PE) and complex (DTPA-PE) were dissolved in chloroform and placed on a TLC plate and developed in the mobile phase. Ninhydrin solution was then sprayed and the spots and their retention times were compared for the formation of the complex.

[00168] 18.5 mg (10.0 mM) of gadolinium (III) chloride hexahydrate (Sigma) in 0.1 ml of water was then added drop-wise to the 100 mg of DTPA-PE complex dissolved in 20 ml of DMSO and the reaction mixture was stirred (400 rpm) for 1 hr.

[00169] An Arsenazo III assay was used to monitor the reaction and formation of the Gd-DTPA-PE complex. 10 μΐ of reaction mixture was added to 0.2 mM of Arsenazo III (Pointe Scientific) in water and observed for the color change (pink to blue). No change in solution color indicated that Gd conjugated to DTPA-PE (free Gd turns Arsenazo III solution to a blue color).

[00170] The resulting Gd⁺³ -DTPA-PE conjugate was purified by dialysis against deionized distilled water at RT using a 3000 molecular weight cut-off membrane (Spectrapore, Spectrum Laboratories). The purified sample was then transferred into tubes and freeze-dried for 48 hr. The conjugate was stored at -20°C until use.

EXAMPLE 4

Preparation of a Nanoemulsion Formulation

[00171] Flaxseed oil (lg) oil was placed in a scintillation glass vial and was used as the oil phase of this nanoemulsion formulation.

[00172] The aqueous phase of this oil-in-water nanoemulsion was prepared as follows. 120 mg egg lecithin (Lipoid E 80, Lipoid GMBH, Ludwigshafen, Germany), 15 mg PEG₂₀₀₀DSPE (Genzyme, Cambridge, MA), 100 mg of Gd-DTPA-PE and 1.2 mg of Dermorphin-PEG2k-PE were added to 4 ml of 2.21% w/v glycerol (Sigma) solution in a glass scintillation vial made in water for injection. The mixture was stirred (1150 rpm) for 2 hr to achieve complete dissolution of these excipients.

[00173] The aqueous and oil phases from above steps were heated to 60°C for 2 min in a water bath, and the aqueous phase was added to the oil phase, and vortex mixed for 30 sec. The resulting mixture was passed through a LV1 Microfluidizer

(Microfluidics Corp., Newton, MA) at 25,000 psi for 10 cycles. Product entered the microfluidizer system via the inlet reservoir and was powered by a high-pressure pump into the interaction chamber at speeds up to 400 m/s. It was then effectively cooled and collected in the output reservoir. [00174] These steps resulted in the production of a μ-opioid receptor targeted nanoemulsion formulation.

[00175] The quantity of ingredients for other representative nanoemulsion formulations (NE) of the present disclosure and made according to the procedure above are listed in Tables IX.

Table IX

EXAMPLE 5

Effect of Nanoemulsion Formulations on Cancer Cells in Vitro

[00176] In order to determine if the nanoemulsion formulations according to the disclosure produce a cytotoxic effect on cancer cells, including brain cancer cells, the following experiments are done on U87 human primary glioblastoma, Ul 18 human glioblastoma, C6 rat glial, and bEND3 endothelialpolyoma middle T antigen transformed cells.

[00177] A tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) assay is performed, which measures the activity of cellular enzymes that reduce the MTT dye to insoluble formazan. Polyethylenimine at 50 μg/ml is used as a positive control for cytotoxicity. The effect of chemotherapeutic agents in solution as a control and nanoemulsion formulations of the disclosure on the viability of cancer cells are studied and measured after 72 hr treatment. After the completion of treatment, cells are incubated with MTT reagent (50 μg/well) for 2 hr. The resulting formazan crystals is dissolved in dimethyl sulfoxide (150 μg/well) and measured at 570 nm in the Plate reader (Synergy HT, Biotek Instruments, Winooski, VT).

[00178] The concentration of drug that inhibits 50% of growth is known as the 50% growth inhibitory concentration (IC5₀). Using the dose response curves, the IC5₀ values are calculated. All IC5₀ values are obtained by analyzing the MTT assays results using Graphpad Prism 5 scientific data analysis software.

[00179] The IC5₀ are decreased when non-targeted nanoemulsion formulations of the present disclosure are used. The most significant decrease in IC50 values is observed when cells expressing the appropriate receptor are treated with the reciprocal ligand targeted nanoemulsion formulations of the present disclosure. The nanoemulsions containing no chemotherapeutic agent do not affect cell viability.

[00180] The optimum concentration of multiple chemotherapeutic agents combined in the nanoemulsion formulations of the present disclosure are determined by calculating the combination index from the dose response curves of the single agents (Chou (2006) Pharmacol. Rev. 58(3):621-681). This method uses the isobologram equation below to determine combination index (CI):

CI = (a /A) + (b/B) where, "a" is the primary therapeutic IC5₀ in combination with secondary therapeutic at concentration "b." "A" is the primary therapeutic IC5₀ without secondary therapeutic; and "B" is the secondary therapeutic IC5₀ in the absence of primary therapeutic. The CI represents the degree of interaction between two drugs regardless of mechanism. A CI value lower than 1.0 indicates synergy, while a CI value greater than 1.0 indicates that the drugs are antagonists. If drugs are synergistic the relative dose needed to get the same effect is reduced and is known as the "dose reduction index" (DRI). DRI is a measure of decrease in drug concentration for a synergistic combination as compared with the concentration of each drug alone. EXAMPLE 6

Targeting of Gd-Labeled Nanoemulsion Formulations to the Brain in Vivo

[00181] That a brain receptor-targeted, Gd-based MRI contrasting agent-containing nanoemulsion formulation is useful in targeting the presently disclosed nanoemulsion formulations to a specific region of the brain was demonstrated as follows:

[00182] Male Sprague-Dawley rats (Charles River Laboratories, Cambridge, MA) each weighing 300 g to 350 g were used as test subjects. Each rat was anesthetized using 1% to 2% isoflurane before being intravenously injected through a tail vein catheter with 0. lmL of a heparinized saline solution. The catheter remained in the tail, and was later used for the injection of brain receptor-targeted, Gd³⁺ labeled nanoemulsions containing a 0.072 mmol/Kg dose of the gadolinium-based MRI contrasting agent Gd-DTPA-PE. All of the animal's heads were scanned and imaged using a Bruker Biospec 20/70 USR MRI machine. A pre-scan was taken before the injection of the nanoemulsion formulation, and 3 time point scans were taken in a row beginning 5 min after the injection of a nanoemulsion, giving the 25 min long scans mid-points at 17 min, 42 min, and 67 min.

[00183] Representations of the resulting MRIs of rats injected with dermorphin- targeted nanoemulsion formulation are shown in FIGS. 10A - 10E and in Table XI.

[00184] The representation of the MRI data in FIGS. 10A - 10D shows that the untargeted NE spreads widely throughout the brain (FIGS. 10B and 10D); whereas the dermorphin-targeted NE concentrates in regions of the brain expressing μ-opioid receptors (FIG. 10A and IOC). The whole brain accumulation of targeted and non- targeted nanoemulsion formulations is approximately the same. In FIG. 10E the shading is related to the actual concentration of dermorphin-targeted NE in areas of interest throughout the brain. In contrast, in FIGS. 10A - 10D, the representation of the areas showing significant NMI signal from the Gd labeled nanoemulsion formulation are not shaded to be representative of the actual concentration of the nanoemulsion in a particular area.

[00185] Table XI lists the specificity index over the entire cohort in a region of interest and preferential accumulation of the μ-opioid receptor targeted, Gd³⁺- containing nanoemulsion formulations of the present disclosure in the cerebral cortex, basal forebrain, and diencephalon regions of the brain as compared to that of the control non-targeted nanoemulsions in Table X (n=7, completed in the same manner as above), which showed nanoemulsion accumulation in the metencephalon and medul Cere_lla oblongata regions of the brain.

