WO2012040513A1

WO2012040513A1 - Compositions and methods for the delivery of beta lapachone

Info

Publication number: WO2012040513A1
Application number: PCT/US2011/052836
Authority: WO
Inventors: David Boothman; Jinming Gao; Erik Bey; Ying Dong; Huabing Chen; Kejin Zhou; Xiaonan Huang; Yiguang Wang
Original assignee: The Board Of Regents Of The University Of Texas System
Priority date: 2010-09-22
Filing date: 2011-09-22
Publication date: 2012-03-29

Abstract

Disclosed herein are methods and micelle compositions for delivering an encapsulated therapeutic agent, such as beta-lapachone, for cancer therapy. Also provided herein are micelle compositions comprising hydrophobic superparamagnetic iron oxide nanoparticles and a beta-lapachone compound encapsulated with the hydrophobic core of the micelle.

Description

COMPOSITIONS AND METHODS FOR THE DELIVERY OF BETA LAPACHONE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial Number 61/385,400 filed September 22, 2010, the contents of which are incorporated herein by reference in their entirety. This application also claims priority to PCT Application Serial Number PCT/US2011/001418, filed August 11, 2011, which claims priority to U.S.

Provisional Applications Serial Numbers 61/470,441 filed March 31, 2011, 61/471,054 filed on April 1, 2011, and 61/385,422 filed on September 22, 2010, the contents of each of which are incorporated herein by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under NIH ROl CA12994 and NIH ROl CA102792-08, awarded by the National Institutes of Health, and DOD W81XWH- 06-1-0198, awarded by the Department of Defense. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Superparamagnetic iron oxide nanoparticles (SPION) have received considerable attention in such fields as magnetic resonance imaging (MRI), drug delivery, biosensors, cell tracking, tissue engineering and so on (See, e.g., H.K. Sajja et al. Curr. Drug Discov.

TechnoL, 2009, 6, 43-51; J. Gao et al., Acc. Chem. Res., 2009, 42, 1097-1107; M.E. Gindy et al., Expert Opin. Drug Deliv. 2009, 6, 865-878; R. Hao et al., Adv. Mater., 2010, 22, 2729- 2942). Moreover, SPION, as a highly important nanoplatform, displays an increasing role in theranostic nanomedicine. The hydrophobic SPION and drugs can generally be encapsulated into one system for achieving cancer imaging and simultaneous targeted drug delivery.

Polymeric micelles composed of SPION and drugs have been established as an emerging nanoplatform for improving cancer imaging and therapy, in which SPION clusters were able to provide an ultrasensitive MRI signal and drug could achieve the enhanced targeted efficiency via functionalized micelles.

[0004] For a long time, SPION clinically have shown no short- or long-term toxicity and are generally considered to be abiocompatible and biologically inert agent. The release of iron ions from SPION in cells had no influence on the cell metabolism and did not show any toxicity to cancer cells or stem cells. Even though SPION were found to have potential catalytic activity at their surface in solution, there has been only limited exploration of the intracellular activity of SPION for medicinal purposes. In addition, the interaction between SPION and anticancer drugs has not been explored for cancer therapy, due to the inert properties of SPION.

[0005] For a number of anticancer antibiotics, such as bleomycin, streptonigrin, and mitomycin C, free -radical reactions are considered to act as a key role in their mechanisms of anticancer action. It has been shown that a highly tumor- selective natural anticancer compound, β -lapachone ( β -lap) has initiating killing effect from the generation of reactive oxygen species (ROS). β -lap can interact with the tumor- selective marker,

NAD(P)H:quinone Oxidoreductase 1 (NQOl) in the cells, and induce free radicals including superoxide (0₂ ^~) and hydrogen proxide (H₂0₂). These radicals could further lead to DNA damage via the Fenton reaction, which is a highly efficient pathway to generate hydroxyl radicals in biological systems.

[0006] It is possible to escalate the free radical-mediated DNA damage by facilitating Fenton reaction in order to improve the inherent efficiency of anticancer drugs (e.g. β -lap). However, iron ions in cells are generally stored in specific proteins and only small amounts of reactive iron are available for the Fenton reaction. The generation of ROS formation is thus limited due to the shortage of reactive iron ions in the cells.

[0007] Therefore, there is a need for methods of increasing the amount of reactive iron ions in cells, particularly in the presence of drugs whose mechanism of action involves free- radical reactions.

[0008] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety for all purposes.

SUMMARY OF THE INVENTION

[0009] Beta lapachone (beta lap) is a potent cytotoxic anticancer agent with antitumor activity against a variety of human cancer cells, including drug resistance cell lines. Beta-lap is bioactivated by the intracellular enzyme NQOl. NQOl is differentially overexpressed in several of human cancers over normal cells. At optimal concentrations and duration of exposure to cells, beta-lap causes DNA damage, inhibits DNA repair and induces

programmed cell death. [0010] Despite its selectivity and potency, poor aqueous solubility limits it therapeutic use. Beta lap formulated with hydroxyl propyl cyclodextrin overcomes aqueous solubility issues. However, this formulation has limited half life in blood circulation (24mins), far shorter than the minimally required duration of drug exposure needed to achieve cytotoxicity. In addition, the use of cyclodextrin excipient causes hemolysis of red blood cells limiting its therapeutic use.

[0011] Disclosed herein, in certain embodiments, are methods and compositions comprising injectable micelles for delivering an encapsulated therapeutic agent, such as betalapachone, for cancer therapy. The formulations result in useful drug solubility, drug yield, shelf life, plasma stability and plasma circulation time. The formulations also result in improved safety with no hemolysis, enhanced drug delivery into tumor cells, improved drug tissue distribution at tumor tissue and significantly enhanced cytotoxicity.

