WO2010111620A1

WO2010111620A1 - Ascorbate-linked nanosystems for brain delivery

Info

Publication number: WO2010111620A1
Application number: PCT/US2010/028872
Authority: WO
Inventors: Vladimir Torchilin; Tiziana Musacchio; Stefano Salmaso
Original assignee: Northeastern University; University Of Padua
Priority date: 2009-03-26
Filing date: 2010-03-26
Publication date: 2010-09-30
Also published as: AU2010229700A1; CN102458365A; EP2410992A4; CA2756581A1; EP2410992A1

Abstract

Ascorbate-conjugated liposomes and micelles, methods of making such liposomes and micelles, and methods of using such liposomes and micelles, such as for delivery of therapeutic and detection agents to the brain, are described.

Description

ASCORBATE-LINKED NANOSYSTEMS FOR BRAIN DELIVERY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 61/163,577, filed March 26, 2009, the contents of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The invention is in the field of medicine, and more specifically, to therapeutic and diagnostic liposomes and micelles for medical screening and treatment.

BACKGROUND OF THE INVENTION

[0003] In the past decades, a great deal of research has focused on targeting the central nervous system for therapeutic and diagnostic purposes. The goal appeared to be extremely challenging due to the physiological architecture of the biological barriers, namely, the blood- brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier, which have poor permeability and allow for only restricted access to selected molecules. Such barriers limit the permeation to the brain compartment, protecting the nervous systems from toxic compounds, metabolites, viruses, and bacteria. By virtue of these physiologic barriers, most of the targeting strategies for drug delivery to the brain have shown limited efficiency. Thus, there is a need for systems that can specifically target and deliver compounds to the brain.

SUMMARY OF THE INVENTION

[0004] The invention is based, at least in part, on the discovery of a method for targeting liposomes and micelles to brain cells. This discovery was exploited to develop the invention, which, in one aspect, features a method of delivering a lipid composition to a brain cell, comprising contacting the brain cell with the lipid composition, the lipid composition comprising ascorbate or an ascorbate derivative on an outer surface of the lipid composition, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter (SVCT) on the brain cell to thereby deliver the lipid composition to the brain cell. In certain embodiments, the lipid composition is a liposome or a micelle. In particular embodiments, the ascorbate linked to the liposome or micelle is not bound by a glucose transporter (GLUT), or has reduced binding to a GLUT relative to free ascorbate.

[0005] In some embodiments, the liposome or the micelle comprises a phospholipid conjugated to the ascorbate or the ascorbate derivative at the C6 position of the ascorbate or the ascorbate derivative. In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyethanolamine (PE), or phosphatidylserine (PS). In particular embodiments, the phospholipid is dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetyl phosphatidylethanolamine - polyglycerin 8G, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphoethanolamine, or dioleoyl phosphatidylethanolamine.

[0006] In some embodiments, the phospholipid comprises a derivatized phospholipid. In particular embodiments, the derivatized phospholipid comprises polyethylene glycol (PEG). In certain embodiments, the ascorbate or the ascorbate derivative is conjugated to the derivatized phospholipid via the PEG. In yet other embodiments, the the derivatized phospholipid is PEG- l,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0007] In some embodiments, the liposome or the micelle comprises 6-ascorbate-PEG-l,2- distearoyl-sft-glycero-3-phosphoethanolamine.

[0008] In some embodiments, about 50% to about 100% of the outer surface area of the liposome or the micelle comprises ascorbate. In other embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about

100% of the outer surface area of the liposome or the micelle comprises ascorbate.

[0009] In some embodiments, the ascorbate comprises about 20% to about 60% of the liposome or the micelle by weight. In particular embodiments, the ascorbate comprises about

10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the liposome or micelle by weight.

[0010] In some embodiments, the brain cell is an epithelial cell of the choroid plexus or an ependymal cell of the blood-brain barrier. In some embodiments, the brain cell transports the liposome or the micelle into the cerebrospinal fluid (CSF) of the brain. In other embodiments, after entering the CSF, the liposome or micelle contacts a second brain cell. In particular embodiments, the second brain cell is a neuron, a glial cell, or an astrocyte. In certain embodiments, the second brain cell is a brain tumor cell.

[0011] In some embodiments, the liposome or micelle comprises a targeting agent. In certain embodiments, the targeting agent is an antibody, a signal peptide, or a nucleic acid. [0012] In certain embodiments, the liposome or the micelle further comprises a therapeutic agent. In some embodiments, the compound is a cancer therapeutic described herein. In some embodiments, the compound is paclitaxel, tamoxifen, camptothecin, topotecan, irinotecan, KRN 5500 (KRN), meso-tetraphenylporphine, dexamethasone, a benzodiazepine, allopurinol, acetohexamide, benzthiazide, chlorpromazine, chlordiazepoxide, haloperidol, indomethacine, lorazepam, methoxsalen, methylprednisone, nifedipine, oxazepam, oxyphenbutazone, prednisone, prednisolone, pyrimethamine, phenindione, sulfϊsoxazole, sulfadiazine, temazepam, sulfamerazine, ellipticin, or trioxsalen.

[0013] In certain embodiments, the liposome or the micelle further comprises a detection agent. In certain embodiments, the detection agent is a magnetic resonance imaging (MRI) contrast agent, a computed tomography (CT scan) imaging agent, an optical imaging agent, or a radioisotope.

[0014] In another aspect, the invention features a method of delivering a therapeutic agent or a detection agent to a brain cell, comprising contacting the brain cell with a lipid composition comprising (i) a therapeutic agent or a detection agent; and (ii) ascorbate or an ascorbate derivative on an outer surface of the lipid composition, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter (SVCT) on the brain cell to thereby deliver the therapeutic agent or the detection agent to the brain cell. In certain embodiments, the lipid composition is a liposome or a micelle. In particular embodiments, the ascorbate linked to the liposome or micelle is not bound by a glucose transporter (GLUT), or has reduced binding to a GLUT relative to free ascorbate.

[0015] In some embodiments, the liposome or the micelle comprises a phospholipid conjugated to the ascorbate or the ascorbate derivative at the C6 position of the ascorbate or the ascorbate derivative. In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyethanolamine (PE), or phosphatidylserine (PS). In particular embodiments, the phospholipid is dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetyl phosphatidylethanolamine - polyglycerin 8G, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphoethanolamine, or dioleoyl phosphatidylethanolamine.

[0016] In some embodiments, the phospholipid comprises a derivatized phospholipid. In particular embodiments, the derivatized phospholipid comprises polyethylene glycol (PEG). In certain embodiments, the ascorbate or the ascorbate derivative is conjugated to the derivatized phospholipid via the PEG. In yet other embodiments, the the derivatized phospholipid is PEG- l,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0017] In some embodiments, the liposome or the micelle comprises 6-ascorbate-PEG-l,2- distearoyl-sft-glycero-3-phosphoethanolamine.

[0018] In some embodiments, about 50% to about 100% of the outer surface area of the liposome or the micelle comprises ascorbate. In other embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about

[0019] In some embodiments, the ascorbate comprises about 20% to about 60% of the liposome or the micelle by weight. In particular embodiments, the ascorbate comprises about

[0020] In some embodiments, the brain cell is an epithelial cell of the choroid plexus or an ependymal cell of the blood-brain barrier. In some embodiments, the brain cell transports the liposome or the micelle into the cerebrospinal fluid (CSF) of the brain. In other embodiments, after entering the CSF, the liposome or micelle contacts a second brain cell. In particular embodiments, the second brain cell is a neuron, a glial cell, or an astrocyte. In certain embodiments, the second brain cell is a brain tumor cell.

[0021] In some embodiments, the liposome or micelle comprises a targeting agent. In certain embodiments, the targeting agent is an antibody, a signal peptide, or a nucleic acid.

[0022] In some embodiments, the therapeutic agent is a therapeutic agent described herein, such as paclitaxel, tamoxifen, camptothecin, topotecan, irinotecan, KRN 5500 (KRN), meso- tetraphenylporphine, dexamethasone, a benzodiazepine, allopurinol, acetohexamide, benzthiazide, chlorpromazine, chlordiazepoxide, haloperidol, indomethacine, lorazepam, methoxsalen, methylprednisone, nifedipine, oxazepam, oxyphenbutazone, prednisone, prednisolone, pyrimethamine, phenindione, sulfϊsoxazole, sulfadiazine, temazepam, sulfamerazine, ellipticin, or trioxsalen.

[0023] In certain embodiments, the detection agent is a magnetic resonance imaging (MRI) contrast agent, a computed tomography (CT scan) imaging agent, an optical imaging agent, or a radioisotope.

[0024] In another aspect, the invention features a method of treating a brain disease or disorder described herein, comprising administering to a subject in need thereof a lipid composition comprising (i) a therapeutic agent; and (ii) ascorbate or an ascorbate derivative on an outer surface of the lipid composition, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter on the brain tumor, thereby delivering the therapeutic agent to a brain cell and treating the brain disease or disorder. In certain embodiments, the lipid composition is a liposome or a micelle. In particular embodiments, the ascorbate linked to the liposome or micelle is not bound by a glucose transporter (GLUT), or has reduced binding to a GLUT relative to free ascorbate.

[0025] In some embodiments, the liposome or the micelle comprises a phospholipid conjugated to the ascorbate or the ascorbate derivative at the C6 position of the ascorbate or the ascorbate derivative. In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyethanolamine (PE), or phosphatidylserine (PS). In particular embodiments, the phospholipid is dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetyl phosphatidylethanolamine - polyglycerin 8G, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphoethanolamine, or dioleoyl phosphatidylethanolamine.

[0026] In some embodiments, the phospholipid comprises a derivatized phospholipid. In particular embodiments, the derivatized phospholipid comprises polyethylene glycol (PEG). In certain embodiments, the ascorbate or the ascorbate derivative is conjugated to the derivatized phospholipid via the PEG. In yet other embodiments, the the derivatized phospholipid is PEG- l,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0027] In some embodiments, the liposome or the micelle comprises 6-ascorbate-PEG-l,2- distearoyl-sft-glycero-3-phosphoethanolamine.

[0028] In some embodiments, about 50% to about 100% of the outer surface area of the liposome or the micelle comprises ascorbate. In other embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about

[0029] In some embodiments, the ascorbate comprises about 20% to about 60% of the liposome or the micelle by weight. In particular embodiments, the ascorbate comprises about

[0030] In some embodiments, the brain cell is an epithelial cell of the choroid plexus or an ependymal cell of the blood-brain barrier. In some embodiments, the brain cell transports the liposome or the micelle into the cerebrospinal fluid (CSF) of the brain. In other embodiments, after entering the CSF, the liposome or micelle contacts a second brain cell. In particular embodiments, the second brain cell is a neuron, a glial cell, or an astrocyte. In certain embodiments, the second brain cell is a brain tumor cell.

[0031] In some embodiments, the liposome or micelle comprises a targeting agent. In certain embodiments, the targeting agent is an antibody, a signal peptide, or a nucleic acid.

[0032] In some embodiments, the brain disease or disorder is a brain tumor, a migraine, convulsions, an infection, schizophrenia, depression, hypoxia, a cerebral ischemia, cerebral palsy, a degenerative brain disease, a cerebrovascular disease, dyspnea, or encephalopathy.