[00186] Tables XII and XIII (n=3, completed in the same manner as above) show the accumulation of the brain receptor targeted, Gd³ - labeled nanoemulsion formulations in various regions of the brain that express a-7 nicotinic receptors and bradykinin receptors, respectively. Significant accumulations of nanoemulsion are indicated in the tables by a box surrounding the bold numbers.

Table X

The Specificity Index of an Untarge ;tteedd,, GGdd³⁴ -Labeled Nanoemulsion in Various

Regions of the Brain

A* Section Region of Intrest (ROI) SPI 20 SPI 40 SPI 60

Glomerular Layer 1.232 1.267 1.335

Olfactory Bulb External Plexiform Layer 1.133 1.147 1.222

Granular Cell Layer 1.089 1.114 1.180

Tenia Tecta 1.159 1.175 1.231

Anterior Olfactory

Olfactory Nucleus 1.064 1.099 1.168 Areas

Caudal Piriform Ctx 1.085 1.110 1.154

Rostral Piriform Ctx 1.134 1.173 1.222

Anterior Cingulate Area 1.176 1.202 1.237

Limbic Lobe Retrosplenial Caudal Ctx 1.196 1.298 1.308

Retrosplenial Rostral Ctx 1.311 1.344 1.375

Insular Cortex Insular Ctx 1.101 1.135 1.182

Visual 1 Ctx 1.234 1.241 1.263

Occipital Lobe

Visual 2 Ctx 1.103 1.148 1.206

Temporal Ctx 1.162 1.214 1.259

O o Entorhinal Ctx 1.256 1.290 1.322 π Temporal Lobe Auditory Ctx 1.088 1.124 1.168

Ectorhinal Ctx 1.045 1.123 1.171

Perirhinal Ctx 1.193 1.238 1.298

Parietal Ctx 1.095 1.162 1.221

Primary Somatosensory

Ctx Barrel Field 1.091 1.11 1 1.167

Primary Somatosensory

Ctx Forelimb 1.094 1.126 1.156

Parietal Lobe

Primary Somatosensory

Ctx Hindlimb 1.093 1.114 1.151

Primary Somatosensory

Ctx Jaw 1.098 1.126 1.174

Primary Somatosensory

Ctx Shoulder 1.044 1.090 1.1 15 Primary Somatosensory

Ctx Trunk 1.103 1.118 1.162

Primary Somatosensory

Ctx Upper Lip 1.099 1.120 1.157 Secondary

Somaotsensory Ctx 1.097 1.125 1.169

Ventral Orbital Ctx 1.098 1.106 1.162

Medial Orbital Ctx 1.137 1.148 1.205

Lateral Orbital Ctx 1.094 1.126 1.163

Infralimbic Ctx 1.101 1.126 1.160

Frontal Lobe

Prelimbic Ctx 1.109 1.129 1.176

Frontal Association Ctx 1.109 1.143 1.218

Primary Motor Ctx 1.098 1.133 1.174

Secondary Motor Ctx 1.131 1.173 1.218

CA1 Dorsal 1.095 1.141 1.159 CA1 Hippocampus

Ventral 1.085 1.115 1.144

CA2 1.056 1.130 1.156

CA3 Dorsal 1.060 1.116 1.156

Hippocampal CA3 Hippocampus

Formation Ventral 1.124 1.220 1.257

Subiculum Dorsal 1.130 1.183 1.205

Ventral Subiculum 1.175 1.213 1.250

Dentate Gyrus Dorsal 1.140 1.211 1.245

Dentate Gyrus Ventral 1.197 1.253 1.297

Basal Amygdaloid

Nucleus 1.059 1.088 1.132

Central Amygdaloid

Nucleus 1.120 1.129 1.167

Anterior Amygdaloid

Nucleus 1.104 1.109 1.140

Lateral Amygdaloid

Amygdaloid Nucleus 1.079 1.109 1.164 Nuclear

Complex Intercalated Amygdaloid

Nucleus 1.105 1.169 1.150

Cortical Amygdaloid

a Nucleus 1.128 1.181 1.197

Medial Amygdaloid

£.

* Nucleus 1.293 1.322 1.368 oi

Extended Amydala 1.092 1.064 1.090 S. Dorsal Lateral Striatum 1.107 1.121 1.151

Dorsal Medial Striatum 1.120 1.139 1.166

Ventral Lateral Striatum 1.101 1.107 1.139

Ventral Medial Striatum 1.095 1.104 1.134

Olfactory Tubercles 1.140 1.161 1.197

Basal Ganglia

Accumbens Core 1.084 1.096 1.139

Accumbens Shell 1.075 1.090 1.134

Globus Pallidus 1.084 1.068 1.100

Ventral Pallidum 1.074 1.086 1.132

Subthalamic Nucleus 1.014 1.072 1.078 Zona Incerta 1.061 1.086 1.1 14p _ice_] Prerubral Field 1.067 1.099 1.1 13

Endopiriform Nucleus 1.090 1.107 1.145

Substantia Innominata 1.248 1.094 1.128

Claustrum 1.089 1.109 1.139

Bed nucleus Stria

Basal Terminalis 1.097 1.088 1.1 13 Forebrain

Lateral Septal Nucleus 1.122 1.139 1.171

Triangular Septal Nucleus 1.276 1.083 1.127

Medial Septum 1.101 1.136 1.173

Septum

Diagonal Band of Broca 1.086 1.091 1.128

Anterior Hypothalamic

Area 1.070 1.068 1.131

Lateral Hypothalamus 1.088 1.109 1.151

Premammillary Nucleus 1.094 1.183 1.167

Supramammillary

Nucleus 1.142 1.191 1.241

Suprachiasmatic Nucleus 1.039 1.106 1.169

Paraventricular Nuclus 1.094 1.079 1.165

Arcuate Nucleus 1.154 1.137 1.169

Dorsal Medial Nucleus 1.060 1.1 17 1.1 18

Hypothalamus Posterior Hypothalamic

Area 1.046 1.069 1.086

Magnocellular Preoptic

Nucleus 1.1 18 1.133 1.184

Medial Mammillary

Nucleus 1.244 1.370 1.426

Supraoptic Nucleus 1.072 1.1 10 1.137

Ventral Medial Nucleus 1.124 1.088 1.151 β

Lateral Preoptic Area 1.062 1.087 1.105 a

Medial Preoptic Area 1.079 1.096 1.1 15

»