[0012] One aspect of the invention is a polymeric micelle composition comprising: a pH- sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle and a beta-lapachone compound encapsulated within the hydrophobic core of the micelle, wherein the block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment; wherein the hydrophilic polymer segment comprises a polymer selected from the group consisting of: poly(ethylene oxide) (PEO),

poly(methacrylate phosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP); wherein the hydrophobic polymer segment comprises

wherein R' is -H or -CH₃; wherein R is -NR 1 R2 , wherein R 1 and R2 are alkyl groups, wherein R 1 and R2 are the same or different, wherein R 1 and R2 together have from 5 to 16 carbons, wherein R 1 and R 2 may optionally join to form a ring; wherein n is 1 to about 10; wherein x is about 20 to about 200 in total; and wherein the block copolymer optionally comprises a labeling moiety. In some embodiments, the hydrophilic polymer segment comprises PEO. In some embodiments, n is 1 to 4._In some embodiments, R' is -CH₃. In some embodiments, R' is -H. In some embodiments, x is about 40 to about 100 in total. In some embodiments, x is about 50 to about 100 in total. In some embodiments, x is about 40 to about 70 in total. In some embodiments, x is about 60 to about 80 in total. In some embodiments, x is about 70 in total. In some embodiments, R 1 and R 2 are each straight or branched alkyl. In some embodiments, R 1 and R 2 join to form a ring. In some embodiments,

R 1 and R2" are the same. In some embodiments, R 1 and R2 are different. In some

embodiments, R 1 and R2 each have 3 to 8 carbons. In some embodiments, R 1 and R2 together form a ring having 5 to 10 carbons. In some embodiments, R 1 and R 2 are propyl. In some embodiments, propyl is iso-propyl. In some embodiments, R 1 and R 2 are butyl. In some embodiments, butyl is n-butyl. In some embodiments, R 1 and R 2 together are -(CH₂)₅-. In some embodiments, R 1 and R 2 together are -(CH₂)₆-. In some embodiments, the micelle has a size of about 10 nm to about 200 nm. In some embodiments, the micelle has a pH transition of less than about 1 pH unit. In some embodiments, the micelle has a pH transition value of about 5 to about 8. In some embodiments, the micelle further comprises a targeting moiety. In some embodiments, the beta-lapachone compound is beta-lapachone, or a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the loading of beta-lapachone compound is between about 3 and about 8%. In some

embodiments, the superparamagnetic iron oxide nanoparticle (SPION) has an average particle size of about 3 nm to about 20 nm. In some embodiments, the micelle comprises about 1% to about 20% by weight of the superparamagnetic iron oxide nanoparticle. In some embodiments, the block copolymer is poly(ethylene glycol)-P-poly(2-(2-diisopropylamino) ethyl methacrylate). In some embodiments, the block copolymer is ΡΕΟι ΐ4-β-ΡϋΡΑι₂ο.

[0013] Another aspect of the invention is a polymeric micelle composition comprising: a pH- sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle encapsulated within the hydrophobic core of the micelle, wherein the block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophilic polymer segment comprises a polymer selected from the group consisting of: poly(ethylene oxide) (PEO), poly(methacrylate phosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP), wherein the hydrophobic polymer segment comprises

wherein R' is -H or -CH₃; wherein R is -NR 1 R2 , wherein R 1 and R2 are alkyl groups, wherein R 1 and R2 are the same or different, wherein R 1 and R2 together have from 5 to 16 carbons, wherein R 1 and R 2 may optionally join to form a ring; wherein n is 1 to about 10; wherein x is about 20 to about 200 in total; and wherein the block copolymer optionally comprises a labeling moiety. In some embodiments, the hydrophilic polymer segment comprises PEO. In some embodiments, n is 1 to 4. In some embodiments, R' is -CH₃. In some embodiments, R' is -H. In some embodiments, x is about 40 to about 100 in total. In some embodiments, x is about 50 to about 100 in total. In some embodiments, x is about 40 to about 70 in total. In some embodiments, x is about 60 to about 80 in total. In some embodiments, x is about 70 in total. In some embodiments, R 1 and R 2 are each straight or branched alkyl. In some embodiments, R 1 and R 2 join to form a ring. In some embodiments,

embodiments, R 1 and R2 each have 3 to 8 carbons. In some embodiments, R 1 and R2 together form a ring having 5 to 10 carbons. In some embodiments, R 1 and R 2 are propyl. In some embodiments, propyl is iso-propyl. In some embodiments, R 1 and R 2 are butyl. In some embodiments, butyl is n-butyl. In some embodiments, R 1 and R 2 together are -(CH₂)₅-. In some embodiments, R 1 and R 2 together are -(CH₂)₆-. In some embodiments, the micelle has a size of about 10 nm to about 200 nm. In some embodiments, the micelle has a pH transition of less than about 1 pH unit. In some embodiments, the micelle has a pH transition value of about 5 to about 8. In some embodiments, the micelle further comprises a targeting moiety. In some embodiments, the block copolymer is poly(ethylene glycol)-P-poly(2-(2- diisopropylamino) ethyl methacrylate. In some embodiments, the block copolymer is PEGii4-P-PDPAi2o.

[0014] Another aspect of the invention is a method of preparing the polymeric micelle composition of any one of the polymeric micelle compositions described herein, comprising mixing a mixture of the beta-lapachone compound, the superparamagnetic iron oxide nanoparticle, and the block copolymer with aqueous solution to form a stable micelle composition.