[0033] In some embodiments, the therapeutic agent is a therapeutic agent described herein, such as paclitaxel, tamoxifen, camptothecin, topotecan, irinotecan, KRN 5500 (KRN), meso- tetraphenylporphine, dexamethasone, a benzodiazepine, allopurinol, acetohexamide, benzthiazide, chlorpromazine, chlordiazepoxide, haloperidol, indomethacine, lorazepam, methoxsalen, methylprednisone, nifedipine, oxazepam, oxyphenbutazone, prednisone, prednisolone, pyrimethamine, phenindione, sulfϊsoxazole, sulfadiazine, temazepam, sulfamerazine, ellipticin, or trioxsalen.

[0034] In some embodiments, the subject is a vertebrate. In certain embodiments, the subject is a mammal. In particular embodiments, the subject is a human. [0035] In certain embodiments, the liposome or micelle is administered in combination with a second therapy for the brain disease or disorder.

[0036] In certain embodiments, the liposome or micelle further comprises a detection agent described herein.

[0037] In another aspect, the invention features a method of imaging a brain cell, comprising administering to a subject in need thereof a lipid composition comprising: (i) a detection agent; and (ii) ascorbate or an ascorbate derivative on an outer surface of the lipid composition, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter on the brain tumor; and detecting the detection agent to thereby image the brain cell. In certain embodiments, the lipid composition is a liposome or a micelle. In particular embodiments, the ascorbate linked to the liposome or micelle is not bound by a glucose transporter (GLUT), or has reduced binding to a GLUT relative to free ascorbate. [0038] In some embodiments, the liposome or the micelle comprises a phospholipid conjugated to the ascorbate or the ascorbate derivative at the C6 position of the ascorbate or the ascorbate derivative. In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyethanolamine (PE), or phosphatidylserine (PS). In particular embodiments, the phospholipid is dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetyl phosphatidylethanolamine - polyglycerin 8G, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphoethanolamine, or dioleoyl phosphatidylethanolamine.

[0039] In some embodiments, the phospholipid comprises a derivatized phospholipid. In particular embodiments, the derivatized phospholipid comprises polyethylene glycol (PEG). In certain embodiments, the ascorbate or the ascorbate derivative is conjugated to the derivatized phospholipid via the PEG. In yet other embodiments, the the derivatized phospholipid is PEG- l,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0040] In some embodiments, the liposome or the micelle comprises 6-ascorbate-PEG-l,2- distearoyl-sft-glycero-3-phosphoethanolamine. [0041] In some embodiments, about 50% to about 100% of the outer surface area of the liposome or the micelle comprises ascorbate. In other embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about

[0042] In some embodiments, the ascorbate comprises about 20% to about 60% of the liposome or the micelle by weight. In particular embodiments, the ascorbate comprises about

[0043] In some embodiments, the brain cell is an epithelial cell of the choroid plexus or an ependymal cell of the blood-brain barrier. In some embodiments, the brain cell transports the liposome or the micelle into the cerebrospinal fluid (CSF) of the brain. In other embodiments, after entering the CSF, the liposome or micelle contacts a second brain cell. In particular embodiments, the second brain cell is a neuron, a glial cell, or an astrocyte. In certain embodiments, the second brain cell is a brain tumor cell.

[0044] In some embodiments, the liposome or micelle comprises a targeting agent. In certain embodiments, the targeting agent is an antibody, a signal peptide, or a nucleic acid.

[0045] In certain embodiments, the detection agent is a magnetic resonance imaging (MRI) contrast agent, a computed tomography (CT scan) imaging agent, an optical imaging agent, or a radioisotope.

[0046] In another aspect, the invention features a liposome or micelle described herein. In particular embodiments, the liposome or micelle comprises 6-ascorbate-PEG-l,2-distearoyl-sn- glycero-3-phosphoethanolamine.

[0047] In another aspect, the invention features the use of a liposome or a micelle described herein for the treatment, detection, or diagnosis of a brain disease or disorder described herein.

[0048] The following figures are presented for the purpose of illustration only, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. IA is a graphic representation of fluorescence-activated cell sorting (FACS) analysis of rat C6 cells treated with control antibody.

[0050] FIG. IB is a graphic representation of FACS analysis of rat F98 cells treated with control antibody. [0051] FIG. 1C is a graphic representation of FACS analysis of mouse NIH/3T3 cells treated with control antibody.

[0052] FIG. ID is a graphic representation of FACS analysis of rat C6 cells treated with goat polyclonal anti-SVCT2 antibody.

[0053] FIG. IE is a graphic representation of FACS analysis of rat F98 cells treated with goat polyclonal anti-SVCT2 antibody.

[0054] FIG. IF is a graphic representation of FACS analysis of mouse NIH/3T3 cells treated with goat polyclonal anti-SVCT2 antibody.

[0055] FIG. 2A is a graphic representation of FACS analysis of untreated rat C6 cells.

[0056] FIG. 2B is a graphic representation of FACS analysis of rat C6 cells treated with rhodamine-labeled PEG-liposomes.

[0057] FIG. 2C is a graphic representation of FACS analysis of rat C6 cells treated with ascorbate-PEG-liposomes.

[0058] FIG. 2D is a graphic representation of FACS analysis of untreated rat C6 cells.

[0059] FIG. 2E is a graphic representation of FACS analysis of rat C6 cells treated with mPEG2kDa-PE micelles.

[0060] FIG. 2F is a graphic representation of FACS analysis of rat C6 cells treated with acrobate-PEG-PE micelles.

[0061] FIG. 3A is a graphic representation of FACS analysis of untreated rat F98 cells.

[0062] FIG. 3B is a graphic representation of FACS analysis of rat F98 cells treated with rhodamine-labeled PEG-liposomes.

[0063] FIG. 3C is a graphic representation of FACS analysis of rat F98 cells treated with ascorbate-PEG-liposomes.

[0064] FIG. 3D is a graphic representation of FACS analysis of untreated rat F98 cells.

[0065] FIG. 3E is a graphic representation of FACS analysis of rat F98 cells treated with mPEG2kDa-PE micelles.

[0066] FIG. 3F is a graphic representation of FACS analysis of rat F98 cells treated with acrobate-PEG-PE micelles.

[0067] FIG. 4A is a representation of an epifluorescence microscopy image of nuclear staining with Hoechst 33342 in rat C6 cells treated with fluorescently labeled PEG-liposomes.

[0068] FIG. 4B is a representation of an epifluorescence microscopy image of Rh-PE- labeled liposomes in rat C6 cells treated with fluorescently labeled PEG-liposomes. [0069] FIG. 4C is a representation of an epifluorescence microscopy image of the superimposition of FIG. 4A and FIG. 4B.

[0070] FIG. 4D is a representation of an epifluorescence microscopy image of nuclear staining with Hoechst 33342 in rat C6 cells treated with ascorbate-PEG-liposomes.

[0071] FIG. 4E is a representation of an epifluorescence microscopy image of Rh-PE- labeled liposomes in rat C6 cells treated with ascorbate-PEG-liposomes.

[0072] FIG. 4F is a representation of an epifluorescence microscopy image of the superimposition of FIG. 4D and FIG. 4E.

[0073] FIG. 5A is a representation of an epifluorescence microscopy image of nuclear staining with Hoechst 33342 in rat C6 cells treated with fluorescently labeled PEG-PE micelles.

[0074] FIG. 5B is a representation of an epifluorescence microscopy image of rhodamine- labeled micelles in rat C6 cells treated with fluorescently labeled PEG-PE micelles.

[0075] FIG. 5C is a representation of an epifluorescence microscopy image of the superimposition of FIG. 5 A and FIG. 5B.

[0076] FIG. 5D is a representation of an epifluorescence microscopy image of nuclear staining with Hoechst 33342 in rat C6 cells treated with ascorbate -PEG-PE micelles.

[0077] FIG. 5E is a representation of an epifluorescence microscopy image of rhodamine- labeled micelles in rat C6 cells treated with ascorbate-PEG-PE micelles.

[0078] FIG. 5F is a representation of an epifluorescence microscopy image of the superimposition of FIG. 5D and FIG. 5E.

[0079] FIG. 6A is a representation of an epifluorescence microscopy image of rat C6 cells treated with fluorescently labeled ascorbate-PEG-liposomes without ascorbic acid preincubation.

[0080] FIG. 6B is a representation of an epifluorescence microscopy image of rat C6 cells treated with fluorescently labeled ascorbate-PEG-liposomes preincubated with ascorbic acid.

[0081] FIG. 6C is a representation of a light microscopy image of rat C6 cells treated with fluorescently labeled ascorbate-PEG-liposomes without ascorbic acid preincubation.

[0082] FIG. 6D is a representation of a light microscopy image of rat C6 cells treated with fluorescently labeled ascorbate-PEG-liposomes preincubated with ascorbic acid.

[0083] FIG. 7 is a graphic representation of relative fluorescence intensity associated with rat C6 cells after treatment with rhodamine-labeled ascorbate-PEG-liposomes with or without preincubation with free ascorbic acid. DETAILED DESCRIPTION OF THE INVENTION

[0084] All publications, patent applications, patents, and other references mentioned herein, including GenBank database sequences, are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

[0085] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Definitions

[0086] As used herein, a "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus.

[0087] As used herein, the term "biodegradable" refers to a substance that can be decomposed (e.g., chemically or enzymatically) or broken down in component molecules by natural biological processes (e.g., in vertebrate animals such as humans).

[0088] As used herein, the term "biocompatible" refers to a substance that has no unintended toxic or injurious effects on biological functions in a target organism.

[0089] The term "about", as used herein, means a numeric value having a range of ± 10% around the cited value.

[0090] As used herein, "treat," "treating" or "treatment" refers to administering a therapy in an amount, manner (e.g., schedule of administration), and/or mode (e.g., route of administration), effective to improve a disorder (e.g., a disorder described herein) or a symptom thereof, or to prevent or slow the progression of a disorder (e.g. , a disorder described herein) or a symptom thereof. This can be evidenced by, e.g. , an improvement in a parameter associated with a disorder or a symptom thereof, e.g. , to a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. By preventing or slowing progression of a disorder or a symptom thereof, a treatment can prevent or slow deterioration resulting from a disorder or a symptom thereof in an affected or diagnosed subject.

[0091] The term "polymer," as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, polysaccharides and polyalkylene glycols. Polymers can also be biodegradable and/or biocompatible.

[0092] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids. Additionally, such polypeptides, peptides, and proteins include amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0093] The terms "therapeutic agent" and "drug" are used interchangeably herein, and refer to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.

[0094] The term "lipid composition", as used herein, refers to amphoteric compounds that are capable of liposome formation, vesicle formation, micelle formation, emulsion formation, and are substantially non-toxic when administered. The lipid composition may include, without limitation, egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), soy phosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG),

Dipalmitoylphosohatidyl-choline (DPPC), disteroylphosphatidylglycerol (DSPG), dipalmitoylphosphatidyl-glycerol (DMPG), cholesterol (Choi), cholesterol sulfate and its salts

(CS), cholesterol hemisuccinate and its salts (Chems), cholesterol phosphate and its salts (CP), cholesterylphospholine and other hydroxycholesterol or amino cholesterol derivatives.

[0095] The term "liposome", as used herein, means a vesicle including one or more concentrically ordered lipid bilayer(s) encapsulating an aqueous phase, when in an aqueous environment.