o Retrochiasmatic Nucleus 1.124 1.1 14 1.189

=

Anterior Lobe Pituitary 1.197 1.270 1.330

Pituitary

Neural Lobe Pituitary 1.271 1.321 1.380

Anterior Thalamic Nuclei 1.089 1.108 1.162

Central Medial Thalamic

Nucleus 1.075 1.101 1.143

Lateral Dorsal Thalamic

Nucleus 1.088 1.142 1.170

Lateral Posterior

Thalamic Nucleus 1.130 1.197 1.208

Thalamus Medial Dorsal Thalamic

Nucleus 1.094 1.1 18 1.152

Parafascicular Thalamic

Nucleus 1.070 1.085 1.088

Reuniens Nucleus 1.087 1.097 1.134

Ventral Anterior

Thalamic Nucleus 1.043 1.082 1.124 Ventrolateral Thalamic

Nucleus 1.049 1.090 1.137 Ventromedial Thalamic

Nucleus 1.059 1.082 1.091

Ventral Posteriolateral

Thalamic Nucleus 1.060 1.098 1.117

Ventral Posteriolmedial

Thalamic Nucleus 1.054 1.095 1.100

Paraventricular Nucleus 1.195 1.211 1.262

Posterior Thalamic

Nucleus 1.066 1.085 1.102

Medial Geniculate 1.175 1.251 1.276

Lateral Geniculate 1.125 1.183 1.247

Reticular Nucleus 1.070 1.104 1.1 17

Habenula Nucleus 1.220 1.262 1.299

Epithalamus

Pineal Gland 1.530 1.764 1.597

1 st Cerebellar Lobule 1.142 1.185 1.215

2nd Cerebellar Lobule 1.131 1.147 1.179

3rd Cerebellar Lobule 1.132 1.164 1.183

4th Cerebellar Lobule 1.174 1.212 1.255

5th Cerebellar Lobule 1.251 1.308 1.318

6th Cerebellar Lobule 1.197 1.251 1.314

7th Cerebellar Lobule 1.130 1.218 1.280

8th Cerebellar Lobule 1.126 1.166 1.233

9th Cerebellar Lobule 1.180 1.185 1.231

10th Cerebellar Lobule 1.188 1.246 1.308

Interposed Nucleus 1.228 1.241 1.247

Cerebellum Simple Lobule

Cerebellum 1.210 1.323 1.352

Copula of the Pyramis 1.312 1.263 1.370

Paraflocculus Cerebellum 1.268 1.282 1.307

Cms 1 of Ansiform

Lobule 1.173 1.263 1.300

Cms 2 of Ansiform

Lobule 1.209 1.257 1.315

Flocculus Cerebellum 1.181 1.204 1.229

Lat (Lateral Cerebellar

Nucleus) 1.218 1.189 1.245

Medial Cerebellar

Nucleus Fastigial 1.222 1.246 1.267

Paramedian Lobule 1.276 1.303 1.370

Pontine Nuclei 1.096 1.113 1.147

Motor Trigeminal

Nucleus 1.113 1.133 1.175

Principal Sensory

Nucleus Trigeminal 1.157 1.208 1.207

Pons Dorsomedial Tegmental

Area 1.166 1.225 1.287

Trapezoid Body 1.062 1.102 1.144

Lemniscal Nucleus 1.107 1.159 1.160

Reticulotegmental

Nucleus 1.103 1.106 1.128 Olivary Nucleus 1.243 1.258 1.227

Periolivary Nucleus 1.158 1.187 1.206 p Me_se nce_ha_lon

Pontine Reticular Nucleus

Caudal 1.085 1.124 1.147

Pontine Reticular Nucleus

Oral 1.067 1.134 1.139

Gigantocellular Reticular

Nucleus Pons 1.129 1.175 1.173

Parabrachial Nucleus 1.103 1.134 1.160

Root of Trigeminal Nerve 1.203 1.235 1.204

Facial Nucleus 1.138 1.166 1.174

Locus Ceruleus 1.217 1.223 1.213

Sub Coeruleus Nucleus 1.099 1.142 1.179

Median Raphe Nucleus 1.075 1.120 1.144

Inferior Colliculus 1.205 1.243 1.267

Tectum

Superior Colliculus 1.158 1.179 1.187

Anterior Pretectal

Nucleus 1.106 1.104 1.155

Medial Pretectal Area

Substantia Nigra

Compacta 1.085 1.098 1.152 Substantia Nigra

Reticularis 1.094 1.133 1.199

Red Nucleus 1.080 1.087 1.126

Ventral Tegmental Area 1.098 1.059 1.1 16

Cerebral Interpeduncular Nucleus 1.070 1.131 1.064 Peduncle Periaqueductal Gray

Thalamus 1.077 1.127 1.145

Central Gray 1.168 1.189 1.220

Reticular Nucleus

Midbrain 1.090 1.119 1.136

Pedunculopontine

Tegmental Area 1.068 1.149 1.186

Dorsal Raphe 1.119 1.105 1.141

Raphe Linear 1.051 1.075 1.1 1 1

Inferior Olivary Complex 1.344 1.285 1.348

Solitary Tract Nucleus 1.177 1.183 1.188

Cochlear Nucleus 1.241 1.335 1.324

Vestibular Nucleus 1.224 1.269 1.286

0. Raphe Magnus 1.053 1.1 13 1.105

ΕΓ Raphe Obscurus Nucleus 1.220 1.246 1.317

O

PCRt 1.158 1.214 1.210

=

DPGi 1.349 1.327 1.353

Precuniform Nucleus 1.088 1.104 1.141

Ventricle 1.250 1.281 1.307

White Matter 1.091 1.121 1.146

White Matter 1.068 1.112 1.128

*A = non-targeted nanoemulsion Table XI

Th_{be r}ra_le Specificity Index of a μ Opioid Receptor-Targeted, Gd³⁺-Labeled

Nanoemulsion in Various Regions of the Brain μ-Opioid

Region of Intrest Receptor SPI SPI SPI

A2* Section (ROI) Expression 20 40 60

Glomerular Layer L 1.183 1.249 1.170

External Plexiform

Olfactory Bulb

Layer L 1.228 1.430 1.110

Granular Cell Layer L 1.475 1.580 1.260

Tenia Tecta L 1.236 1.531 1.374

Anterior Olfactory

Olfactory Nucleus L 1.566 1.365 1.030

Areas

Caudal Piriform Ctx L 1.619 1.668 1.439

Rostral Piriform Ctx L 0.692 0.759 0.820

Anterior Cingulate Area M 0.893 1.112 1.142

Retrosplenial Caudal

Limbic Lobe Ctx M-L 1.272 1.025 1.229

Retrosplenial Rostral

Ctx M-L 0.492 0.549 0.630

Insular Cortex Insular Ctx L 1.174 1.212 1.149

Visual 1 Ctx M 0.995 1.086 1.206

Occipital Lobe

Visual 2 Ctx L 1.320 1.207 1.101

O n Temporal Ctx L 0.934 0.945 0.852

Entorhinal Ctx M-L 0.747 0.893 0.922

Temporal

Auditory Ctx L 1.100 1.131 1.088

O Lobe

e Ectorhinal Ctx L 3.352 1.464 1.346 π Perirhinal Ctx L 0.673 0.841 0.844

Parietal Ctx * 1.163 0.868 0.913

Primary Somatosensory

Ctx Barrel Field * 1.057 1.193 1.068

Primary Somatosensory

Ctx Forelimb * 1.047 1.142 1.168

Primary Somatosensory

Ctx Hindlimb * 1.085 1.236 1.193

Primary Somatosensory

Parietal Lobe Ctx Jaw * 1.094 1.207 1.183

Primary Somatosensory

Ctx Shoulder * 1.884 1.239 1.401

Primary Somatosensory

Ctx Trunk * 0.787 1.203 1.241

Primary Somatosensory

Ctx Upper Lip * 1.110 1.168 1.166

Secondary

Somaotsensory Ctx * 1.139 1.179 1.114

Ventral Orbital Ctx * 1.258 1.637 1.333

Frontal Lobe

Medial Orbital Ctx * 1.226 1.417 1.197 Lateral Orbital Ctx * 1.214 1.356 1.283

Infralimbic Ctx L 1.478 1.574 1.570 _bra Prelimbic Ctx L 1.188 1.388 1.271

Frontal Association Ctx M 1.567 1.397 1.072

Primary Motor Ctx * 0.994 1.117 1.149

Secondary Motor Ctx * 1.029 1.228 1.215