[0015] Another aspect of the invention is a method for treating cancer in an individual in need thereof, comprising administering to the individual an effective amount of any of the compositions described herein. In some embodiments, the composition is a polymeric micelle composition comprising a pH-sensitive micelle comprising a block copolymer, a hydrophobic superparamagnetic iron oxide nanoparticle, and a beta-lapachone compound encapsulated within the hydrophobic core of the micelle. In some embodiments, the cancer is a solid tumor with cancer cells expressing NQOl. In some embodiments, the cancer is lung, prostate, breast, pancreatic, colon, or melanoma cancer. In some embodiments, the composition is administered once a day, once every two days, or once every three days. In some embodiments, the composition comprising: a pH-sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle encapsulated within the hydrophobic core of the micelle is administered to the individual before the administration of the composition comprising a pH-sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle and a beta- lapachone compound encapsulated within the hydrophobic core of the micelle. In some embodiments, the period between the two treatments is about 6 hours to about 48 hours.

[0016] Another aspect of the invention is a polymeric micelle formulation of comprising: a. a hydrophobic core component, including polylactide; b. an hydrophilic poly(ethylene glycol) segment as shell layers; c. a hydrophobic anti-cancer drug encapsulated within the

hydrophobic cores; d. one of: i. a injectable lipid solubilizer for enhancing drug loading of hydrophobic drug, or ii. poly(lactide)-poly (ethylene glycol) maleimide or lipid-poly(ethylene glycol) maleimide with post-modification of cancer-targeting ligand such as cRGD; and e. hydrophobic superparamagnetic iron oxide nanoparticles encapsulated within the

hydrophobic core component. Another aspect of the invention is a polymeric micelle formulation comprising: a. an amphiphilic polymer including monomethoxy poly (ethylene glycol)-b-polylactide or 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol); b. an hydrophobic anti-cancer drug such as beta-lapachone encapsulated within hydrophobic cores; and c. one of: i. one or two injectable solubilizers for enhancing drug loading and stability of hydrophobic drug in the micellar cores; ii. a multifunctional hydrophobic synergist encapsulated within hydrophobic cores for further improving cytotoxicity of beta lapachone and also as a contrast imaging agent for cancer diagnosis and monitoring; or iii. poly(lactide)-poly (ethylene glycol) maleimide or 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) maleimide with post- modification of cancer-targeted ligand. In some embodiments, the therapeutic agent is a hydrophobic agent. In some embodiments, the therapeutic agent is beta-lap. In some embodiments, the hydrophilic monomethoxy poly (ethylene glycol) of amphiphilic polymers has an average molecular weight of between 1000 and 10000. In some embodiments, the hydrophobic polylactide segments have an average molecular weight of between 2000 and 8000. In some embodiments, the hydrophobic polylactide segments is selected from: 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) include 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] or 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)5000] . In some embodiments, the biodegradable hydrophobic polylactide is synthesized from monomers selected from: D,L-lactide, D-lactide, and L-lactide. In some embodiments, the injectable solubilizers are selected from: benzyl alcohol, polyoxyethylene (660) esters of 12- hydroxystearic acid, and phosphatidyl choline. In some embodiments, the polymeric micelles comprise about 0.5-20% by weight of amphiphilic polymers and 60-95% by weight of solubilizers in total inactive ingredients and drug. In some embodiments, the micelle has a size range between 10 and 200 nm. In some embodiments, the drug dispersed in the total inactive ingredient and drug has a content of 0.5-20% weight percent. In some embodiments, the formulation further comprises magnetic nanoparticles and/or targeting moieties. In some embodiments, the magnetic nanoparticles are superparamagnetic iron oxide nanoparticles. In some embodiments, the superparamagnetic iron oxide nanoparticles have the average particle size between 3-20 nm. In some embodiments, the surface of superparamagnetic iron oxide nanoparticles is coated by oleic acid. In some embodiments, the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles. In some

embodiments, the single or multiple superparamagnetic iron oxide nanoparticles are encapsulated within the micellar cores. In some embodiments, the transverse relaxivity of solution ranges from 50 Fe mM^"1 s^"1 to 600.0 Fe mM^"1 s In some embodiments, the superparamagnetic iron oxide nanoparticles increase of cytotoxicity of beta-lapachone. In some embodiments, the cytotoxicity of the composition is increased by up to 2-8 times induced by the increase of reactive oxygen species induced by superparamagnetic iron oxide nanoparticles, when compared with the micelles without superparamagnetic iron oxide nanoparticles. In some embodiments, the targeting moiety is conjugated with poly (lactide)- poly(ethylene glycol) maleimide. In some embodiments, the molecular weight of

poly(ethylene glycol) moiety is between 2000-10000 and the molecular weight of poly (lactide) moiety is between 2000-5000. In some embodiments, the targeting moiety including cyclic (RGDfK (SEQ ID NO. 1)) peptide, and lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL (SEQ ID NO. 2)). In some embodiments, the formulations disclosed herein result in any of the following: (a) improvement in yield of micelles (e.g., a yield of about 60-98%); (b) improvement in storage stability (e.g., storage stability of about 24 months); (c) improvement in infusion fluid stability (e.g., infusion stability of about 48 hours); (d) improvement in safety (e.g., reduction in hemolysis); (e) improvement in plasma pharmacokinetics (e.g., a circulation time of >20 h); (f) improvement in tumor distribution; and (g) improvement in efficacy.

[0017] Another aspect of the invention is a method of treating a cancer, comprising administering to an individual in need thereof a composition disclosed herein. In some embodiments, the composition is administered before, during, or simultaneously with the administration of a cytotoxic agent.

[0018] Another aspect of the invention is a method of preparing a formulation comprising micelles in aqueous solution, comprising: (a) preparing a mixture of drug, polymers and solubilizers; and (b) mixing the mixture with aqueous solution to form a stable micelle solution. In some embodiments, preparing a mixture of drug, polymers and solubilizers comprises (a) mixing the amphiphilic polymers and solubilizers and a hydrophobic drug, or (b) dissolving the amphiphilic polymers and solubilizers and a hydrophobic drug in an organic solvent followed by evaporation of solvent. In some embodiments, mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing. In some embodiments, the method further comprises filtering the solution through filter paper with 0.22 μπι pore size.