[0096] The term "micelle", as used herein, means a vesicle including a single lipid monolayer encapsulating an aqueous phase.

[0097] The term "polyethylene glycol (PEG)" includes polymers of lower alkylene oxide, in particular ethylene oxide (polyethylene glycols) having an esterifϊable hydroxyl group at least at one end of the polymer molecule, as well as derivatives of such polymers having esterifiable carboxy groups. Polyethylene glycols include those having an average molecular weight ranging from about 200 to about 20,000.

[0098] As used herein, the term "brain cell" refers to cells that are found in, about, or associated with, cells of the central nervous system and the brain, whether healthy or diseased, including the lower, mid and upper cortex, immune cells, and support cells associated therewith. Brain cells include all types of neurons, e.g., afferent neurons, efferent neurons, and interneurons, whether pseudounipolar, bipolar, multipolar and the like. Brain cells also include glial cells, astrocytes, Schwann cells, Purkinje cells, epithelial cells of the choroid plexus, ependymal cells of the blood-brain barrier, and the like. Brain cells also include tumor cells within the brain.

[0099] The present disclosure provides, in part, lipid compositions for delivery of an agent (e.g., a therapeutic agent or a detection agent), to a brain cell. The lipid compositions include liposomes and micelles that are conjugated or linked to ascorbate or to an ascorbate derivative.

Liposomes

[0100] Liposomes are vesicles that include one or more concentrically ordered lipid bilayer(s) encapsulating an aqueous phase, when in an aqueous environment. Such vesicles are formed in the presence of "vesicle-forming lipids", which are defined herein as amphipathic lipids capable of either forming or being incorporated into a bilayer structure. The term includes lipids that are capable of forming a bilayer by themselves or when in combination with another lipid or lipids. An amphipathic lipid is incorporated into a lipid bilayer by having its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane and its polar head moiety oriented towards an outer, polar surface of the membrane. Hydrophilicity arises from the presence of functional groups, such as hydroxyl, phosphate, carboxyl, sulfate, amino or sulfhydryl groups. Hydrophobicity results from the presence of a long chain of aliphatic hydrocarbon groups.

[0101] Liposomes include multilamellar vesicles, multivesicular liposomes, unilamellar vesicles, and giant liposomes. Multilamellar liposomes (also known as multilamellar vesicles ("MLV")) contain multiple concentric bilayers within each liposome particle, resembling the layers of an onion. Multivesicular liposomes consist of lipid membranes enclosing multiple non-concentric aqueous chambers. Unilamellar liposomes enclose a single internal aqueous compartment. Single bilayer (or substantially single bilayer) liposomes include small unilamellar vesicles ("SUV") and large unilamellar vesicles ("LUV"). LUVs and SUVs can range in size from about 50 nm to about 500 nm and about 20 nm to about 50 nm, respectively. Giant liposomes can range in size from about 5000 nm to about 50,000 nm (Needham et al., Colloids and Surfaces B: Biointerfaces 18:183-195 (2000)).

[0102] Any suitable vesicle-forming lipid {e.g., naturally occurring lipids and synthetic lipids) can be utilized in the liposomes and micelles described herein. Suitable lipids include, without limitation, phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyethanolamine (PE), phosphatidylserine (PS), and phosphoethanolamine; sterols such as cholesterol; glycolipids; sphingolipids such as sphingosine, ceramides, sphingomyelin, and glycosphingolipids (such as cerebrosides and gangliosides). Particular lipids include dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetyl phosphatidylethanolamine -polyglycerin 8G, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoyl phosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphoethanolamine, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, galactosyl ceramides, glycosyl ceramides, lactosyl ceramides, phosphatides, globosides, GMl (Galβl, 3GalNAcβl, 4(NeuAa2,3)Galβl, 4Glcβl, l'Cer), ganglioside GDIa, ganglioside GDIb, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, distearoyl-glycero-phosphoethanolamine, and 1,2-dioleoyl-sn- glycero-3-phsophoethanolamine. Suitable phospholipids can include one or two acyl chains having any number of carbon atoms, such as about 6 to about 24 carbon atoms, selected independently of one another and with varying degrees of unsaturation. Thus, combinations of phospholipid of different species and different chain lengths in varying ratios can be used. Mixtures of lipids in suitable ratios, as judged by one of skill in the art, can also be used. [0103] Liposomes can be generated using a variety of techniques known in the art. These techniques include, without limitation, ether injection (Deamer et al., Acad. Sci. 308:250 (1978)); surfactant (Brunner et al, Biochim. Biophys. Acta 455:322 (1976)); Ca²⁺ fusion (Paphadjopoulos et al., Biochim. Biophys. Acta 394:483 (1975)); freeze-thaw (Pick et al., Arach. Biochim. Biophys. 212:186 (1981)); reverse-phase evaporation (Szoka et al, Biochim. Biophys. Acta 601 :559 (1980)); ultrasonic treatment (Huang et al, Biochemistry 8:344 (1969)); ethanol injection (Kremer et al, Biochemistry 16:3932 (1977)); extrusion (Hope et al, Biochim. Biophys. Acta 812:55 (1985)); French press (Barenholz et al, FEBS Lett. 99:210 (1979)); thin film hydration (Bangham et al, J. MoI Biol. 13:238-252 (1965)); and any other methods described herein or known in the art. Liposomes can also be generated using commercially available kits {e.g. , from Boehringer-Mannheim, ProMega, and Life Technologies (Gibco)).

[0104] Different techniques can be used depending on the type of liposome desired. For example, small unilamellar vesicles (SUVs) can be prepared by the ultrasonic treatment method, the ethanol injection method, or the French press method, while multilamellar vesicles (MLVs) can be prepared by the reverse-phase evaporation method or by the simple addition of water to a lipid film, followed by dispersal by mechanical agitation (Bangham et al, J. MoI Biol 13:238-252 (1965)). LUVs can be prepared by the ether injection method, the surfactant method, the Ca²⁺ fusion method, the freeze-thaw method, the reverse-phase evaporation method, the French press method, or the extrusion method.

[0105] Average liposome size can be determined by known techniques, such as quasi- elastic light scattering, photon correlation spectroscopy, dynamic light scattering, or various electron microscopy techniques (such as negative staining transmission electron microscopy, freeze fracture electron microscopy or cryo-transmission electron microscopy). In some instances, the resulting liposomes can be run down a Sephadex™ G50 column or similar size exclusion chromatography column equilibrated with an appropriate buffer in order to remove unencapsulated therapeutic agents or detection agents described herein. [0106] Liposomes can range in size, such as from about 50 nm to about 1 μm in diameter. For example, liposomes described herein can be less than about 200 nm in diameter, less than about 160 nm in diameter, or less than about 140 nm in diameter. In some embodiments, liposomes described herein can be substantially uniform in size, for example, 10% to 100%, or more generally at least 10%, 20%, 30%, 40%, 50, 55% or 60%, or at least 65%, 75%, 80%, 85%, 90%, or 95%, or as much as 96%, 97%, 98%, 99%, or 100% of the liposomes can have the same size. In some instances, liposomes can be sized by extrusion through a filter {e.g., a polycarbonate filter) having pores or passages of the desired diameter. [0107] In some instances, liposomes can include a hydrophilic moiety. Attaching a hydrophilic moiety to the surface of liposomes can sterically stabilize liposomes and can increase the circulation longevity of the liposome. This can enhance blood stability and increase circulation time, reduce uptake into healthy tissues, and increase delivery to disease sites such as solid tumors (see, e.g., U.S. Nos. 5,013,556 and 5,593,622; and Patel et ah, Crit. Rev. Ther. Drug Carrier Syst. 9:39 (1992)). The hydrophilic moiety can be conjugated to a lipid component of the liposome, forming a hydrophilic polymer- lipid conjugate. The term "hydrophilic polymer- lipid conjugate", as used herein, refers to a lipid (e.g., a vesicle-forming lipid) co valently joined at its polar head moiety to a hydrophilic polymer, and can be made by attaching the polymer to a reactive functional group at the polar head moiety of the lipid. The covalent linkage can be releasable, such that the polymer dissociates from the lipid (at, e.g. , physiological pH or after a variable length of time (see, e.g., Adlakha-Hutcheon et al, Nat. Biotechnol. 17:775-779 (1999)). Nonlimiting suitable reactive functional groups include, e.g., amino, hydroxyl, carboxyl, and formyl groups. The lipid can be any lipid described in the art for use in such conjugates. For example, the lipid can be a phospholipid having one or two acyl chains including between about 6 to about 24 carbon atoms in length with varying degrees of unsaturation.

[0108] In some circumstances, the lipid in the conjugate can be a phosphatidyethanolamine, such as of the distearoyl form. The polymer can be a biocompatible polymer. In some instances, the polymer has a solubility in water that permits polymer chains to extend away from a liposome surface with sufficient flexibility that produces uniform surface coverage of a liposome. Such a polymer can be a polyalkylether, including PEG, polymethylene glycol, polyhydroxy propylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyacrylic acid and copolymers thereof, as well as those disclosed in U.S. Nos. 5,013,556 and 5,395,619. The polymer can have an average molecular weight between about 350 daltons and about 10,000 daltons.

[0109] In some instances, the phospholipids can be derivatized phospholipids, such as a PEG-modifϊed phospholipid. The average molecular weight of the PEG can be about 200 daltons to about 20,000 daltons. The liposomes described herein can also be composed of combinations of PEG phospholipids of different species and different chain lengths in varying ratios. Combinations of phospholipids and PEG phospholipids can also be used in forming the liposomes described herein. The derivatized phospholipid can be prepared to include a releasable lipid-polymer linkage such as a peptide, ester, or disulfide linkage.

Micelles [0110] Micelles are vesicles that include a single lipid monolayer encapsulating an aqueous phase. Micelles can be spherical or tubular and form spontaneously about the critical micelle concentration ("CMC"). In general, micelles are in equilibrium with the monomers under a given set of physical conditions such as temperature, ionic environment, concentration, etc. [0111] Micelles are formed in the presence of "micelle-forming compounds", which include amphipathic lipids {e.g., a vesicle-forming lipid as described herein or known in the art), lipoproteins, detergents, non-lipid polymers, or any other compound capable of either forming or being incorporated into a monolayer vesicle structure. Thus, a micelle-forming compound includes compounds that are capable of forming a monolayer by themselves or when in combination with another compound, and may be polymer micelles, block co-polymer micelles, polymer-lipid mixed micelles, or lipid micelles. A micelle-forming compound, in an aqueous environment, generally has a hydrophobic moiety in contact with the interior of the vesicle, and a polar head moiety oriented outwards into the aqueous environment. Hydrophilicity generally arises from the presence of functional groups, such as hydroxyl, phosphate, carboxyl, sulfate, amino or sulfhydryl groups. Hydrophobicity generally results from the presence of a long chain of aliphatic hydrocarbon groups.

[0112] A micelle can be prepared, e.g., from lipoproteins or artificial lipoproteins including low density lipoproteins, chylomicrons and high density lipoproteins. Micelles can be generated using a variety of known techniques, including, without limitation, simple dispersion by mixing in aqueous or hydroalcoholic media or media containing surfactants or ionic substances; sonication; solvent dispersion; or any other technique described herein or known in the art. Different techniques can be used, depending on the type of micelle desired and the physicochemical properties of the micelle-forming components, such as solubility, hydrophobicity and behavior in ionic or surfactant-containing solutions. [0113] Micelles can range in size, such as between about 5 nm to about 50 nm in diameter. In some instances, micelles can be less than about 50 nm in diameter, less than about 30 nm in diameter, or less than about 20 nm in diameter.