CA1 Dorsal M 1.242 1.181 1.216

CA1 Hippocampus

Ventral M 1.145 1.254 1.229

CA2 M 1.484 1.143 1.193

CA3 Dorsal M 1.565 1.113 1.074

Hippocampal CA3 Hippocampus

Formation Ventral M 0.678 0.625 0.707

Subiculum Dorsal M 1.014 0.945 1.002

Ventral Subiculum M 0.922 1.094 0.990

Dentate Gyrus Dorsal L 1.013 0.863 0.944

Dentate Gyrus Ventral M 0.976 0.966 0.998

Basal Amygdaloid

Nucleus M-H 1.456 1.544 1.229

Central Amygdaloid

Nucleus M 0.940 1.010 0.929

Anterior Amygdaloid

Nucleus M-H 0.776 1.165 1.103

Amygdaloid Lateral Amygdaloid

Nuclear Nucleus M-H 1.463 1.432 1.231

Complex Intercalated

Amygdaloid Nucleus H 0.877 0.910 0.836 Cortical Amygdaloid

Nucleus M-H 0.948 0.991 1.049

Medial Amygdaloid

Nucleus H 0.633 0.693 0.670

Extended Amydala M-H 0.769 1.865 1.516 w

Dorsal Lateral Striatum H 1.066 1.266 1.253

Dorsal Medial Striatum H 0.934 1.219 1.202 o

n Ventral Lateral Striatum H 1.136 1.434 1.338

Ventral Medial Striatum H 1.186 1.507 1.457

Olfactory Tubercles L 1.070 1.162 1.207

Accumbens Core H 1.329 1.585 1.401

Accumbens Shell H 1.436 1.687 1.460

Basal Ganglia

Globus Pallidus M 1.122 2.223 1.564

Ventral Pallidum M 1.137 1.474 1.234

Subthalamic Nucleus None 4.959 1.535 1.741

Zona Incerta None 1.367 1.319 1.232

Prerubral Field N/A 1.465 1.233 1.079

Endopiriform Nucleus M 1.240 1.407 1.344

Substantia Innominata L 0.344 1.678 1.214

Claustrum H

Basal 1.267 1.501 1.470 Forebrain Bed nucleus Stria

Terminalis M 1.037 1.804 1.532 Lateral Septal Nucleus L 1.003 1.246 1.198

Triangular Septal

Nucleus N/A 0.306 0.133 0.294

Medial Septum H 0.572 0.680 0.789

Septum

Diagonal Band of Broca H 1.203 1.627 1.450

Anterior Hypothalamic

Area L 1.302 1.928 1.299

Lateral Hypothalamus L 1.455 1.394 1.176

Premammillary Nucleus L 0.199 0.128 0.137

Supramammillary

Nucleus M 1.565 1.043 1.166

Suprachiasmatic

Nucleus None 6.665 2.359 1.275

Paraventricular Nuclus L 1.083 1.831 0.999

Arcuate Nucleus L 0.687 0.772 0.804

Dorsal Medial Nucleus L 1.886 1.396 1.405

Hypothalamus Posterior Hypothalamic

Area M 1.989 1.956 1.908

Magnocellular Preoptic

Nucleus L 0.605 1.119 0.787

Medial Mammillary

Nucleus L 0.636 0.272 0.499

Supraoptic Nucleus L 1.354 0.284 0.135

Ventral Medial Nucleus M-L 0.615 1.068 0.835

Lateral Preoptic Area M-L 1.120 1.527 1.321

Medial Preoptic Area L 0.880 1.520 1.209

Retrochiasmatic

Nucleus L 0.952 1.361 0.730

ST

B Anterior Lobe Pituitary L 0.555 0.774 0.729

Pituitary

Neural Lobe Pituitary L 0.535 0.836 0.694

Anterior Thalamic

Nuclei H 1.147 1.170 0.948

Central Medial

Thalamic Nucleus M 1.130 1.127 1.088

Lateral Dorsal Thalamic

Nucleus M 1.319 0.946 0.980

Lateral Posterior

Thalamic Nucleus M 0.883 0.929 0.960 Medial Dorsal Thalamic

Nucleus H 1.088 0.950 1.014

Thalamus Parafascicular Thalamic

Nucleus L 1.399 1.777 1.612

Reuniens Nucleus H 0.906 1.192 1.039

Ventral Anterior

Thalamic Nucleus H 2.053 1.304 1.151

Ventrolateral Thalamic

Nucleus L 1.777 1.123 1.037

Ventromedial Thalamic

Nucleus H 1.318 1.217 1.409

Ventral Posteriolateral

Thalamic Nucleus M-L 1.259 0.976 1.097 Ventral Posteriolmedial

Thalamic Nucleus L 1.651 1 289 1. 394

Paraventricular Nucleus H 0.687 1 097 1 043

Posterior Thalamic

Nucleus H 1.425 1 566 1. 414

Medial Geniculate M-H 0.310 0 374 0 573

Lateral Geniculate H 0.587 0 517 0 671

Reticular Nucleus M 1.032 1 045 1 212

Habenula Nucleus H 0.615 0 698 0 730

Epithalamus

Pineal Gland N/A 1.333 1 493 1 800

1 st Cerebellar Lobule N/A 0.819 0 970 0 969

2nd Cerebellar Lobule N/A 0.731 0 940 0 988

3rd Cerebellar Lobule N/A 0.743 0 973 1 141

4th Cerebellar Lobule N/A 0.601 0 828 0 911

5th Cerebellar Lobule N/A 0.654 0 769 0 892

6th Cerebellar Lobule N/A 0.962 0 967 0 912

7th Cerebellar Lobule N/A 1.005 0 866 0 771

8th Cerebellar Lobule N/A 1.023 1 201 0 959

9th Cerebellar Lobule N/A 0.710 0 881 0 914

10th Cerebellar Lobule N/A 0.780 0 707 0 831

Interposed Nucleus N/A 0.612 1 014 1 198

Simple Lobule

Cerebellum

Cerebellum N/A 0.628 0 681 0 748

Copula of the Pyramis N/A 0.725 1 157 0 889

Paraflocculus

Cerebellum N/A 0.851 1 027 1 073

Crus 1 of Ansiform

Lobule N/A 1.028 0 992 0 986

Crus 2 of Ansiform

v

sr Lobule N/A 0.912 1 030 0 903

ST Flocculus Cerebellum N/A 0.767 0 889 1 153

B Lat (Lateral Cerebellar

Nucleus) N/A 0.198 0 829 0 872

Medial Cerebellar

Nucleus Fastigial None 0.653 0 895 0 979

Paramedian Lobule N/A 0.731 0 949 0 802

Pontine Nuclei None 0.782 1 002 0 956

Motor Trigeminal

Nucleus L 0.470 0 909 0 913

Principal Sensory

Nucleus Trigeminal L 0.391 0 731 0 707

Dorsomedial Tegmental

Pons Area L 0.459 0 641 0 752

Trapezoid Body L 0.622 0 856 0 890

Lemniscal Nucleus L 0.457 0 626 0 932

Reticulotegmental

Nucleus N/A 0.124 0 538 0 895

Olivary Nucleus None 0.200 0 378 0 667

Periolivary Nucleus None 0.360 0 668 0 805 Pontine Reticular

Nucleus Caudal L 0.298 0.649 0.882

Pontine Reticular

p_esecen_ha_l

go _lna_ta Nucleus Oral L 0.928 0.690 1.069

Gigantocellular

Reticular Nucleus Pons L 0.602 0.900 1.014

Parabrachial Nucleus M-H 0.618 0.822 1.008

Root of Trigeminal

Nerve N/A 0.903 1.064 1.165

Facial Nucleus L 0.824 0.936 1.169

Locus Ceruleus M-H 0.759 1.019 1.148

Sub Coeruleus Nucleus M 0.541 0.794 0.906

Median Raphe Nucleus M-H 0.637 0.644 0.946

Inferior Colliculus M 0.356 0.466 0.728

Tectum

Superior Colliculus M-H 0.603 0.968 1.031

Anterior Pretectal

Nucleus N/A 0.741 0.972 0.908

Medial Pretectal Area N/A 0.000 0.000 0.000

Substantia Nigra

Compacta M 1.173 1.047 1.204

Substantia Nigra

Reticularis M-L 1.512 1.278 1.049

Red Nucleus None 1.011 1.271 1.376

Ventral Tegmental Area M 1.410 0.613 1.899