[0019] Another aspect of the invention is a method of preparing the stable micelles with superparamagnetic iron oxide nanoparticles in aqueous solution, comprising: (a) preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles; and (b) mixing the film with aqueous solution. In some embodiments, preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles comprises dissolving the amphiphilic copolymers, solubilizers, superparamagnetic iron oxide nanoparticles and a hydrophobic drug in an organic solvent, followed by evaporating the micelle solution at room temperature to remove the organic solvent and form a film. In some embodiments, mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing. In some embodiments, the method further comprised filtering the solution through a filter device with 0.22 μπι pore size. [0020] Another aspect of the invention is a method of preparing stable micelles with targeting ligand in aqueous solution, comprising: (a) preparing a mixture of drug and polymers; (b) mixing the mixture with aqueous solution to form a stable micelle solution; and (c) conjugating cRGD peptide with micelle solution. In some embodiments, preparing a mixture of drug and polymers comprises dissolving the amphiphilic polymers, solubilizers, or multifunctional synergist and poly(lactide)-poly(ethylene glycol) maleimide and hydrophobic drug in an organic solvent, followed by evaporation of solvent. In some embodiments, mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing. In some embodiments, the method further comprises filtering the solution through a filter device with a 0.22 μπι pore size.

[0021] Disclosed herein, in certain embodiments, is a polymeric micelle formulation of comprising:

a. a hydrophobic core component, including polylactide;

b. an hydrophilic poly(ethylene glycol) segment as shell layers;

c. a hydrophobic anti-cancer drug encapsulated within the hydrophobic cores; d. one of:

i. a injectable lipid solubilizer for enhancing drug loading of

hydrophobic drug, or

ii. poly(lactide)-poly (ethylene glycol) maleimide or lipid-poly(ethylene glycol) maleimide with post-modification of cancer-targeting ligand such as cRGD; and

e. hydrophobic superparamagnetic iron oxide nanoparticles encapsulated within the hydrophobic core component.

[0022] In some embodiments, the agent is beta-lap. In some embodiments, the hydrophilic monomethoxy poly (ethylene glycol) of amphiphilic polymers has an average molecular weight of between 1000 and 10000. In some embodiments, the hydrophobic polylactide segments have an average molecular weight of between 2000 and 8000. In some

embodiments, the hydrophobic polylactide segments is selected from: 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) include 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] or 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)5000]. In some embodiments, the biodegradable hydrophobic polylactide is synthesized from monomers selected from: D,L-lactide, D-lactide, and L-lactide. In some embodiments, the injectable solubilizers are selected from: benzyl alcohol, polyoxyethylene (660) esters of 12- hydroxystearic acid, and phosphatidyl choline. In some embodiments, the polymeric micelles comprise about 0.5-20% by weight of amphiphilic polymers and 60-95% by weight of solubilizers in total inactive ingredients and drug. In some embodiments, the micelle has a size range between 10 and 200 nm. In some embodiments, the drug dispersed in the total inactive ingredient and drug has a content of 0.5-20% weight percent. In some embodiments, the polymeric micelles are pH-sensitive. In some embodiments, the formulation further comprises magnetic nanoparticles and/or targeting moieties. In some embodiments, the magnetic nanoparticles are superparamagnetic iron oxide nanoparticles. In some

embodiments, the superparamagnetic iron oxide nanoparticles have the average particle size between 3-20 nm. In some embodiments, the surface of superparamagnetic iron oxide nanoparticles is coated by oleic acid. In some embodiments, the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles. In some

[0023] Disclosed herein, in certain embodiments, is a polymeric micelle formulation comprising: f. an amphiphilic polymer including monomethoxy poly (ethylene glycol)-b- polylactide or 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol);

g. an hydrophobic anti-cancer drug such as beta lapachone encapsulated within hydrophobic cores; and

h. one of:

i. one or two injectable solubilizers for enhancing drug loading and

stability of hydrophobic drug in the micellar cores;

ii. a multifunctional hydrophobic synergist encapsulated within

hydrophobic cores for further improving cytotoxicity of beta lapachone and also as a contrast imaging agent for cancer diagnosis and monitoring; or

iii. poly(lactide)-poly (ethylene glycol) maleimide or 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-poly(ethylene glycol) maleimide with post-modification of cancer-targeted ligand.

[0024] In some embodiments, the therapeutic agent is a hydrophobic agent. In some embodiments, the agent is beta-lap. In some embodiments, the hydrophilic monomethoxy poly (ethylene glycol) of amphiphilic polymers has an average molecular weight of between 1000 and 10000. In some embodiments, the hydrophobic polylactide segments have an average molecular weight of between 2000 and 8000. In some embodiments, the hydrophobic polylactide segments is selected from: l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol) include l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)2000] or l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)5000]. In some embodiments, the biodegradable

hydrophobic polylactide is synthesized from monomers selected from: D,L-lactide, D-lactide, and L-lactide. In some embodiments, the injectable solubilizers are selected from: benzyl alcohol, polyoxyethylene (660) esters of 12-hydroxy stearic acid, and phosphatidyl choline. In some embodiments, the polymeric micelles comprise about 0.5-20% by weight of

amphiphilic polymers and 60-95% by weight of solubilizers in total inactive ingredients and drug. In some embodiments, the micelle has a size range between 10 and 200 nm. In some embodiments, the drug dispersed in the total inactive ingredient and drug has a content of 0.5-20% weight percent. In some embodiments, the formulation further comprises magnetic nanoparticles and/or targeting moieties. In some embodiments, the magnetic nanoparticles are superparamagnetic iron oxide nanoparticles. In some embodiments, the superparamagnetic iron oxide nanoparticles have the average particle size between 3-20 nm. In some