[0114] In some situations, micelles described herein can include a hydrophilic polymer- lipid conjugate, as described herein or known in the art. Ascorbate Attachment to Liposomes or Micelles for Brain Delivery

[0115] Ascorbate is taken up by a sodium-dependent- vitamin C transporter (SVCT2), and the expression of SVCT2 is limited to choroid plexus epithelial cells, some neurons, tanycytes, astrocytes, and the arachnoid membrane (see, e.g., Tsukaguchi et ah, Nature 399:70-75 (1999)). Thus, to target a liposome or micelle described herein to a brain cell, the liposome or micelle is attached to ascorbate. The ascorbate is attached to the liposome or micelle and is exposed on an outer surface of the liposome or micelle, where it can contact an SVCT2 transporter. In particular instances, the ascorbate linked to a liposome or micelle described herein is not bound by a glucose transporter (GLUT), or has reduced binding to a GLUT relative to free ascorbate.

[0116] The ascorbate is not limited with respect to its form, and any known ascorbate or ascorbate derivative can be used. For example, ascorbate, ascorbic acid, or any pharmaceutically acceptable salt, hydrate, and solvate thereof, can be used. Ascorbate can be linked at its 6 position to a phospholipid described herein or to a hydrophilic polymer-lipid conjugate described herein using methods known in the art. For example, the ascorbate can be linked to a phospholipid via a covalent bond, such as by a sulfur atom, an oxygen atom, a nitrogen atom, or a hydrocarbon linking group, using known techniques. [0117] In particular instances, about 10% to about 100% of the phospholipids of the liposome or micelle are attached to ascorbate. For example, about 20% to about 95%, about 30% to about 90%, about 40% to about 80%, about 50% to about 95%, about 60% to about 90%, about 70% to about 100%, or about 80% to about 95% of the phospholipids are attached to ascorbate.

[0118] The ascorbate can be used to deliver a liposome or micelle described herein to the brain. For example, in some instances, ascorbate within an outer surface of a liposome or micelle described herein can contact an SVCT2 on a brain cell of the choroid plexus, where the liposome or micelle can release a therapeutic agent or diagnostic agent at the membrane of a brain cell of the choroid plexus. The therapeutic agent or diagnostic agent can subsequently be transported by the brain cell into the cerebrospinal fluid (CSF) within the ventricles of the brain. In other instances, ascorbate within an outer surface of a liposome or micelle described herein can contact an SVCT2 on a brain cell of the choroid plexus, which can transport the liposome or micelle into the brain cell. The liposome or micelle can then release a therapeutic agent or diagnostic agent within the brain cell of the choroid plexus, and the therapeutic agent or diagnostic agent can subsequently be transported or diffuse out of the brain cell into the CSF of the brain. In other situations, ascorbate-linked liposomes or micelles described herein can contact a receptor, e.g., an SVCT2, in a brain cell of the choroid plexus and be transported into the brain cell. The liposome or micelle can subsequently be transported or diffuse out of the brain cell into the cerebrospinal fluid (CSF) within the ventricles of the brain, where the liposome or micelle can release a therapeutic agent or diagnostic agent into the CSF, or be subsequently targeted to a brain cell using a targeting agent described herein.

Targeting Agents

[0119] An outer surface of a liposome or micelle of the disclosure can include, in addition to ascorbate or an ascorbate derivative, a targeting agent. As described herein, ascorbate on a liposome or micelle can mediate its transport into the brain, e.g., into the CSF. Once the liposome or micelle has entered the brain, e.g., the CSF of the brain, a targeting agent can direct the liposome or micelle to a particular target within the brain. Nonlimiting examples of brain targets include tumor cells, bacteria, viruses, cell surface proteins, cell surface receptors, cell surface polysaccharides, extracellular matrix proteins, intracellular proteins and intracellular nucleic acids. The targeting agents can be, for example, various specific ligands, such as antibodies, monoclonal antibodies and their fragments, folate, mannose, galactose and other mono-, di-, and oligosaccharides, and RGD peptide.

[0120] The liposomes and micelles described herein are not limited to any particular targeting agent, and a variety of targeting agents can be used. Examples of such targeting agents include, but are not limited to, nucleic acids (e.g., RNA and DNA), polypeptides (e.g., receptor ligands, signal peptides, avidin, Protein A, and antigen binding proteins), polysaccharides, biotin, hydrophobic groups, hydrophilic groups, drugs, and any organic molecules that bind to receptors. In some instances, a liposome or micelle described herein can be conjugated to one, two, or more of a variety of targeting agents. For example, when two or more targeting agents are used, the targeting agents can be similar or dissimilar. Utilization of more than one targeting agent on a particular liposome or micelle can allow the targeting of multiple biological targets or can increase the affinity for a particular target. [0121] The targeting agents can be associated with the liposomes or micelles in a number of ways. For example, the targeting agents can be associated (e.g., covalently or noncovalently bound) to a phospholipid of the liposome or micelle with either short (e.g., direct coupling), medium (e.g., using small-molecule bifunctional linkers such as SPDP (Pierce Biotechnology, Inc., Rockford, IL)), or long (e.g., PEG bifunctional linkers (Nektar Therapeutics, Inc., San Carlos, CA)) linkages.

[0122] In addition, a liposome or micelle can also incorporate reactive groups (e.g., amine groups such as polylysine, dextranemine, pro famine sulfate, and/or chitosan). The reactive group can allow for further attachment of various specific ligands or reporter groups (e.g., ¹²⁵I, ¹³¹I, I, Br, various chelating groups such as DTPA, which can be loaded with reporter heavy metals such as ¹¹¹In, ^99mTc, Gd, Mn, fluorescent groups such as FITC, rhodamine, Alexa, and quantum dots), and/or other moieties (e.g., ligands, antibodies, and/or portions thereof).

Antibodies as Targeting Agents

[0123] In some instances, the targeting agents are antigen binding proteins or antibodies or binding portions thereof. Antibodies can be generated to allow for the specific targeting of antigens or immunogens (e.g., tumor, tissue, or pathogen specific antigens) on various biological targets (e.g., pathogens, tumor cells, normal tissue). Such antibodies include, but are not limited to, polyclonal antibodies; monoclonal antibodies or antigen binding fragments thereof; modified antibodies such as chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof (e.g., Fv, Fab', Fab, F(ab')2); or biosynthetic antibodies, e.g., single chain antibodies, single domain antibodies (DAB), Fvs, or single chain Fvs (scFv). [0124] Methods of making and using polyclonal and monoclonal antibodies are well known in the art, e.g. , in Harlow et al. , Using Antibodies: A Laboratory Manual: Portable Protocol I. Cold Spring Harbor Laboratory (December 1, 1998). Methods for making modified antibodies and antibody fragments (e.g., chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof, e.g., Fab', Fab, F(ab')₂ fragments); or biosynthetic antibodies (e.g., single chain antibodies, single domain antibodies (DABs), Fv, single chain Fv (scFv), and the like), are known in the art and can be found, e.g. , in Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Springer Verlag (December 15, 2000; 1st edition).

[0125] Antibody attachment can be performed through standard covalent binding to free amine groups (see, e.g., Torchilin et al. (1987) Hybridoma, 6:229-240; Torchilin, et al., (2001) Biochim. Biovhvs. Acta, 1511 :397-411; Masuko, et al., (2005), BiomacromoL 6:800-884). Signal Peptides as Targeting Agents

[0126] In some instances, the targeting agents include a signal peptide. These peptides can be chemically synthesized or cloned, expressed and purified using known techniques. Signal peptides can be used to target the liposomes or micelles described herein to a discreet region within a brain cell.

Nucleic Acids as Targeting Agents

[0127] In other instances, the targeting agent is a nucleic acid (e.g., RNA or DNA). In some examples, the nucleic acid targeting agents are designed to hybridize by base pairing to a particular nucleic acid (e.g., chromosomal DNA, mRNA, or ribosomal RNA). In other situations, the nucleic acids bind a ligand or biological target. For example, the nucleic acid can bind reverse transcriptase, Rev or Tat proteins of HIV (Tuerk et al, Gene, 137(l):33-9 (1993)); human nerve growth factor (Binkley et al, Nuc. Acids Res., 23(16):3198-205 (1995)); or vascular endothelial growth factor (Jellinek et al, Biochem., 83(34): 10450-6 (1994)). Nucleic acids that bind ligands can be identified by known methods, such as the SELEX procedure (see, e.g., U.S. 5,475,096; 5,270,163; and 5,475,096; and WO 97/38134; WO 98/33941; and WO 99/07724). The targeting agents can also be aptamers that bind to particular sequences.

Other Targeting Agents

[0128] The targeting agents can recognize a variety of epitopes on preselected biological targets (e.g., pathogens, tumor cells, or normal cells). For example, in some instances, the targeting agent can be sialic acid to target HIV (Wies et al., Nature, 333:426 (1988)), influenza (White et al, Cell, 56:725 (1989)), Chlamydia (Infect. Immunol, 57:2378 (1989)), Neisseria meningitidis, Streptococcus suis, Salmonella, mumps, newcastle, reovirus, Sendai virus, and myxovirus; and 9-OAC sialic acid to target coronavirus, encephalomyelitis virus, and rotavirus; non- sialic acid glycoproteins to target cytomegalovirus {Virology, 176:337 (1990)) and measles virus (Virology, 172:386 (1989)); CD4 (Khatzman et al, Nature, 312:763 (1985)), vasoactive intestinal peptide (Sacerdote et al, J. ofNeuroscience Research, 18:102 (1987)), and peptide T (Ruff et al , FEBS Letters, 211 : 17 ( 1987)) to target HIV; epidermal growth factor to target vaccinia (Epstein et al, Nature, 318: 663 (1985)); acetylcholine receptor to target rabies (Lentz et al, Science 215: 182 (1982)); Cd3 complement receptor to target Epstein-Barr virus (Carel et al, J. Biol. Chem., 265:12293 (1990)); .beta. -adrenergic receptor to target reovirus (Co et al, Proc. Natl. Acad. Sci. USA, 82:1494 (1985)); ICAM-I (Marlin et al, Nature, 344:70 (1990)), N-CAM, and myelin-associated glycoprotein MAb (Shephey et ah, Proc. Natl. Acad. Sci. USA, 85:7743 (1988)) to target rhinovirus; polio virus receptor to target polio virus (Mendelsohn et ah, Cell, 56:855 (1989)); fibroblast growth factor receptor to target herpes virus (Kaner et ah, Science, 248:1410 (1990)); oligomannose to target Escherichia coli; and ganglioside G_MI to target Neisseria meningitides .

[0129] In other instances, the targeting agent targets nanoparticles to factors expressed by oncogenes. These can include, but are not limited to, tyrosine kinases (membrane-associated and cytoplasmic forms), such as members of the Src family; serine/threonine kinases, such as Mos; growth factor and receptors, such as platelet derived growth factor (PDDG), small GTPases (G proteins), including the ras family, cyclin-dependent protein kinases (cdk), members of the myc family members, including c-myc, N-myc, and L-myc, and bcl-2 family members.