Cerebral

Interpeduncular Nucleus H

Peduncle 1.574 0.955 3.314 o

B Periaqueductal Gray

Thalamus M 0.980 1.020 1.201

Central Gray M 0.665 0.968 1.002

Reticular Nucleus

Midbrain L 0.871 1.002 1.253

Pedunculopontine

Tegmental Area L 0.914 0.762 0.862

Dorsal Raphe L 0.377 0.820 0.961

Raphe Linear N/A 1.169 0.946 1.439

Inferior Olivary

Complex L 0.468 0.541 0.698

Solitary Tract Nucleus M-H 0.151 0.571 0.816

Cochlear Nucleus L 0.686 0.723 0.820

Vestibular Nucleus L 0.551 0.814 1.009 a Raphe Magnus L 0.601 1.173 1.346

ST Raphe Obscurus -

O

=r Nucleus L 0.029 0.593 0.436

PCRt N/A 0.899 0.967 1.006

DPGi L 0.555 0.801 0.941

Precuniform Nucleus 0 0.540 0.617 1.072

Ventricle 0 1.001 1.062 1.062

White Matter 0 0.909 0.994 1.109

White Matter 0 1.482 1.316 1.468 Inferior Colliculus M 0.356 0.466 0.728

Tectum

Superior Colliculus M-H 0.603 0.968 1.031_g O_bo _lla _lna_ta Anterior Pretectal

p _>eceon _iha_lr

Nucleus N/A 0.741 0.972 0.908

Medial Pretectal Area N/A 0.000 0.000 0.000 Substantia Nigra

Compacta M 1.173 1.047 1.204

Substantia Nigra

Reticularis M-L 1.512 1.278 1.049

Red Nucleus None 1.011 1.271 1.376

Ventral Tegmental

Area M 1.410 0.613 1.899

Cerebral Interpeduncular

Peduncle Nucleus H 1.574 0.955 3.314

Periaqueductal Gray

Thalamus M 0.980 1.020 1.201

Central Gray M 0.665 0.968 1.002

Reticular Nucleus

Midbrain L 0.871 1.002 1.253

Pedunculopontine

Tegmental Area L 0.914 0.762 0.862

Dorsal Raphe L 0.377 0.820 0.961

Raphe Linear N/A 1.169 0.946 1.439

Inferior Olivary

Complex L 0.468 0.541 0.698

Solitary Tract Nucleus M-H 0.151 0.571 0.816

Cochlear Nucleus L 0.686 0.723 0.820

Vestibular Nucleus L 0.551 0.814 1.009 a Raphe Magnus L 0.601 1.173 1.346

Raphe Obscurus

Nucleus L 0.029 0.593 0.436

PCRt N/A 0.899 0.967 1.006

DPGi L 0.555 0.801 0.941

Precuniform Nucleus 0 0.540 0.617 1.072

Ventricle 0 1.001 1.062 1.062

White Matter 0 0.909 0.994 1.109

White Matter 0 1.482 1.316 1.468mulsions targeted with the μ-opioid receptor ligand dermorphin; Experiment performed on seven rats.

Table XII

C_eer

The Specificity Index of an Alpha-7 Receptor-Targeted, Gd³⁺-Labeled

Nanoemulsion in Various Regions of the Brain

Alpha-7

Receptor SPI

A3* Section Region of Intrest (ROI) Expression SPI 20 SPI 40 60

Glomerular Layer N/A 1.090 1.150 1.050

Olfactory Bulb External Plexiform Layer N/A 1.200 1.700 1.320

Granular Cell Layer N/A 1.160 1.900 1.240

Tenia Tecta H 0.890 1.260 0.970

Olfactory Anterior Olfactory Nucleus H 1.130 1.540 1.190

Areas Caudal Piriform Ctx N/A 1.640 1.630 0.970

Rostral Piriform Ctx N/A 1.300 1.250 1.080

Anterior Cingulate Area N/A 1.220 1.440 1.290

Limbic Lobe Retrosplenial Caudal Ctx N/A 2.220 1.330 1.850

Retrosplenial Rostral Ctx N/A 0.870 0.900 0.950

Insular Cortex Insular Ctx N/A 1.460 1.480 1.180

Visual 1 Ctx M-H 1.290 1.450 1.530

Occipital Lobe

Visual 2 Ctx M-H 1.940 1.540 1.830

Temporal Ctx M 3.450 1.700 1.740

Entorhinal Ctx M-L 1.350 1.520 1.430

Temporal Lobe Auditory Ctx M 2.460 1.900 1.450

Ectorhinal Ctx M 1.400 0.880 1.120

Perirhinal Ctx N/A 1.310 1.300 1.040

Parietal Ctx N/A 1.050 1.040 1.400

Primary Somatosensory Ctx

Barrel Field N/A 1.100 1.570 1.310

Primary Somatosensory Ctx

Forelimb N/A 1.300 1.470 1.290

Primary Somatosensory Ctx

Hindlimb N/A 0.780 1.270 1.240

Primary Somatosensory Ctx

Parietal Lobe Jaw N/A 1.000 1.550 1.160

Primary Somatosensory Ctx

Shoulder N/A 1.870 1.580 1.480

Primary Somatosensory Ctx

Trunk N/A 0.780 1.670 1.070

Primary Somatosensory Ctx

Upper Lip N/A 1.450 1.730 1.320

Secondary Somaotsensory

Ctx N/A 1.430 1.490 1.160

Ventral Orbital Ctx H 1.170 2.250 1.330

Frontal Lobe Medial Orbital Ctx H 0.890 1.360 1.300

Lateral Orbital Ctx H 1.170 1.850 1.470 Infralimbic Ctx H 1.450 1.890 1.350 ra_in

Prelimbic Ctx H 1.060 1.720 1.270

Frontal Association Ctx H 0.850 1.050 0.890

Primary Motor Ctx H 0.860 1.230 1.090

Secondary Motor Ctx H 1.030 1.130 1.140

CA1 Dorsal H 1.190 1.100 1.270

CA1 Hippocampus Ventral H 0.900 1.380 1.210

CA2 H 3.070 1.140 1.210

CA3 Dorsal H 1.700 1.070 1.120

Hippocampal

Formation CA3 Hippocampus Ventral H 1.160 0.880 0.780

Subiculum Dorsal N/A 1.230 0.710 0.970

Ventral Subiculum N/A 1.010 1.050 1.220

Dentate Gyrus Dorsal H 1.840 1.060 1.210

Dentate Gyrus Ventral H 1.000 0.990 1.050

Basal Amygdaloid Nucleus H 1.380 1.680 1.170

Central Amygdaloid

Nucleus H 1.010 1.100 0.930

Anterior Amygdaloid

Nucleus H 2.020 1.910 1.000

Amygdaloid

Lateral Amygdaloid Nucleus H 1.350 1.350 0.970 Nuclear

Intercalated Amygdaloid

Complex

Nucleus H 1.270 1.080 0.830

Cortical Amygdaloid

Nucleus H 1.280 1.310 1.120

Medial Amygdaloid Nucleus H 0.410 0.430 0.360

Extended Amydala N/A 1.170 17.020 2.130

Dorsal Lateral Striatum N/A 1.310 1.680 1.270

Dorsal Medial Striatum N/A 1.410 1.780 1.290

Ventral Lateral Striatum N/A 1.120 1.990 1.260

Ventral Medial Striatum N/A 1.360 2.330 1.480

Olfactory Tubercles N/A 0.910 1.190 0.930

Accumbens Core N/A 1.740 2.690 1.330

Accumbens Shell N/A 1.480 2.590 1.210

Basal Ganglia

Globus Pallidus N/A 0.910 3.150 1.460

Ventral Pallidum N/A 1.460 2.220 1.170

Subthalamic Nucleus L 9.490 0.840 1.070

Zona Incerta L 0.990 1.090 0.950

Prerubral Field N/A 0.570 1.350 1.220

Endopiriform Nucleus M-H 1.010 1.920 0.960

Substantia Innominata N/A 0.380 2.950 1.230

Claustrum N/A 1.300 2.180 1.350

Basal Bed nucleus Stria Terminalis N/A 0.990 2.770 1.170

Forebrain Lateral Septal Nucleus N/A 1.510 2.100 1.300

Triangular Septal Nucleus N/A 1.080 2.280 1.130

Medial Septum N/A 2.320 1.970 1.390

Septum

Diagonal Band of Broca N/A 1.750 3.140 2.310 Anterior Hypothalamic Area M 0.800 1.610 1.070