embodiments, the surface of superparamagnetic iron oxide nanoparticles is coated by oleic acid. In some embodiments, the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles. In some embodiments, the single or multiple superparamagnetic iron oxide nanoparticles are encapsulated within the micellar cores. In some embodiments, the transverse relaxivity of solution ranges from 50 Fe mM^"1 s^"1 to 600.0 Fe mM^"1 s In some embodiments, the superparamagnetic iron oxide nanoparticles increase of cytotoxicity of beta-lapachone. In some embodiments, the cytotoxicity of the composition is increased by up to 2-8 times induced by the increase of reactive oxygen species induced by superparamagnetic iron oxide nanoparticles, when compared with the micelles without superparamagnetic iron oxide nanoparticles. In some embodiments, the targeting moiety is conjugated with poly (lactide)-poly(ethylene glycol) maleimide. In some embodiments, the molecular weight of poly(ethylene glycol) moiety is between 2000-10000 and the molecular weight of poly (lactide) moiety is between 2000-5000. In some embodiments, the targeting moiety including cyclic (RGDfK (SEQ ID NO. 1)) peptide, and lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL (SEQ ID NO. 2)). In some embodiments, the formulations disclosed herein result in any of the following: (a) improvement in yield of micelles (e.g., a yield of about 60-98%); (b) improvement in storage stability (e.g., storage stability of about 24 months); (c) improvement in infusion fluid stability (e.g., infusion stability of about 48 hours); (d) improvement in safety (e.g., reduction in hemolysis); (e) improvement in plasma pharmacokinetics (e.g., a circulation time of >20 h); (f) improvement in tumor distribution; and (g) improvement in efficacy.

[0025] Disclosed herein, in certain embodiments, is a method of treating a cancer, comprising administering to an individual in need thereof a composition disclosed herein. In some embodiments, the composition is administered before, during, or simultaneously with the administration of a cytotoxic agent.

[0026] Disclosed herein, in certain embodiments, is a method of preparing the stable micelles in aqueous solution, comprising the process of: (a) preparing a mixture of drug, polymers and solubilizers by mixing the amphiphilic polymers and solubilizers and a hydrophobic drug, or by dissolving the amphiphilic polymers and solubilizers and a hydrophobic drug in an organic solvent followed by evaporation of solvent; (b) mixing the mixture with aqueous solution under ultrasonication or high speed mixing to form a stable micelle solution; and (c) filtering through filter with 0.22 μπι pore size. [0027] Disclosed herein, in certain embodiments, is a method of preparing the stable micelles with superparamagnetic iron oxide nanoparticles in aqueous solution, comprising the process of: (a) preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles by dissolving the amphiphilic copolymers, solubilizers, superparamagnetic iron oxide nanoparticles and a hydrophobic drug in an organic solvent, followed by evaporating the micelle solution at room temperature to remove the organic solvent and form a film; (b) mixing the film with aqueous solution under ultrasonication or high speed mixing to form a stable micelle solution; and (c) filtering through a filter with 0.22 μπι pore size.

[0028] Disclosed herein, in certain embodiments, is a method of preparing stable micelles with targeting ligand in aqueous solution, comprising the process of: (a) preparing a mixture of drug and polymers by dissolving the amphiphilic polymers, solubilizers, or multifunctional synergist and poly(lactide)-poly(ethylene glycol) maleimide and hydrophobic drug in an organic solvent, followed by evaporation of solvent; (b) mixing the mixture with aqueous solution under ultrasonication or high speed stirring to form a stable micelle solution; (c) conjugating cRGD peptide with micelle solution; and (d) filtering through filter with 0.22 μπι pore size.

BRIEF DESCRIPTION OF THE FIGURES

[0029] Figure 1. Schematic illustration of the mechanism of the synergistic effect of mSPION on cytotoxicity of anticancer drug, β-Lapachone, in cells.

[0030] Figure 2. A. TEM image of SPION. B. TEM image of mSPION with 20% SPIO loading. C. Dynamic light scattering analysis of size distribution for mSPION in aqueous solution. D. Release profiles of iron ions from mSPION at various pH values.

[0031] Figure 3. A. Intracellular distribution of fluorescent mSPION in A549 cells.

Optical image (1), distribution of red fluorescent mSPION (2), distribution of green endosomes/lysosomes (3), and co-localization of fluorescent mSPION and

endosomes/lysosomes (4) in LysoTracker Green DND26-labelled cells after 4 h incubation with fluorescent mSPION (bar, 30 μπι). B. Synergistic killing effects of 3 μΜ free β-lap and mSPION after 48 h pretreatment of cells with mSPION at different concentrations. C.

Synergistic killing effects of different concentration of free β-lap and 0.14 mM mSPION after 48 h pretreatment.

[0032] Figure 4. A. Cytotoxicity of 3 μΜ β-lap on A549 cells (4 h incubation) with pretreatment of 0.07 mM and 0.14 mM mSPION for 4 h and 48 h. B. Cytotoxicity of 3 μΜ β-lap on A549 cells (4 h incubation) with 48 h pretreatment of 0.07 mM and 0.14 mM mSPION in the presence or absence of deferoxamine (Def, 5 μg/mL).