Therapeutic and Detection Agents

[0130] A liposome or micelle described herein can include a therapeutic agent or a detection agent. Such liposomes or micelles containing a therapeutic agent or detection agent can be prepared by conventional active or passive loading methods. For example, a therapeutic agent can be mixed with vesicle-forming lipids and be incorporated within a lipid film, such that when the liposome is generated, the therapeutic agent is incorporated or encapsulated into the liposome. Thus, if the therapeutic agent is substantially hydrophobic, it will be encapsulated in the bilayer of the liposome. Alternatively, if the therapeutic agent is substantially hydrophilic, it will be encapsulated in the aqueous interior of the liposome. The therapeutic agent can be soluble in aqueous buffer or aided with the use of detergents or ethanol. The liposomes can subsequently be purified, for example, through column chromatography or dialysis to remove any unincorporated therapeutic agent. [0131] Nonlimiting examples of therapeutic agents useful for inclusion in a liposome or micelle include, e.g., steroids, analgesics, local anesthetics, antibiotic agents, chemotherapeutic agents, immunosuppressive agents, anti-inflammatory agents, antiproliferative agents, antimitotic agents, angiogenic agents, antipsychotic agents, central nervous system (CNS) agents, anticoagulants, fibrinolytic agents, growth factors, antibodies, ocular drugs, and metabolites, analogs, derivatives, fragments, and purified, isolated, recombinant and chemically synthesized versions of these species, and combinations thereof. [0132] Representative useful therapeutic agents include, but are not limited to, tamoxifen, paclitaxel, low soluble anticancer drugs, camptothecin and its derivatives, e.g., topotecan and irinotecan, KRN 5500 (KRN), meso-tetraphenylporphine, dexamethasone, benzodiazepines, allopurinol, acetohexamide, benzthiazide, chlorpromazine, chlordiazepoxide, haloperidol, indomethacine, lorazepam, methoxsalen, methylprednisone, nifedipine, oxazepam, oxyphenbutazone, prednisone, prednisolone, pyrimethamine, phenindione, sulfϊsoxazole, sulfadiazine, temazepam, sulfamerazine, ellipticin, porphine derivatives for photo-dynamic therapy, and/or trioxsalen, as well as all mainstream antibiotics, including the penicillin group, fluoroquinolones, and first, second, third, and fourth generation cephalosporins. These agents are commercially available from, e.g., Merck & Co., Barr Laboratories, Avalon Pharma, and Sun Pharma, among others.

[0133] In some instances, the liposomes or micelles described herein can be used to detect or image cells, e.g., using a liposome or a micelle that includes a detection agent. The detection agent can be used to qualitatively or quantitatively analyze the location and/or the amount of a liposome or micelle at a particular locus. The detection agent can also be used to image a liposome, micelle, and/or a cell or tissue target of a liposome or micelle using standard methods.

[0134] The liposome or micelle according to the disclosure, such as a phospholipid therein, can be modified or derivatized (or labeled) with a detection agent. Examples of detection agents include, but are not limited to, magnetic resonance imaging (MRI) contrast agents, computed tomography (CT scan) imaging agents, optical imaging agents and radioisotopes. Nonlimiting examples of detection agents include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms, (e.g., europium (Eu)), radioactive isotopes, quantum dots, electron-dense reagents, and haptens. The detection reagent can be detected using various means including, but are not limited to, spectroscopic, photochemical, radiochemical, biochemical, immunochemical, or chemical means.

[0135] Nonlimiting exemplary fluorescent detection agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, and the like. A detection agent can also be a detectable enzyme, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase and the like. When a liposome or micelle according to the disclosure is derivatized with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detection agent is horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine leads to a detectable colored reaction product. A liposome or micelle can also be derivatized with a prosthetic group (e.g. , streptavidin/biotin and avidin/biotin). For example, a liposome or micelle can be derivatized with biotin and detected through indirect measurement of avidin or streptavidin binding. Nonlimiting examples of fluorescent compounds that can be used as detection reagents include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Luminescent materials include, e.g., luminol, and bio luminescent materials include, e.g. , luciferase, luciferin, and aequorin. [0136] A detection agent useful for modification of the liposome or micelle can also be a radioactive isotope, such as, but not limited to, α-, β-, or γ-emitters; or β- and γ-emitters. Radioactive isotopes can be used in diagnostic or therapeutic applications. Such radioactive isotopes include, but are not limited to, iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium (¹⁴²Pr or ¹⁴³Pr), astatine (²¹¹At), rhenium (¹⁸⁶Re or ¹⁸⁷Re), bismuth (²¹²Bi or ²¹³Bi), indium (¹¹¹In), technetium (^99mTc), phosphorus (³²P), rhodium (¹⁸⁸Rh), sulfur (³⁵S), carbon (¹⁴C), tritium (³H), chromium (⁵¹Cr), chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron (⁵⁹Fe), selenium (⁷⁵Se), and gallium (⁶⁷Ga).

[0137] The liposome or micelle can be radiolabeled using techniques known in the art. In some situations, a liposome or micelle described herein is contacted with a chelating agent, e.g., l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), to thereby produce a conjugated liposome or micelle. The conjugated liposome or micelle is then radiolabeled with a radioisotope, e.g., ¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁷Re, or ^99mTc, to thereby produce a labeled liposome or micelle. In other methods, the liposome or micelle can be labeled with ¹¹¹In and ⁹⁰Y using weak transchelators such as citrate (see, e.g., Khaw et al., Science 209:295-297 (1980)) or ^99mTc after reduction in reducing agents such as Na Dithionite (see, e.g., Khaw et al., J. Nucl. Med. 23:1011-1019 (1982)) or by SnCl₂ reduction (see, e.g., Khaw et al, J. Nucl. Med. 47:868-876 (2006)). Other methods are described in, e.g., Lindegren et al, Bioconjug. Chem. 13:502-509 (2002); Boyd et al., Mol. Pharm. 3:614-627 (2006); and del Rosario et al., J. Nucl. Med. 34:1147-1151 (1993).

Brain Diseases and Disorders

[0138] The liposomes or micelles described herein can be used to target and/or treat (e.g. , mediate the translocation of therapeutic agents into) brain cells or brain tissue, such as diseased brain cells and tissues. In this regard, various diseases and disorders are amenable to treatment using the liposomes and micelles and methods described herein.

[0139] In certain instances, the liposomes or micelles of the disclosure are useful for the treatment of a disease or disorder characterized by abnormal brain tissue. Abnormal brain regions may include, for example, regions of brain tissue characterized by abnormal cell proliferation (e.g., malignant brain tumors), as well as regions of brain tissue physiologically affected by physical or biochemical injury, such as degenerative brain disease (e.g., Alzheimer's disease, Parkinson's disease), stroke, brain ischemia, infection or trauma. [0140] In some situations, the abnormal brain region is characterized by abnormal cell proliferation, such as associated with a neoplastic disease or malignancy (such as a cancer or a tumor). In some instances, the abnormal brain region is a malignant brain tumor. Among malignant brain tumors for which the inventive methods are effective are gliomas, which include any malignant glial tumor, i.e., a tumor derived from a transformed glial cell. About half of all primary brain tumors are gliomas. A glial cell is a cell that has one or more glial- specifϊc features, associated with a glial cell type, including a morphological, physiological and/or immunological feature specific to a glial cell (e.g. astrocyte or oligodendrocyte), for example, expression of the astroglial marker fibrillary acidic protein (GFAP) or the oligodendroglial marker 04. Non-limiting examples of neoplastic diseases or malignancies treatable with the composition of the present invention in combination and/or alternation with an antiproliferative agent include gliomas, glioblastomas, glioblastoma multiforme (GBM; i.e., astrocytoma grade IV), oligodendroglioma, primitive neuroectodermal tumor, low, mid and high grade astrocytoma (i.e., astrocytoma grade II, anaplastic astrocytoma grade III, astrocytoma with oligodendrogliomal component), ependymoma (e.g., myxopapillary ependymoma papillary ependymoma, subependymoma, anaplastic ependymoma), oligodendroglioma, medulloblastoma, meningioma (i.e., atypical meningioma, malignant meningioma), pituitary tumors (i.e., pituitary adenoma), neuroblastoma, and craniopharyngioma .

[0141] Other nonlimiting examples of brain tumors that can be treated using the liposomes and micelles described herein include acoustic neuroma (e.g., Neurilemmoma, Schwannoma, Neurinoma), chordoma, chordoma, CNS lymphoma, cysts, dermoid cysts, gangliocytoma, ganglioglioma, and hemangioblastoma.

[0142] In certain instances, the abnormal brain tissue is a secondary or metastatic brain tumor (i.e., tumors that have spread to the brain from another part of the body). Nonlimiting examples of metastatic brain tumors include cancers originating in breast, lung, kidney, colon, prostate, and skin (malignant melanoma).

[0143] Other nonlimiting examples of brain diseases or disorders include migraines, convulsions, infections, metal illnesses (e.g., schizophrenia, depression), hypoxias, cerebral ischemias, cerebral palsy, degenerative brain diseases, cerebrovascular diseases, dyspnea, or encephalopathy. A brain disease or disorder can also be the result of physical or biochemical injury, such as trauma.

[0144] Migraines include, for example, migraine with aura, migraine without aura, masilar artery migraine, carotidynia, headache-free migraine, ophthalmoplegic migraine, and status migraine.

[0145] The liposomes and micelles described herein can also be used to treat a convulsive disease or disorder. The term convulsion (i.e., seizure) refers to a sudden change in behavior due to an excessive electrical activity in the brain. Causes include, for example, epilepsy, head injury, infection or stroke. Types of epilepsy include, for example, general epilepsy, generalized cryptogenic or symptomatic epilepsies, generalized symptomatic epilepsies of nonspecific etiology, focal or partial epilepsy, temporal lobe epilepsies and frontal lobe epilepsies.

[0146] Cerebrovascular diseases can also be treated using a liposome or micelle described herein. Cerebrovascular diseases include diseases in which neurological symptoms and signs result from disorders or diseases of the blood vessels (e.g., congenital anomalies and atherosclerosis). Nonlimiting examples include ischemic syndromes and hemorrhagic syndromes. Ischemic syndromes are disorders caused by insufficient cerebral circulation, and including for example, transient ischemic attacks (TIAs) and ischemic stroke. Hemorrhagic syndromes involve bleeding into brain tissue, including the epidural, subdural, or subarachnoid space, or a combination of these sites. Intracerebral hemorrhages can occur almost anywhere in the brain, including for example, near the basal ganglia, internal capsule, thalamus, cerebellum, or brain stem. Head trauma is the most common cause of subarachnoid hemorrhage. In a particular embodiment of the invention, the abnormal brain region is a region of brain tissue physiologically affected by stroke.

[0147] Other brain diseases or disorders include neurodegenerative diseases, which are characterized by progressive nervous system dysfunction in which neurons in particular structures or regions of the brain deteriorate or die over time. Nonlimiting degenerative brain diseases include, e.g., Alzheimer's, cerebellar atrophies, triplet repeat diseases (e.g., Huntington's disease), Parkinson's disease, Niemann-Pick Type C Disease (NP-C), prior disorders (e.g., Creutzfeldt Jakob Disease), olivopontocerebellar degeneration, motor neuron disease, cerebellar degenerations, Amyotrophic Lateral Sclerosis (i.e., Lou Gehrig's Disease), dementia (e.g. , dementia with lewy bodies), as well as diseases involving neurological autoimmune disease (e.g., multiple sclerosis). Other neurodegenerative diseases are described in Williams, BMJ 324: 1465-1466 (2002).