Lateral Hypothalamus M 0.640 0.820 0.870

Premammillary Nucleus M 1.380 1.000 0.230

Supramammillary Nucleus M -0.160 0.820 0.940

Suprachiasmatic Nucleus M 2.460 1.900 0.920

Paraventricular Nuclus M 0.640 1.020 0.970

Arcuate Nucleus M 1.810 1.340 0.720

Dorsal Medial Nucleus M 1.020 1.060 0.860

Hypothalamus Posterior Hypothalamic Area M 0.490 1.240 1.250

Magnocellular Preoptic

Nucleus M 1.000 1.450 0.850

Medial Mammillary Nucleus M 1.780 0.980 0.940

Supraoptic Nucleus M 0.990 -0.660 0.680

Ventral Medial Nucleus M 0.410 1.480 0.920

Lateral Preoptic Area M 1.430 1.720 1.120

Medial Preoptic Area M 1.700 2.400 1.050

Retrochiasmatic Nucleus M -0.120 -0.130 0.430

Anterior Lobe Pituitary M 0.970 1.310 0.970

Pituitary

Neural Lobe Pituitary M 0.660 0.660 0.450

Anterior Thalamic Nuclei L 1.200 1.310 1.140

Central Medial Thalamic

Nucleus L 0.870 0.900 0.820

Lateral Dorsal Thalamic

v Nucleus L 1.270 1.010 1.500 sr

ST Lateral Posterior Thalamic

B Nucleus L 1.700 1.500 1.620

Medial Dorsal Thalamic

Nucleus L 0.350 0.630 0.550

Parafascicular Thalamic

Nucleus L 0.660 1.110 1.200

Reuniens Nucleus L 0.700 1.000 0.940

Ventral Anterior Thalamic

Nucleus L 1.470 1.010 1.270

Thalamus Ventrolateral Thalamic

Nucleus L 2.050 1.020 1.090

Ventromedial Thalamic

Nucleus L 0.730 0.990 1.080

Ventral Posteriolateral

Thalamic Nucleus L 1.390 0.990 1.190

Ventral Posteriolmedial

Thalamic Nucleus L 1.150 1.060 1.280

Paraventricular Nucleus L 1.520 1.790 1.220

Posterior Thalamic Nucleus L 0.880 1.390 1.380

Medial Geniculate L 2.120 0.980 1.070

Lateral Geniculate L 4.700 1.460 1.320

Reticular Nucleus L 1.470 1.090 1.350

Habenula Nucleus L 0.940 1.100 1.010

Epithalamus

Pineal Gland N/A 0.590 0.660 0.710

Rostral Piriform Ctx H 1.530 1.230 1.000

Anterior Cingulate Area H 0.667 0.844 0.727

Limbic Lobe Retrosplenial Caudal Ctx H 1.460 0.955 1.450

Retrosplenial Rostral Ctx H 0.962 0.996 1.060

Insular Cortex Insular Ctx H 1.580 1.480 1.090

Visual 1 Ctx 0.675 0.902 0.935

Occipital Lobe

Visual 2 Ctx 1.490 1.340 1.610

Temporal Ctx P 2.900 1.600 1.540 Entorhinal Ctx H 1.430 1.710 1.440

Temporal

Lobe Auditory Ctx P 1.690 1.500 1.240

Ectorhinal Ctx P 3.670 3.010 2.860 Perirhinal Ctx P 0.719 0.822 0.670

Parietal Ctx 0.494 0.667 0.726

Primary Somatosensory Ctx

Barrel Field 0.802 1.180 0.988

Primary Somatosensory Ctx

Forelimb 0.745 1.020 0.933

Primary Somatosensory Ctx

Hindlimb 0.545 1.010 0.899

Primary Somatosensory Ctx

Parietal Lobe Jaw 1.130 1.330 0.933

Primary Somatosensory Ctx

Shoulder 0.747 0.848 0.830

Primary Somatosensory Ctx

Trunk 0.361 1.060 1.240

Primary Somatosensory Ctx

Upper Lip 0.984 1.260 1.010

Secondary Somaotsensory

Ctx 1.260 1.260 1.130

Ventral Orbital Ctx P 0.848 1.240 0.813

Medial Orbital Ctx P 0.834 1.110 0.834

Lateral Orbital Ctx P 0.868 1.060 0.854

Infralimbic Ctx P 1.350 1.390 1.160

Frontal Lobe

Prelimbic Ctx P 0.911 1.170 0.807

Frontal Association Ctx P 0.901 1.080 0.858

Primary Motor Ctx P 0.704 0.883 0.762

Secondary Motor Ctx P 0.719 0.797 0.755

CA1 Dorsal 0.596 0.695 1.010

CA1 Hippocampus Ventral 0.878 0.924 0.981

H Molecular

CA2 Layer 1.940 0.830 0.967

H Molecular

Hippocampal CA3 Dorsal Layer 1.400 0.994 0.941 Formation

H Molecular

CA3 Hippocampus Ventral Layer 1.910 1.190 1.210 Subiculum Dorsal P 1.120 1.030 1.160 Ventral Subiculum P 0.657 0.839 0.920 Dentate Gyrus Dorsal H 1.870 I 1.100 1.230 Dentate Gyrus Ventral H 0.710 0.724 0.909 ra_in Basal Amygdaloid Nucleus H 1.590 1.800 1.440 poha_ln Central Amygdaloid

Nucleus H 1.050 1.040 1.250

Anterior Amygdaloid

Nucleus H 2.660 2.700 1.680

Lateral Amygdaloid

Amygdaloid

Nucleus H 0.650 0.859 0.929 Nuclear

Intercalated Amygdaloid

Complex

Nucleus H 1.120 0.888 0.987

Cortical Amygdaloid

Nucleus H 2.420 2.240 1.850

Medial Amygdaloid

Nucleus H 0.832 0.789 0.821

Extended Amydala H 0.865 12.800 1.790

Dorsal Lateral Striatum P Within 0.892 1.340 1.100 Dorsal Medial Striatum P Within 0.897 1.330 1.090 w Ventral Lateral Striatum P Within 0.697 1.470 1.180

— . Ventral Medial Striatum P Within 0.614 1.440 1.010 o Olfactory Tubercles P Within 1.700 1.940 1.350 n "1