[0033] Figure 5. A. The fluorescent images of A549 cells with or without 48 h pretreatment of 0.14 mM mSPION after 10 min, 20 min and 60 min co-treatment of 3.0 μΜ β-lap and 0.14 mM mSPION using DHE staining for superoxide detection. B. The increased fold change of ROS formation in the A549 cells without pretreatment of mSPION after various co-treatment time of 3.0 μΜ β-lap and 0.14 mM mSPION (co-10 for 10 min, co-20 for 20 min and co-60 for 60 min), and with 48 h pretreatment of 0.14 mM mSPION after various co-treatment time of 3.0 μΜ β-lap and 0.14 mM mSPION (pre-10 for 10 min, pre-20 for 20 min, and pre-60 for 60 min), from the fluorescent images in A. C. Cytotoxicity of 3.0 μΜ β-lap on A549 cells after 48 h pretreatment with mSPION with 0.07 mM and 0.14 mM in presence or absence of catalase (1000 u).

[0034] Figure 6. A. Hydrodynamic diameter of β-lap encapsulated PEG-PDPA micelles with different SPIO loadings. B. β-lap loading contents in PEG-PDPA micelles with different SPIO loadings after original 10% β-lap feeding amount.

[0035] Figure 7. A. Hydrodynamic diameters of p-lap/10% SPIO encapsulated PEG- PDPA micelles at various pH values. B. β-lap release profile from 10% SPIO loaded PEG- PDPA micelles under various pH environments. C. Iron ion release profile from 10% SPIO loaded PEG-PDPA micelles under various pH environments.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

[0036] As used herein, "alkyl" indicates any saturated hydrocarbon moiety, including, for example, straight chain, branched chain, or cyclic (including fused and spiro bicyclic and polycyclic) saturated hydrocarbon moieties which may optionally be substituted with one or more additional saturated hydrocarbon moieties.

[0037] As used herein, "pH-sensitive micelle", "pH-activatable micelle" and "pH- activatable micellar (pHAM) nanoparticle" are used interchangeably herein to indicate a micelle comprising one or more block copolymers, which disassociates depending on the pH (e.g. above or below a certain pH). As a non-limiting example, at a certain pH, the block copolymer is substantially in micellar form. As the pH changes (e.g. decreases), the micelles begin to disassociate, and as the pH further changes (e.g. further decreases), the block copolymer is present substantially in disassociated (non-micellar) form. [0038] As used herein, "pH transition range" indicates the pH range over which the micelles disassociate. In some embodiments, the pH transition range is the pH response sharpness. Briefly, the fluorescence intensity versus pH is measured for a block copolymer which comprises a fluorescent label that is sequestered within the micelle (quenching fluorescence) when the block copolymer is in micellar form. As the pH changes (e.g.

decreases), the micelle disassociates, exposing the fluorescent label and resulting in fluorescence emission. Normalized fluorescence intensity (NFI) vs. pH curves permit quantitative assessment of the pH responsive properties of the micelle. NFI is calculated as the ratio of [F-F_min]/[F_max-F_min], where F is the fluorescence intensity of the micelle at any given pH, and F_max and F_min are the maximal and minimal fluorescence intensities at the ON/OFF states, respectively. pH response sharpness is ΔρΗιο-90%, the pH range in which the NFI value varies from 10% to 90%. For label-free copolymers, dynamic light scattering (DLS) or an external fluorophore (e.g. pyrene) can be used to characterize the pH-dependent micellization behaviors, for instance, as described in PCT/US2011/001418.

[0039] As used herein, "pH transition value" (pH_t) indicates the pH at which half of the micelles are disassociated. Briefly, for a block copolymer which comprises a fluorescent label that is sequestered within the micelle (quenching fluorescence) when the block copolymer is in micellar form, the pH transition value is the pH at which the fluorescence emission measured is 0.5 x (F_max+F_mi_n), where F_max and F_mj_n are the maximal and minimal fluorescence intensities at the ON/OFF states, respectively. For label-free copolymers, dynamic light scattering (DLS) or an external fluorophore (e.g. pyrene) can be used to characterize the pH-dependent micellization behaviors, for instance, as described in

PCT/US2011/001418.

COMPOSITIONS

MICELLE FORMULATIONS

[0040] Beta lapachone (beta lap) is a potent cytotoxic anticancer agent with antitumor activity against a variety of human cancer cells, including drug resistance cell lines. Beta lap is bioactivated by the intracellular enzyme NQOl. NQOl is differentially overexpressed in several of human cancers over normal cells. At optimal concentrations and duration of exposure to cells, beta lap causes DNA damage, inhibits DNA repair and induces programmed cell death. [0041] Despite its selectivity and potency, poor aqueous solubility limits it therapeutic use. Beta lap formulated with hydroxyl propyl cyclodextrin overcomes aqueous solubility issues. However, this formulation has limited half life in blood circulation (24mins), far shorter than the minimally required duration of drug exposure needed to achieve cytotoxicity. In addition, the use of cyclodextrin excipient causes hemolysis of red blood cells limiting its therapeutic use.

[0042] Disclosed herein, in certain embodiments, are methods and compositions comprising injectable micelles for delivering an encapsulated therapeutic agent, such as beta- lapachone, for cancer therapy. The formulations result in useful drug solubility, drug yield, shelf life, plasma stability and plasma circulation time. The formulations also result in improved safety with no hemolysis, enhanced drug delivery into tumor cells, improved drug tissue distribution at tumor tissue and significantly enhanced cytotoxicity.

[0043] Disclosed herein, in certain embodiments, are polymeric micelle formulations of active agents comprising: (a) a hydrophobic core component including polylactide; (b) an hydrophilic poly(ethylene glycol) segment as shell layers; (c) a hydrophobic anti-cancer drug encapsulated within the hydrophobic cores; and (d) (i) a injectable lipid solubilizer for enhancing drug loading of hydrophobic drug, or (ii) poly(lactide)-poly (ethylene glycol) maleimide or lipid-poly(ethylene glycol) maleimide with post-modification of cancer- targeting ligand such as cRGD (f) hydrophobic superparamagnetic iron oxide nanoparticles encapsulated within hydrophobic cores for enhancing drug loading, improving cytotoxicity and as a contrast imaging agent.