[0148] Yet other brain diseases or disorders include brain infections. Infections of the brain can be caused by, for example, a bacteria, virus or virus-like agent. Infections can include both acute and chronic conditions. Nonlimiting bacterial infections include, e.g., Streptococcus pneumonia, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Enterobacteriacea, Propionibacterium, Pseudomonoas aeruginosa, Neisseria meningitis, Haemophilus influenzae and Listeria moncytogenes . Nonlimiting examples of acute neurological syndromes associated with viral infection include acute viral encephalitis, flaccid paralysis, aspectic meningitis, and post infectious encephalomyelitis. Acute viral encephalitis can be caused by, for example, herpes simplex virus, cytomegalovirus, varicella, rabies or an arbovirus. Chronic neurological diseases attributable to viral infection include, without limitation, subacute sclerosing pan encephalitis (caused by persistent measles infection), progressive multifocal leuco-encephalopathy (caused by members of the papovavirus family) spongiform encephalopathies (prion diseases) (e.g., Creutzfeldt- Jakob disease (CJD), Gerstmann-Streussler Syndrome), and retroviral diseases (e.g., HIV-I and HIV- 2) characterized by paralysis, wasting, and ataxia.

[0149] Other brain diseases and disorders include, e.g., senile dementia, autonomic function disorders such as hypertension and sleep disorders (e.g., insomnia, hypersomnia, parasomnia, and sleep apnea), neuropsychiatric disorders (e.g. , schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, and obsessive-compulsive disorder), psychoactive substance use disorders, anxiety, panic disorder, bipolar affective disorder (e.g. , severe bipolar affective disorder and bipolar affective disorder with hypomania and major depression), disorders associated with developmental, cognitive, and autonomic neural and neurological processes (such as pain, appetite, long term memory, and short term memory), focal brain disorders including aphasia, apraxia, agnosia, and amnesias (e.g., posttraumatic amnesia, transient global amnesia, and psychogenic amnesia), and global-diffuse cerebral disorders such as coma, stupor, obtundation, and disorders of the reticular formation. [0150] Brain diseases and disorders also include metabolic disorders, including, without limitation, Abetalipoproteinemi, Central pontine myelinolysis, Galactosemia, Gaucher, Homocystinuria, Kernicterus, Leigh's Disease, Lesch-Nyhan Syndrome, Menkes' Syndrome, Niemann-Pick Type C disease, Reye's Syndrome, Korsakoffs disease, and Tay-Sach's disease. [0151] Yet other brain diseases and disorders include, for example, Batten Disease, Canavan disease, Charcot-Marie-Tooth disorder (CMT), dystonia, Neurofibromatosis (NF), Tuberous sclerosis complex (TSC), Aicardi Syndrome, Akinetic Mutism, Amblyopia, Bardet- Biedl Syndrome, cerebral abscess, cerebral edema, Corticobasal Degeneration, Familial Mediterranean Fever, Glycogen Storage Disease Type II, Hallervorden-Spatz Syndrome, intracranial hypertension, intracranial hypotension, Joubert Syndrome, Kluver-Bucy Syndrome, Laurence-Moon Syndrome, Lowe Syndrome, Machado-Joseph, Miller Fisher Syndrome, Moyamoya, olivopontocerebellar atrophy, phenylketonuria, Schizencephaly, transient global amnesia, and Zellweger Syndrome.

Therapeutic Administration

[0152] The route and/or mode of administration of a liposome or micelle described herein can vary depending upon the desired results. One with skill in the art, i.e., a physician, is aware that dosage regimens can be adjusted to provide the desired response, e.g., a therapeutic response.

[0153] Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin. The mode of administration is left to the discretion of the practitioner.

[0154] In some instances, a liposome or micelle described herein (e.g., a pharmaceutical formulation of a liposome or a micelle) can effectively cross the blood brain barrier and enter the brain. In other instances, a liposome or micelle can be delivered using techniques designed to permit or to enhance the ability of the formulation to cross the blood-brain barrier. Such techniques are known in the art (e.g., WO 89/10134; Cloughesy et αl, J. Neurooncol. 26:125-

132 (1995); and Begley, J. Phαrm. Pharmacol. 48:136-146 (1996)). Components of a formulation can also be modified (e.g. , chemically) using methods known in the art to facilitate their entry into the CNS.

[0155] For example, in some instances, a liposome or micelle described herein is administered locally. This is achieved, for example, by local infusion during surgery, topical application (e.g., in a cream or lotion), by injection, by means of a catheter, by means of a suppository or enema, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In some situations, a liposome or micelle described herein is introduced into the central nervous system, circulatory system or gastrointestinal tract by any suitable route, including intraventricular, intrathecal injection, paraspinal injection, epidural injection, enema, and by injection adjacent to a peripheral nerve.

[0156] Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.

[0157] A liposome or micelle described herein can be formulated as a pharmaceutical composition that includes a suitable amount of a physiologically acceptable excipient (see, e.g., Remington's Pharmaceutical Sciences pp. 1447-1676 (Alfonso R. Gennaro, ed., 19th ed. 1995)). Such physiologically acceptable excipients can be, e.g., liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The physiologically acceptable excipients can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one situation, the physiologically acceptable excipients are sterile when administered to an animal. The physiologically acceptable excipient should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms. Water is a particularly useful excipient when a liposome or micelle described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable physiologically acceptable excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Other examples of suitable physiologically acceptable excipients are described in Remington's Pharmaceutical Sciences pp. 1447-1676 (Alfonso R. Gennaro, ed., 19th ed. 1995). The pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0158] Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, and elixirs. A liposome or micelle described herein can be suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier can contain other suitable pharmaceutical additives including solubilizers, emulsifϊers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (particular containing additives described herein, e.g., cellulose derivatives, including sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. The liquid carriers can be in sterile liquid form for administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0159] In other instances, a liposome or micelle described herein is formulated for intravenous administration. Compositions for intravenous administration can comprise a sterile isotonic aqueous buffer. The compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic such as lignocaine to lessen pain at the site of the injection. The ingredients can be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where a liposome or micelle described herein is administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where a liposome or micelle described herein is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0160] A liposome or micelle described herein can be administered rectally or vaginally in the form of a conventional suppository. Suppository formulations can be made using methods known to those in the art from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water-soluble suppository bases, such as polyethylene glycols of various molecular weights, can also be used. [0161] The amount of a liposome or micelle described herein that is effective for treating disorder or disease can be determined using standard clinical techniques known to those with skill in the art. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration, the condition, the seriousness of the condition being treated, as well as various physical factors related to the individual being treated, and can be decided according to the judgment of a health-care practitioner. For example, the dose of a liposome or micelle described herein can each range from about 0.001 mg/kg to about 250 mg/kg of body weight per day, from about 1 mg/kg to about 250 mg/kg body weight per day, from about 1 mg/kg to about 50 mg/kg body weight per day, or from about 1 mg/kg to about 20 mg/kg of body weight per day. Equivalent dosages can be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The number and frequency of dosages corresponding to a completed course of therapy can be determined according to the judgment of a health-care practitioner. [0162] In some instances, a pharmaceutical composition described herein is in unit dosage form, e.g., as a tablet, capsule, powder, solution, suspension, emulsion, granule, or suppository. In such form, the pharmaceutical composition can be sub-divided into unit doses containing appropriate quantities of a nanoparticle described herein. The unit dosage form can be a packaged pharmaceutical composition, for example, packeted powders, vials, ampoules, pre- fϊlled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. Such unit dosage form can contain from about 1 mg/kg to about 250 mg/kg, and can be given in a single dose or in two or more divided doses.

Kits

[0163] A liposome or micelle described herein can be provided in a kit. In some instances, the kit includes (a) a container that contains a liposome or micelle and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the liposome or micelle, e.g., for therapeutic benefit.

[0164] The informational material of the kits is not limited in its form. In some instances, the informational material can include information about production of the liposome or micelle, molecular weight of the liposome or micelle, concentration, date of expiration, batch or production site information, and so forth. In other situations, the informational material relates to methods of administering the liposome or micelle, e.g. , in a suitable amount, manner, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). The method can be a method of treating a subject having a disorder.

[0165] In some cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. The informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In other instances, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about the nanoparticles therein and/or their use in the methods described herein. The informational material can also be provided in any combination of formats.

[0166] In addition to the liposome or micelle, the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The kit can also include other agents, e.g., a second or third agent, e.g., other therapeutic agents. The components can be provided in any form, e.g., liquid, dried or lyophilized form. The components can be substantially pure (although they can be combined together or delivered separate from one another) and/or sterile. When the components are provided in a liquid solution, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. When the components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

[0167] The kit can include one or more containers for the liposomes or micelles or other agents. In some cases, the kit contains separate containers, dividers or compartments for the liposomes or micelles and informational material. For example, the liposomes or micelles can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other situations, the separate elements of the kit are contained within a single, undivided container. For example, the liposomes or micelles can be contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some cases, the kit can include a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the liposomes or micelles. The containers can include a unit dosage, e.g., a unit that includes the liposomes or micelles. For example, the kit can include a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a unit dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. [0168] The kit can optionally include a device suitable for administration of the liposomes or micelles, e.g., a syringe or other suitable delivery device. The device can be provided preloaded with liposomes or micelles, e.g., in a unit dose, or can be empty, but suitable for loading.

[0169] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

EXAMPLES

EXAMPLE 1 Preparation and Characterization of Ascorbate-Linked Liposomes and Micelles

[0170] Macromolecular systems aimed at brain targeting, which take advantage of the SVCT2 transporter features, e.g., molecular selectivity and physiological disposition, are unknown. In order to set up a new in vitro cellular model to exploit for brain delivery studies, immunological characterization of a panel of immortalized cell lines was carried out to assess which of them express the SVCT2 transporter. The potential of pharmaceutical nanocarriers, e.g., liposomes and lipid-core polymeric micelles, to target such cells via the SVCT transporter was investigated after the nanocarriers were decorated with ascorbate by modifying the 1 ,2- distearoyl-5/?-glycero-3-phosphoethanolamine-PEG2kDa-NH2 with ascorbic acid.

A. Methods

1. Materials

[0171] Cell lines, mouse fibroblast NIH/3T3 and melanoma Bl 6-F 10, human glioblastoma LN-18 and neuroblastoma SK-N-AS, rat glioma C6 and F98, were purchased from the American Type Culture Collection (Manassas, VA). All cell culture media, RPMI, heat- inactivated fetal bovine serum (FBS), and penicillin/streptomycin stock solutions were purchased from Cellgro (Herndon, VA). LAB-TEK 4-well cell culture chambers were purchased from Nunc (Rochester, NY). Goat polyclonal antibody anti-SVCT2 transporter (G- 19) and donkey antigoat IgG-FITC were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology, CA). Goat anti-mouse IgG antibody was from

ICN Biomedical (Aurora, OH). Egg phosphatidylcholine (PC), cholesterol, 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-JV-[methoxy (poly(ethylene glycol))-2000] (mPEG2kDa-PE), 1 ,2-distearoyl-5/?-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (PE- PEG2kDa-amine) and l,2-dioleoyl-5/?-glycero-3-phosphoethanolamine-Λ/-(lissamine rhodamine B sulfonyl) (Pvh-PE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Bovine serum albumin and all other chemicals and buffer solution components were from Sigma (St. Louis, MO) and were of analytical grade. Sephadex G25 superfine medium was from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA).