=T Accumbens Core P Within 1.070 2.000 1.080

Accumbens Shell P Within 0.981 1.790 1.010

Basal Ganglia Globus Pallidus H 0.740 2.580 1.530

Ventral Pallidum H 1.140 2.090 1.020

Subthalamic Nucleus H 16.100 1.480 1.330

Zona Incerta P 1.590 1.390 1.230

Prerubral Field P 0.187 0.824 0.670 Endopiriform Nucleus P 0.796 1.370 0.856

Substantia Innominata P -0.050 0.137 0.403

Claustrum P 1.060 1.780 1.160

Bed nucleus Stria

Basal Terminalis P 0.420 1.730 1.010

Forebrain

Lateral Septal Nucleus M 0.807 1.470 1.040

Triangular Septal Nucleus P 0.556 1.810 1.140

Medial Septum M 1.390 1.440 1.020

Septum

Diagonal Band of Broca P 1.150 2.960 1.670

Anterior Hypothalamic

Area H 0.843 1.450 1.160

Lateral Hypothalamus H 1.770 1.580 1.700

Premammillary Nucleus H 2.290 1.920 2.070

O SupramammiUary Nucleus H 2.670 2.420 2.770

B

r> Suprachiasmatic Nucleus H 2.530 1.950 1.580

Hypothalamus

Paraventricular Nuclus H 1.010 1.340 1.250 Arcuate Nucleus H 1.090 1.070 1.160

Dorsal Medial Nucleus H 1.580 1.370 1.140

Posterior Hypothalamic

Area H -0.513 0.080 0.941

Magnocellular Preoptic H 2.280 2.140 1.620 Nucleus

Medial Mammillary

Nucleus H 1.890 0.851 0.968

Supraoptic Nucleus H 5.700 5.200 2.990

Ventral Medial Nucleus H 0.903 1.780 1.390

Lateral Preoptic Area H 0.875 1.400 1.070

Medial Preoptic Area H 0.971 2.030 1.080

Retrochiasmatic Nucleus H 2.680 3.340 3.560

Anterior Lobe Pituitary H 0.337 0.432 0.251

Pituitary

Neural Lobe Pituitary H 0.002 0.288 0.185

Anterior Thalamic Nuclei M-H 0.645 0.842 0.848

Central Medial Thalamic

Nucleus M-H 0.583 0.923 0.910

Lateral Dorsal Thalamic

Nucleus M-H 1.320 1.030 1.270

Lateral Posterior Thalamic

Nucleus M-H 1.050 0.904 0.941

Medial Dorsal Thalamic

Nucleus M-H 0.752 0.765 0.829

Parafascicular Thalamic

Nucleus M-H 0.271 0.615 0.899

Reuniens Nucleus M-H 0.480 0.832 0.931

Ventral Anterior Thalamic

Nucleus H 1.620 0.994 1.190

Thalamus Ventrolateral Thalamic

Nucleus M-H 1.280 0.803 0.833

Ventromedial Thalamic

Nucleus M-H 0.360 0.622 0.732

Ventral Posteriolateral

Thalamic Nucleus M-H 0.954 0.670 0.890

Ventral Posteriolmedial

Thalamic Nucleus M-H 0.599 0.732 0.794

Paraventricular Nucleus M-H 0.775 1.120 0.985

Posterior Thalamic Nucleus M-H 0.386 0.835 0.846

Medial Geniculate M-H 5.330 1.940 2.140

Lateral Geniculate M-H 5.430 1.860 1.330

Reticular Nucleus M-H 1.190 0.980 1.000

Habenula Nucleus M-H 0.679 0.806 0.764

Epithalamus

Pineal Gland P 1.120 1.240 1.330

1st Cerebellar Lobule M 0.987 1.030 1.320

2nd Cerebellar Lobule M 0.348 0.821 0.725

3rd Cerebellar Lobule M 0.730 0.953 0.970

4th Cerebellar Lobule M 0.400 0.481 0.528

5th Cerebellar Lobule M 0.395 0.406 0.408

Cerebellum

6th Cerebellar Lobule M 0.914 0.758 0.710

ST 7th Cerebellar Lobule M 1.030 1.240 1.070

B

8th Cerebellar Lobule M 2.970 1.630 1.610

9th Cerebellar Lobule M 1.370 0.964 1.200

10th Cerebellar Lobule M 1.830 1.180 1.170 Interposed Nucleus H 2.080 1.620 2.830

Simple Lobule Cerebellum P 0.567 0.481 0.665

Copula of the Pyramis P 1.350 1.700 1.400

Paraflocculus Cerebellum P 1.580 1.490 2.110

Cms 1 of Ansiform Lobule P 1.680 0.815 1.020

Cms 2 of Ansiform Lobule P 2.300 1.420 1.140

Flocculus Cerebellum P 3.980 2.600 3.630

Lat (Lateral Cerebellar

Nucleus) P 1.360 2.170 2.120

Medial Cerebellar Nucleus

Fastigial P 1.750 1.390 2.410

Paramedian Lobule P 1.700 1.340 1.370

Pontine Nuclei P 0.521 0.635 0.586

Motor Trigeminal Nucleus H 0.519 0.999 0.764 Principal Sensory Nucleus

Trigeminal M-H 1.260 0.869 1.060

Dorsomedial Tegmental

Area P 1.690 1.320 1.320

Trapezoid Body P -46.800 1.890 1.040

Lemniscal Nucleus P 1.600 1.260 1.610

Reticulotegmental Nucleus M 0.516 0.972 0.994

Olivary Nucleus H 3.610 2.920 5.120

Periolivary Nucleus H -19.400 2.660 3.110

Pons Pontine Reticular Nucleus

Caudal M 0.084 0.757 0.689

Pontine Reticular Nucleus

Oral M 0.797 0.703 1.020

Gigantocellular Reticular

Nucleus Pons P 0.527 0.402 0.718

Parabrachial Nucleus P -0.201 0.428 0.309

Root of Trigeminal Nerve M-H 0.509 0.710 0.804

Facial Nucleus M-H 3.790 2.130 2.980

Locus Ceruleus M-H 1.530 1.340 3.090

Sub Coemleus Nucleus P 0.538 0.625 0.726

Median Raphe Nucleus M 2.470 1.070 1.240

Inferior Colliculus P 0.827 0.931 0.850

Tectum

Superior Colliculus M 0.374 0.585 0.669

Anterior Pretectal Nucleus P 0.413 0.815 0.665

Medial Pretectal Area P

Substantia Nigra Compacta M 0.427 0.594 0.560

Substantia Nigra Reticularis M 1.170 1.370 1.160

Cerebral Red Nucleus M 0.119 0.301 0.553

ST Peduncle Ventral Tegmental Area P -0.005 0.142 0.269

B

Interpeduncular Nucleus P -0.078 0.249 0.773 Periaqueductal Gray

Thalamus P 0.550 0.785 1.030

Central Gray M 0.799 1.220 1.740

Reticular Nucleus Midbrain M 1.030 0.969 1.250 Pedunculopontine

M _ih_li Tegmental Area P 1.670 0.643 0.800

Dorsal Raphe P 0.400 0.772 0.891

Raphe Linear P -0.026 0.581 2.880

Inferior Olivary Complex M 2.350 2.430 1.720

Solitary Tract Nucleus P 0.359 0.430 0.717

Cochlear Nucleus M 8.320 2.150 3.010

Vestibular Nucleus M 1.070 0.842 0.952 ft)

a Raphe Magnus M 56.400 0.698 1.250

Raphe Obscurus Nucleus M 0.139 0.237 0.196

PCRt M 1.730 0.889 1.280

B

ore DPGi P 0.661 0.779 0.771

Precuniform Nucleus 0.989 1.380 1.890

Ventricle 0.636 0.661 0.704

White Matter 1.060 1.190 1.150

White Matter 1.340 1.080 1.020

*A4 = nanoemulsions targeted with the bradykinin receptor ligand Cereport; Experiment performed on three rats.

[00187] The Specificity Index (SPI) was calculated from the raw Tl relaxation values of Gd³⁺ from the data compiled across a cohort and normalized using the following

equation:

where "A2" is the targeted NE and "A" is the non-targeted NE, "Pre" represents the pre-scan performed before NEs are injected, "t=20,40,60 min" represents a scan done 20 min, 40 min, and 60 min after injection of the NE. When calculating the specificity of the non-targeted NEs, the equation becomes:

[00188] Upon calculation of an SPI for a cohort, the "QUARTILE" function in Excel was used to find the top quartile (or the 75^th percentile) of the array. These SPI values are highlighted by boxing in and bolding the numbers to visualize the data with the biggest positive differences in increased Tl time, which indicates more gadolinium labeled nanoemulsion accumulation in the highlighted area. [00189] To determine statistical significance of the data a paired t-test was used to determine the statistical significance of data within a set, as well as between sets. Specifically, a t-test was used to compare the pre-scans across a cohort with the post scans across a cohort and determine the level of statistical significance.