[0044] Disclosed herein, in certain embodiments, are polymeric micelle formulations of active agents comprising: (a) an amphiphilic polymer including monomethoxy poly (ethylene glycol)-b-polylactide or 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol); (b) an hydrophobic anti-cancer drug such as beta lapachone encapsulated within hydrophobic cores; and (c) (i) one or two injectable solubilizers for enhancing drug loading and stability of hydrophobic drug in the micellar cores, (ii) a multifunctional hydrophobic synergist encapsulated within hydrophobic cores for further improving cytotoxicity of beta lapachone and also as a contrast imaging agent for cancer diagnosis and monitoring; or (iii) poly(lactide)-poly (ethylene glycol) maleimide or 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) maleimide with post- modification of cancer-targeted ligand.

[0045] In some embodiments, the therapeutic agent is a hydrophobic agent. In some embodiments, the agent is beta-lap. [0046] In some embodiments, the hydrophilic monomethoxy poly (ethylene glycol) of amphiphilic polymers has an average molecular weight of between 1000 and 10000.

[0047] In some embodiments, the hydrophobic polylactide segments have an average molecular weight of between 2000 and 8000. For example, the hydrophobic polylactide segments is selected from: l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol) include l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)2000] or l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)5000] .

[0048] In some embodiments, the biodegradable hydrophobic polylactide is synthesized from monomers selected from the groups consisting of D,L-lactide, D-lactide, and L-lactide.

[0049] In some embodiments, the injectable solubilizers are selected from benzyl alcohol, polyoxyethylene (660) esters of 12-hydroxystearic acid, and phosphatidyl choline.

[0050] In some embodiments, the polymeric micelles comprise about 0.5-20% by weight of amphiphilic polymers and 60-95% by weight of solubilizers in total inactive ingredients and drug.

[0051] In some embodiments, the micelle has a size range between 10 and 200 nm.

[0052] In some embodiments, the drug dispersed in the total inactive ingredient and drug has a content of 0.5-20% weight percent.

[0053] In some embodiments, the formulation further comprises magnetic nanoparticles (e.g., as contrast imaging agents) and/or targeting ligands.

[0054] In some embodiments, the magnetic nanoparticles are superparamagnetic iron oxide nanoparticles. In some embodiments, the superparamagnetic iron oxide nanoparticles have the average particle size between 3-20 nm. In some embodiments, the surface of

superparamagnetic iron oxide nanoparticles is coated by oleic acid. In some embodiments, the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles. In some embodiments, the single or multiple superparamagnetic iron oxide nanoparticles are encapsulated within the micellar cores. In some embodiments, the transverse relaxivity of solution ranges from 50 Fe mM^"1 s^"1 to 600.0 Fe mM^"1 s In some embodiments, the superparamagnetic iron oxide nanoparticles increase of cytotoxicity of beta-lapachone. In some embodiments, the cytotoxicity of the composition is increased by up to 2-8 times induced by the increase of reactive oxygen species induced by

superparamagnetic iron oxide nanoparticles, when compared with the micelles without superparamagnetic iron oxide nanoparticles. [0055] In some embodiments, targeting moiety is conjugated with poly (lactide)- poly(ethylene glycol) maleimide. In some embodiments, the molecular weight of

poly(ethylene glycol) moiety is between 2000-10000 and the molecular weight of poly (lactide) moiety is between 2000-5000. In some embodiments, the targeting moiety including cyclic (RGDfK (SEQ ID NO. 1)) peptide, and lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL (SEQ ID NO. 2)).

[0056] In some embodiments, the formulations disclosed herein result in any of the following: (a) improvement in yield of micelles (e.g., a yield of about 60-98%); (b) improvement in storage stability (e.g., storage stability of about 24 months); (c) improvement in infusion fluid stability (e.g., infusion stability of about 48 hours); (d) improvement in safety (e.g., reduction in hemolysis); (e) improvement in plasma pharmacokinetics (e.g., a circulation time of >20 h); (f) improvement in tumor distribution; and (g) improvement in efficacy.

Methods of Preparing

[0057] Disclosed herein, in certain embodiments, are methods of preparing the stable micelles in aqueous solution, comprising the process of: (a) preparing a mixture of drug, polymers and solubilizers by mixing the amphiphilic polymers and solubilizers and a hydrophobic drug, or by dissolving the amphiphilic polymers and solubilizers and a hydrophobic drug in an organic solvent followed by evaporation of solvent; (b) mixing the mixture with aqueous solution under ultrasonication or high speed mixing to form a stable micelle solution; and (c) filtering through filter with 0.22 μπι pore size.

[0058] Disclosed herein, in certain embodiments, are methods of preparing the stable micelles with superparamagnetic iron oxide nanoparticles in aqueous solution, comprising the process of: (a) preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles by dissolving the amphiphilic copolymers, solubilizers, superparamagnetic iron oxide nanoparticles and a hydrophobic drug in an organic solvent, followed by evaporating the micelle solution at room temperature to remove the organic solvent and form a film; (b) mixing the film with aqueous solution under ultrasonication or high speed mixing to form a stable micelle solution; and (c) filtering through a filter with 0.22 μπι pore size.

[0059] Disclosed herein, in certain embodiments, are methods of preparing stable micelles with targeting ligand in aqueous solution, comprising the process of: (a) preparing a mixture of drug and polymers by dissolving the amphiphilic polymers, solubilizers, or multifunctional synergist and poly(lactide)-poly(ethylene glycol) maleimide and hydrophobic drug in an organic solvent, followed by evaporation of solvent; (b) mixing the mixture with aqueous solution under ultrasonication or high speed stirring to form a stable micelle solution; (c) conjugating cRGD peptide with micelle solution; and (d) filtering through filter with 0.22 μπι pore size. pH-SENSITIVE MICELLE FORMULATIONS

[0060] Provided herein are pH-sensitive micelle compositions comprising a block copolymer comprising a hydrophilic polymer segment and a hydrophobic polymer segment. In some embodiments, the micelle composition comprises a hydrophobic superparamagnetic iron oxide nanoparticle and/or a beta-lapachone compound.