2. Synthesis of 6-Bromodeoxy-ascorbic Acid

[0172] The preparation of the 6-bromo derivative of ascorbic acid was performed according to the published procedure (Kiss et al, HeIv. Chim. Acta 63:1728-1739 (1980)) with some modifications. 2 g of ascorbic acid (11.35 mmol) was added to 2.7 mL of 33% w/v hydrobromic acid in glacial acetic acid (17.18 mmol). The suspension was stirred ON at RT. Afterward, hydrobromic and acetic acid were removed by nitrogen flux and rotary evaporation. 5 mL of deionized water was added to the syrup, and the mixture was stirred at 60 ⁰C for 30 min. After reduction of the volume by rotary evaporation, the residue was extracted 6 times with 5 mL of ethylacetate, and traces of water were removed by sodium sulfate treatment. The organic solvent was removed under reduced pressure, and the resulting solid product was dissolved in 3 mL of acetonitrile warmed to 70 ⁰C. The solution was cooled to 4 ⁰C to allow crystallization. The white crystals were redissolved and recrystallized according to the same procedure. The final product was recovered by filtration and dried under the vacuum ON. 6- Bromodeoxy-ascorbic acid dissolved in DMSO-J₆ was analyzed by ¹H NMR and ¹³C NMR using a Varian 500 MHz spectrometer.

[0173] ¹H NMR: δ 3.36 (dd, IH, J = 10.06, δ 7.14 Hz, BrCCHH), δ 3.60 (dd, IH, J = 10.06, 6.56 Hz, BrCCHH), δ 3.96 (t, 1Η, J= 6.67 Hz, BrCH₂CHOH), δ 4.83 (broad s, IH, OCH), δ 5.63 (broad s, 5.63, IH, BrCH₂CHOH), δ 8.42 (s, 1Η, OCOCOΗCOH), «5 11.66 (s, 1Η, OCOCOHCOΗ). ¹³C NMR: δ 34.6 (BrC), 68.2 (BrCC), 75.2 (OCΗ), 152.3 (OCCOΗ), 118.1 (OCCOH=COH), 170.3 (OC=O). Mass spectroscopy analysis was carried out by MALDI-TOF using 2,5-dihydroxybenzoic acid as a matrix. The negative ion was found to have 237.05 mlz.

3. Synthesis of 6-Ascorbate-PEG-PE

[0174] Into a 25 mL round-bottomed flask, 2.8 mL of a 25 mg/mL solution of PE-PEG₂kDa- amine in chloroform was added (25 μmol, 70 mg). The solvent was removed by rotary evaporation, and the solid residue was dissolved in 2 mL of DMF. Then, 60 mg of 6- bromodeoxyascorbic acid (250 μmol) and 35 μL of triethylamine (250 μmol) were added. The solution was stirred ON under argon in the dark. The mixture was precipitated by dropwise addition to cold diethyl ether under stirring. The crude precipitate was washed several times with diethyl ether, and the solid was dried under vacuum. The dry powder was dissolved in 2 mL of deionized water, and the derivative was purified by size exclusion chromatography using a column prepacked with Sephadex G25 superfine medium eluted with milli-Q water. Fractions positive both to UV analysis and iodine test were pooled together and lyophilized. Yield was 40 mg (57%).

[0175] In order to assess the PEG/ascorbate molar ratio in the conjugate, iodine test for PEG (Sims et al., Anal. Biochem. 107:60-63 (1980)), UV spectroscopic analysis for ascorbate (Karlsen et al., J. Chromatogr. B 824:132-138 (2005)), and Snyder colorimetric test for the residual PEG unconjugated amino groups (Snyder et ah, Anal. Biochem. 64:284-288 (1975)) were carried out on a weighed amount of the purified derivative. 6- Ascorbate -PEG-PE dissolved in CDCI3 underwent ¹H NMR spectroscopic analysis. ¹H NMR: δ 3.73 (d, IH, OCHCHOHCH₂ of ascorbate), δ 3.642 (s, ~180 H, -[OCH₂CH₂],,- of PEG), δ 2.308 (m, 4H, OCOCH₂ of phospholipid stearate), 1.249 (m, 56 Η, -[CH₂],,- of phospholipid stearate).

4. Preparation and Characterization of Liposomes and Micelles [0176] Rhodamine-labeled targeted liposomes were prepared by thin film hydration technique. A lipid film was obtained by removing organic solvent from the chloroform solution of 6-ascorbic acid-PEG-PE, egg PC, cholesterol, and Rh-PE in 3/60/30/0.25 molar ratio. The lipid film was suspended in RPMI, pH 7.4, containing 300 μM Tris(2- carboxyethyl)phosphine (TCEP) at a total lipid concentration of 0.435 mg/mL and sonicated in a bath sonicator for 10 min, followed by 11 passages through a mini-extruder equipped with 200 nm pore size polycarbonate filter (Avanti Polar Lipids). Untargeted liposomes were prepared according to the same procedure using mPEG₂kDa-PE instead of 6-ascorbic acid-PEG- PE. Rhodamine-labeled targeted micelles were prepared by dissolving 6-ascorbic acid-PEG- PE in chloroform. To that solution, 1 mol % of Rh-PE was added. The organic solvent was then removed by rotary evaporation, and the film was rehydrated at a final concentration of 1 mg/mL in RPMI containing TCEP 300 μM. Untargeted micelles were prepared using mPEG₂kDa-PE according to the same procedure. [0177] The micelle and liposome sizes and size distributions were measured by dynamic light scattering (DLS) using a Zeta Plus instrument (Brookhaven Instrument Corporation, Holtsville, NY). The sample suspensions were analyzed after the appropriate dilution required for DLS. For each sample, size distribution measurements were performed six cycles per run. [0178] The zeta-potential of liposome and micelle formulations was measured by a Zeta phase Analysis Light scattering (PALS) with an ultrasensitive Zeta Potential Analyzer instrument (Brookhaven Instruments, Holtsville, NY). The micelle and liposome suspensions were diluted properly with a 1 M KCl solution. All zeta-potential measurements were performed in triplicate.

5. Assessment of SVCT2-Expressing Cell Lines by Flow Cytometry

[0179] In order to select immortalized cell lines, which express SVCT2 transporter on plasma membrane, cells (NIH/3T3, B16-F10, LN-18, SK-N-AS, C6, and F98) were grown in RPMI with 10% FBS in 10 cm tissue culture plates for 72 hr. Medium was removed and cells were washed with RPMI and detached by prechilling incubation for 15 min and pipetting. Cells were recovered by centrifugation at 1500 rpm, and 10⁶ cells were resuspended in 50 μL of PBS containing 3% FBS. 3 μL of a 200 μg/mL goat polyclonal anti-SVCT2 transporter (G- 19) or goat anti-mouse IgG antibody was added to each sample and incubated in the dark in an ice-cold bath for 30 min. Cells were then washed once with 1 mL of PBS containing 3% FBS, recovered by centrifugation, resuspended in 50 μL of the same medium, and treated with 3 μL of 200 μg/mL donkey anti-goat IgG-FITC for 30 min in the dark in the ice bath. After washing with PBS containing 3% FBS, samples were fixed with 2% paraformaldehyde in PBS and analyzed by FACS (10⁴ cells in average count) using BD FACSCalibur flow cytometer and BD CellQuest Pro software (Beckton Dickinson Biosciences, San Jose, CA). Cells were live-gated upon acquisition using forward versus side scatter to exclude debris and dead cells.

6. Ascorbate-Mediated Targeting of Pharmaceutical Nanocarriers

[0180] In order to assess the cell-targeting capacity of ascorbate-conjugated nanocarriers, SVCT2-expressing cells incubated with liposomal or micellar formulations were subjected to FACS analysis. SVCT2-expressing cells (C6, F98) were grown in 10 cm cell culture plates for 48 hr. Cells were washed with serum-free medium, detached by prechilling incubation and repetitive pipetting, recovered by centrifugation, and 10⁶ cells were resuspended in serum-free medium containing fluorescently labeled either targeted or untargeted nanocarriers prepared as described above. Samples were incubated at 37 ⁰C in the dark with gentle shaking for 90 min. Afterward, the cell samples were recovered by centrifugation, washed twice with PBS, and gated using forward versus side scatter to exclude debris and dead cells and analyzed (10⁴ cells in average count) using BD FACSCalibur flow cytometer and BD CellQuest Pro software.

[0181] C6 and F98 cells were investigated using fluorescence microscopy after incubation with fluorescently labeled targeted liposomes and micelles or untargeted nanocarriers as a reference. After the initial passage in tissue culture flasks, cells were grown in four-well tissue culture detachable LAB-TEK chambers, at a concentration of 5 x 10⁵ cells per well in RPMI with 10% FBS. After 24 hr, the chambers were washed twice with RPMI and then incubated at 37 ⁰C with 0.5 mL of liposome or micelle formulations prepared as described above in RPMI. After 1 hr incubation, the medium was removed, and the plates were washed with medium three times. Samples were further incubated for 10 min at 37 ⁰C with complete medium containing 0.5 μg/mL of Hoechst 33342 and washed twice with complete medium. Individual slides were covered with coverslips. Cells were observed immediately on a Nikon Eclipse E400 fluorescence microscope equipped with appropriate filters for bright light, Rhodamine and Hoechst detection, and a Nikon N60 camera.

[0182] For competition studies with ascorbic acid, cells grown on LAB-TEK chambers were washed twice with RPMI and incubated at 37 ⁰C with 0.5 mL of 200 μM ascorbic acid in RPMI for 30 min followed by incubation for an additional 1 hr with 6-ascorbate-PEG-PE - containing liposomes (0.464 mg/mL of total lipids). Samples were then washed three times with PBS and mounted with fluormount medium. Cells were observed immediately by fluorescence microscopy as described above, and the fluorescence intensity associated with the cells was quantified by image analysis software (ImageJ).

B. Results

1. Synthesis and Characterization of 6-Ascorbate-PEG-PE and Ascorbate-Conjugated Nanocarriers

[0183] The preparation of the 6-ascorbate-PEG-PE was carried out by a two-step procedure: (1) activation of ascorbic acid with bromine; and (2) synthesis of 6-ascorbate-PEG- PE by PE-PEG2kDa-amine reaction with excess of 6-Br-ascorbic acid. The procedure adopted to synthesize the 6-bromodeoxy ascorbic acid allowed, after the crystallization, a 40.5% yield. The ¹H NMR spectroscopic analysis showed that hydroxyl groups of ascorbate in positions 2, 3, and 5 were unmodified as confirmed by the corresponding signals at δ 8.42, 11.66, and 5.63, respectively. Mass spectrometry analysis of 6-bromo-ascorbic acid showed a signal at 237.05 ml z, which has been attributed to the monodeprotonated ion [6-bromoscorbate-H⁺]^"1. [0184] The reaction of the commercially available PE-PE G_2kDa-amine with a 20-fold molar excess of 6-bromo-ascorbate (as depicted in Scheme 1) allowed for obtaining 6-ascorbate- PEG-PE in 12 hr as determined by the disappearance of the primary PEG amino group by ninhydrin assay. The conjugation was carried out in anhydrous conditions to reduce water deactivation of the 6-bromoascorbate.