[00190] These studies show that the brain targeted Gd-containing nanoemulsion formulations of the present disclosure are useful as MRI agents as well as concentrating the brain receptor targeted nanoemulsions in specific regions of brain tissues that express the receptors for the targeting ligands.

EQUIVALENTS

[00191] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

What is claimed is:

1. A nanoemulsion formulation comprising:

a targeting agent;

a drug delivery system comprising:

an oil phase; and

an interfacial surface membrane; and

an aqueous phase; and

a therapeutic agent, an imaging agent, or a combination thereof.

2. The nanoemulsion formulation of claim 1, wherein the targeting ligand comprises a brain tumor targeting ligand, a brain receptor targeting ligand, or a combination thereof.

3. The nanoemulsion formulation of claim 2, wherein the brain receptor targeting ligand comprises an μ-opioid receptor targeting ligand, an N-acetylcholine receptor targeting ligand, an integrin targeting ligand, a neurophilin targeting ligand, a bradykinin targeting ligand, or a combination thereof.

4. The nanoemulsion formulation of claim 3, wherein the brain receptor targeting ligand comprises the μ-opioid receptor targeting ligand dermorphin, the N- acetylcholine receptor targeting ligand candoxin, the integrin and neurophilin targeting ligand cRGD, the bradykinin targeting ligand cereport, or a combination thereof.

5. The nanoemulsion formulation of claim 2, wherein the brain tumor receptor targeting ligand comprises an EGFR-targeting ligand.

6. The nanoemulsion formulation of claim 5, wherein the EGFR-targeting ligand comprises peptide 4, an anti-EGFR immunoglobulin or EGFR-binding fragment thereof, EGal-PEG, or a combination thereof.

7. The nanoemulsion formulation of claim 1, wherein the oil phase of the drug delivery system comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or a combination thereof.

8. The nanoemulsion formulation of claim 1, wherein the interfacial surface membranes phase of the drug delivery system comprises an emulsifier and/or a stabilizer.

9. The nanoemulsion formulation of claim 8, wherein the emulsifier comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, hydrogenated soy

phosphatidylcholine, 1 ,2-distearoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine, or a combination thereof.

10. The nanoemulsion formulation of claim 8, wherein the stabilizer comprises a polyethylene glycol derivative, a phosphatide, a polyglycerol mono oleate, or a combination thereof.

1 1. The nanoemulsion formulation of claim 1, wherein the therapeutic agent is a chemotherapeutic agent comprising a platinum, a taxol, an aurora kinase inhibitor, an EGFR inhibitor, a src-c inhibitor, a PI3K/mTOR inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, or a combination thereof.

12. The nanoemulsion formulation of claim 11, wherein the therapeutic agent comprises the aurora kinase inhibitor NMI-900, the src-c inhibitor Dasatinib, the EGFR inhibitor Erlotinib, or a combination thereof.

13. The nanoemulsion formulation of claim 1, further comprising a

chemopotentiator.

14. The nanoemulsion formulation of claim 13, wherein the chemopotentiator comprises ceramide or a derivative thereof.

15. The nanoemulsion formulation of claim 13, wherein the chemopotentiator comprises C6-ceramide.

16. The nanoemulsion formulation of claim 1 , wherein the imaging agent is an MRI contrasting moiety.

17. The nanoemulsion formulation of claim 16, wherein the MRI contrasting moiety comprises gadolinium, iron oxide, iron platinum, manganese, or a combination thereof.

18. A method of targeting a nanoemulsion formulation to a specific region of the brain of a mammalian subject, the method comprising: administering an effective amount of the nanoemulsion formulation to the subject, the nanoemulsion formulation comprising:

a targeting agent;

a drug delivery system comprising:

an oil phase; and

an interfacial surface membrane; and

an aqueous phase; and

a therapeutic agent, an imaging agent, or a combination thereof; and,

determining if the nanoemulsion is in the targeted region of the brain.

19. The method of claim 18, wherein the nanoemulsion formulation is

administered orally, intranasally, intraperitoneally, intraocularly, or intravenously.

20. The method of claim 19, wherein the nanoemulsion formulation is

administered by injection into the cerebrospinal fluid.

21. The method of claim 18, wherein the targeting agent comprises a brain tumor targeting agent, a brain receptor targeting agent, or combinations thereof.

22. The method of claim 21, wherein the brain receptor targeting agent is an μ- opioid receptor targeting ligand, an N-acetylcholine receptor targeting ligand, an integrin targeting ligand, a neurophilin targeting ligand, a bradykinin targeting ligand, or a combination thereof.

23. The method of claim 22, wherein the brain receptor targeting agent comprises the μ-opioid receptor targeting ligand dermorphin, the N-acetylcholine receptor targeting ligand candoxin, the integrin and neurophilin targeting ligand cRGD, the bradykinin targeting ligand cereport, or combinations thereof.

24. The method of claim 21 , wherein the brain tumor receptor targeting agent comprises an EGFR targeting ligand.

25. The method of claim 18, wherein the oil phase of the drug delivery system of the nanoemulsion formulation comprises flaxseed oil, omega-3 polyunsaturated fish oil, omega-6 polyunsaturated fish oil, safflower oil, olive oil, pine nut oil, cherry kernel oil, soybean oil, pumpkin oil, pomegranate oil, primrose oil, or a combination thereof.

26. The method of claim 18, wherein the interfacial surface membrane of the drug delivery system of the nanoemulsion formulation comprises an emulsifier and/or a stabilizer.

27. The method of claim 26, wherein the emulsifier comprises egg lecithin, egg phosphatidyl choline, soy lecithin, phosphatidyl ethanolamine, phosphatidyl inositol, dimyristoylphosphatidyl choline, dimyristoylphosphatidyl ethyl-N-dimethyl propyl ammonium hydroxide, hydrogenated soy phosphatidylcholine, 1,2-distearoyl-sn- glycero-3-phosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or a combination thereof.

28. The method of claim 18, wherein the nanoemulsion formulation comprises a therapeutic agent, which treats a brain disorder.

29. The method of claim 28, wherein the therapeutic agent comprises a platinum, a taxol, an aurora kinase inhibitor, an EGFR inhibitor, a src-c inhibitor, a PI3K/mTOR inhibitor, a cytoskeletal inhibitor, a kinase inhibitor, an inflammatory signal inhibitor, or a combination thereof.

30. The method of claim 28, wherein the therapeutic agent comprises the aurora kinase inhibitor NMI-900, the src-c inhibitor Dasatinib, the EGFR inhibitor Erlotinib, or a combination thereof.

31. The method of claim 18, wherein the nanoemulsion formulation comprises an imaging agent, which images a region of the brain.

32. The method of claim 31, wherein the imaging agent comprises gadolinium, iron oxide, iron, platinum, manganese, or a combination thereof.

33. The method of claim 18, wherein the nanoemulsion formulation is targeted to a brain cancer in a region of the subject's brain.

34. The method of claim 33, wherein the brain cancer is a Glioblastoma or Glioma.

35. The method of claim 18, wherein the targeting agent of the nanoemulsion formulation comprises dermorphin, and the region of the brain to which the nanoemulsion formulation is targeted is a region which has opioid receptors.

36. A method of inhibiting the growth of, or killing, a cancer cell, comprising contacting the cancer cell with an amount of the nanoemulsion formulation of claim 1 that is toxic to, inhibits the growth of, or kills, the cancer cell.

37. The method of claim 36, wherein the cancer cell is in a mammal, and the contacting step comprises administering to the mammal a therapeutically effective amount of the nanoemulsion formulation.