Scheme 1 :

[0185] The crude product was precipitated in ether and dissolved in water to allow the formation of the micelles. The conjugate was purified by size exclusion chromatography from the unreacted 6-bromo-ascorbate yielding 70% product recovery. UV-vis, iodine, and Snyder colorimetric tests were carried out on a 1 mg/mL water solution of 6-ascorbate-PEG-PE in order to assess ascorbate, PEG, and free amino groups, respectively. According to the tests, the conjugation yield was 98.9%, i.e., practically complete conjugation of the amino-terminating PEG-phospholipid to ascorbate was achieved by the procedure reported. The chemical identity of the conjugate was confirmed by ¹H NMR spectroscopy that showed typical signals of phospholipids at δ 2.308 and 1.249, PEG at δ 3.642, and ascorbate at δ 3.73. [0186] The newly synthesized ascorbate-conjugated moiety was incorporated into liposomes or lipid-core PEG-PE micelles. As shown by the dynamic light scattering analysis, the lipid film rehydration techniques and extrusion yielded liposomal formulations of a similar size: 175.1 ± 21.2 nm and 181.9 ± 24.9 nm for untargeted and targeted liposomes, respectively. Micelle size was found to be 13.65 ± 5.1 nm and 21.3 ± 2.4 nm for mPEG2kDa-PE and 6-ascorbate-PEG2kDa-PE micelles, respectively. A slight increase in the micelle size can be ascribed to the additional presence of ascorbate moieties in the outer surface of the micelles. [0187] Zeta-potential analysis of the lipid-based nanosystems showed that the presence of the ascorbate on their surface enhanced their negative character due to the dissociation of the acid. PEGylated liposomes and PEG-PE-based micelles had zeta-potentials of -12.8 ± 3.2 mV and -13.2 ± 2.3 mV, respectively, while ascorbate-conjugated liposomes and micelles showed a slight increase in the negative surface charge with zeta-potentials of -16.4 ± 4.2 mV and -19.2 ± 7.3 mV, respectively. Liposome and micelle sizes and zeta-potentials were unaffected by the presence of the reducing agent TCEP.

2. Selection of SVCT2-Expressing Cell Lines

[0188] In order to select a proper cell model suitable for further targeting studies, a panel of immortalized cell lines was tested to identify those expressing SVCT2 translocator on the plasma membrane by using saturating amounts of specific antibodies against the extracellular domain of SVCT2 protein. SK-N-AS (human neuroblastoma), LN 18 (human glioblastoma), C6 and Fl 8 (rat gliomas), NIH 3T3 (mouse fibroblasts), and B16-F10 (mouse melanoma) cells were investigated.

[0189] Cell lines were grown in an appropriate medium and detached by prechilling incubation and pipeting to prevent surface proteins from being proteolytically degraded by trypsin treatment. Detached cells were first incubated with goat polyclonal IgG anti-SVCT2 transporter, which has selectivity for the detection of SVCT2 of mouse, rat, and human origin. After washing, cells were incubated with donkey FITC-labeled anti-goat antibody as a secondary antibody, and the degree of binding was determined by FACS analysis. Isotype control was performed by incubating cells with goat anti-mouse IgG in order to detect nonspecific antibody binding on the cell membrane. [0190] As shown in Figure 1, 43.9%, 41.1%, and 28.9% of C6, F98, and NIH/3T3 cells stained positive for the SVCT2 expression, respectively (Figures ID, IE and IF), while nonspecific immunostaining with the isotype control was about 5% only (Figures IA, IB, and 1C, respectively).

[0191] When FACS analysis was carried out with murine LN18, SK-N-AS, and B16F10 cell lines treated with the monoclonal anti-SVCT2 transporter, only a nonspecific weak immunoreactivity was detected, and the cell-associated fluorescence increase accounted for about 5% regardless of cell incubation with goat polyclonal IgG anti-SVCT2 transporter or goat anti-mouse IgG antibody. The FACS experiments demonstrated that such cell lines do not express constitutionally the plasma membrane SVCT2 transporter.

3. Nanocarrier Targeting Studies

[0192] The ability of ascorbate-conjugated nanocarriers to selectively target SVCT2 transporter-expressing cells was evaluated using FACS analysis after an hour and a half incubation with liposomes or micelles. Ascorbate-free PEGylated liposomes or mPEG2kDa-PE micelles were used as control. Two different cell lines of rat origin, C6 and F98 glioma cell lines, both expressing SVCT2 transporter, were incubated with liposomes containing 3% of 6- ascorbate-PEG-PE or with 6-ascorbate-PEG-PE micelles fluorescently labeled with Rh-PE. [0193] As depicted in Figure 2, FACS analysis showed that ascorbate conjugation increases C6 cells targeting by 23.2% and 10.1% when targeted liposomes and micelles were employed, respectively (Figures 2C and 2F), compared to ascorbate-free PEG-liposomes and mPEG2kDa- PE micelles controls. Nonspecific cell targeting was shown to account for about 9% and 3% fluorescence increase for PEGylated liposomes and mPEG2kDa-PE micelles (Figures 2B and 2E, respectively) with respect to untreated cell samples (Figures 2A and 2D, respectively). Cells were analyzed as a whole without gating (data not shown).

[0194] Figure 3 shows the results obtained by FACS analysis to evaluate the targeting efficiency of ascorbate-conjugated nanocarriers toward F98 glioma cell line. The fluorescence of F98 cells was remarkably higher compared to C6 cells when cell samples were treated with ascorbate-decorated liposomes (Figure 3C) and micelles (Figure 3F), (42% and 40.1%, respectively). A stronger nonspecific cell-associated fluorescence was also found on the F98 cell line for nontargeted liposome and micelles, (19.1% and 45.2%, respectively (Figures 3B and 3E)), compared to the C6 cell line, which may be due to higher nonspecific uptake activity of these cells. [0195] To further evaluate whether the ascorbate can be employed as a targeting moiety to deliver pharmaceutical nanocarriers to SVCT transporter-expressing cells, C6 cells were also investigated using fluorescence microscopy after incubation with fluorescently labeled liposomes and micelles. Nuclei were stained by Hoechst 33342 treatment (Figures 4A and 4D). The epifluorescence micrographs obtained clearly support ascorbate-PEG-liposome targeting to the investigated glioma cells. In fact, strongly fluorescent spots associated with the cells were clearly visible (Figure 4E shows the fluorescence of targeted liposomes; Figure 4F shows superimposition of the nuclei and liposomes fluorescence). On the contrary, the treatment with nontargeted PEGylated liposomes did not result in any defined fluorescent spots associated to the glioma cells, indicating the lack of targeting toward this cell line (Figure 4B shows undetectable fluorescence of PEG-liposomes; Figure 4C shows superimposition of nuclei and liposomes fluorescence). It is important to note here that PEGylated liposomes are commonly recognized for their stealth properties, which reduce the interaction with biological surfaces.

[0196] The ascorbate-mediated targeting was investigated also after the treatment of C6 cells with rhodamine-labeled micelles as shown in Figure 5. Figures 5 A and 5D show nuclei staining by Hoechst treatment. Figures 5B and 5E show fluorescence of cells treated with untargeted micelles and ascorbate -PEG-PE micelles, respectively, while Figures 5C and 5F are the superimpositions of the two channels. Cells treated with ascorbate-conjugated micelles showed small punctuate fluorescent spots (Figures 5E and 5F), which may be due to aggregation of micelles and uptake of the aggregates by C6 glioma cells. [0197] Competition studies were performed with epifluorescence microscopy by C6 cells preincubation with free ascorbic acid followed by the treatment with rhodamine-labeled ascorbate-PEG-liposomes. The presence of ascorbic acid in the medium dramatically reduced the cell-associated fluorescence (Figure 6B), compared to cell samples treated with targeted liposomes only (Figure 6A). The preincubation of cell samples with ascorbic acid followed by the treatment with ascorbate-PEG-liposomes reduced the cell-associated fluorescence induced by targeted nanosystems by 7-fold as determined by image analysis (Figure 7).

Equivalents

[0198] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claims:

1. A method of delivering a liposome or a micelle to a brain cell, comprising contacting the brain cell with the liposome or the micelle, the liposome or the micelle comprising ascorbate or an ascorbate derivative on an outer surface of the liposome or the micelle, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter (SVCT) on the brain cell to thereby deliver the liposome or the micelle to the brain cell.

2. The method of claim 1, wherein the liposome or the micelle comprises a phospholipid conjugated to the ascorbate or the ascorbate derivative at the C6 position of the ascorbate or the ascorbate derivative.

3. The method of claim 2, wherein the phospholipid comprises a derivatized phospholipid.

4. The method of claim 3, wherein the derivatized phospholipid comprises polyethylene glycol (PEG).

5. The method of claim 4, wherein the ascorbate or the ascorbate derivative is conjugated to the derivatized phospholipid via the PEG.

6. The method of claim 5, wherein the derivatized phospholipid is PEG-l,2-distearoyl-sn- glycero-3-phosphoethanolamine.

7. The method of claim 1, wherein the liposome or the micelle comprises 6-Ascorbate- PEG- 1 ^-distearoyl-sn-glycero-S-phosphoethanolamine.

8. The method of claim 1, wherein about 50% to about 100% of the outer surface area of the liposome or the micelle comprises ascorbate.

9. The method of claim 1, wherein the ascorbate comprises about 20% to about 60% of the liposome or the micelle by weight.

10. The method of claim 1, wherein the brain cell is a choroid plexus cell.

11. The method of claim 10, wherein the liposome or the micelle crosses the blood-brain barrier to reach the brain cell.

12. The method of claim 1, wherein the liposome or the micelle further comprises a therapeutic agent or a detection agent.

13. The method of claim 12, wherein the liposome or the micelle delivers the therapeutic agent or the detection agent to the brain cell.

14. A method of delivering a therapeutic agent or a detection agent to a brain cell, comprising: contacting the brain cell with a liposome or a micelle comprising: (i) a therapeutic agent or a detection agent; and (ii) ascorbate or an ascorbate derivative on an outer surface of the liposome or the micelle, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter (SVCT) on the brain cell to thereby deliver the therapeutic agent or the detection agent to the brain cell.

15. A method of treating a brain tumor, comprising: administering to a subject in need thereof a liposome or a micelle comprising: (i) a therapeutic agent; and

(ii) ascorbate or an ascorbate derivative on an outer surface of the liposome or the micelle, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter on the brain tumor, thereby delivering the therapeutic agent to the brain tumor and treating the brain tumor.

16. A method of imaging a brain tumor, comprising: administering to a subject in need thereof a liposome or a micelle comprising: (i) a detection agent; and (ii) ascorbate or an ascorbate derivative on an outer surface of the liposome or the micelle, the ascorbate or the ascorbate derivative contacting a sodium-dependent vitamin C transporter on the brain tumor; and detecting the detection agent to thereby image the brain tumor.