Polymers for pH-Sensitive Micelle Formulations

[0061] In some aspects of the invention, the micelle polymer is an amphiphilic block copolymer comprising a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophilic polymer segment comprises a polymer selected from the group consisting of: poly(ethylene oxide) (PEO), poly(methacrylate phosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP), and wherein the hydrophobic polymer segment comprises

wherein R' is -H or -CH₃, wherein R is -NR 1 R2 , wherein R 1 and R2 are alkyl groups, wherein R 1 and R2 are the same or different, wherein R 1 and R2 together have from 5 to 16 carbons, wherein R 1 and R 2 may optionally join to form a ring, wherein n is 1 to about 10, wherein x is about 20 to about 200 in total, and wherein the block copolymer may further optionally comprise a labeling moiety. For example, x may be about 20 to about 200 as a continuous segment (i.e. a continuous segment of about 20 to about 200 monomer units), or other moieties (e.g. moieties comprising a label) may be interspersed between the monomer units, for example as described in more detail below. In any of the embodiments of the invention described herein, the PEO may of lower molecular weight and can include polyethylene glycol (PEG). [0062] Block copolymers include, for example, compounds of Formula I:

wherein L is a labeling moiety, wherein y is 0 to about 6, wherein R" is -H or -CH₃; wherein m is 1 to about 10, wherein z is such that the PEO is about 2 kD to about 20 kD in size, wherein x, n, R, and R' are as defined above, wherein R' " is any suitable moiety, and wherein the following portion of the structure:

may be arranged in any order.

[0063] In some embodiments, R' " is an end group resulting from a polymerization reaction. For example, R' " may be -Br when atom transfer radical polymerization (ATRP) is used, or R" ' may be a sulfur-containing group such as thiolate or a thioester when reversible addition-fragmentation chain transfer (RAFT) is used. In some embodiments, R" ' is -Br. In some embodiments, R" ' is thiolate. In some embodiments, R' " is a thioester. The end group may optionally be further modified following polymerization with an appropriate moiety.

[0064] In some embodiments, the following portion of the structure:

is randomized, i.e.

wherein r indicates a random ordering of the R-containing moieties and the L-containing moieties (i.e. the R-containing moieties and the L-containing moieties are randomly interspersed).

[0065] In some embodiments, the following portion of the structure:

is arranged sequentially. For example, the R-containing moieties may be present as a single block, with the L-containing moieties present as a single block either preceding or following the R-containing moieties. Other arrangements may also be utilized.

[0066] In some embodiments, the hydrophilic polymer segment comprises poly(ethylene oxide) (PEO). In some embodiments, the hydrophilic polymer segment comprises poly(methacrylate phosphatidyl choline) (MPC). In some embodiments, the hydrophilic polymer segment comprises polyvinylpyrrolidone (PVP). In general, the PEO, MPC, or PVP polymer in the hydrophilic polymer segment is about 2 kD to about 20 kD in size. In some embodiments, the polymer is about 2 kD to about 10 kD in size. In some embodiments, the polymer is about 2 kD to about 5 kD in size. In some embodiments, the polymer is about 3 kD to about 8 kD in size. In some embodiments, the polymer is about 4 kD to about 6 kD in size. In some embodiments, the polymer is about 5 kD in size. In some embodiments, the polymer has about 100 to about 130 monomer units. In some embodiments, the polymer has about 110 to about 120 monomer units. In some embodiments, the polymer has about 114 monomer units. In some embodiments, the polydispersity index (PDI) of the polymer is less than about 1.2. In some embodiments, the polydispersity index (PDI) of the polymer is less than about 1.1.

[0067] Suitable PEO, MPC, and PVP polymers may be purchased (for example, PEO polymers may be purchased from Aldrich Sigma) or may be synthesized according to methods known in the art. In some embodiments, the hydrophilic polymer can be used as an initiator for polymerization of the hydrophobic monomers to form a block copolymer.

[0068] For example, MPC polymers (e.g. narrowly distributed MPC polymers) can be prepared by atom transfer radical polymerization (ATRP) with commercially available small molecule initiators such as ethyl 2-bromo-2-methylpropanoate (Sigma Aldrich). These resulting MPC polymers can be used as macromolecular ATRP initiators to further copolymerize with other monomers to form block polymers such as MPC-b-PDPA. PEO-b- PR block copolymers can be synthesized using atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) methods (See e.g. Australian Journal of Chemistry Volume: 58 Issue: 6 Pages: 379-410 (2005); Progress in Polymer Science Volume: 32 Issue: 1 Pages: 93-146 (2007). ATRP or RAFT allows for living polymerization which can yield PEO-b-PR copolymers with narrow polydispersity (<1.1). Different methacrylate or acrylate monomers can be used to produce PR segments with different pH sensitivity.

[0069] In some embodiments, the hydrophobic polymer segment comprises:

wherein R' is -H or -CH₃, wherein R is -NR 1 R2 , wherein R 1 and R2 are alkyl groups, wherein R 1 and R2 are the same or different, wherein R 1 and R2 together have from 5 to 16 carbons, wherein R 1 and R 2 may optionally join to form a ring, wherein n is 1 to about 10, and wherein x is about 20 to about 200 in total.

[0070] In some embodiments, n is 1 to 4. In some embodiments, n is 2. In various embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0071] In some embodiments, R' is -CH₃. In some embodiments, R' is -H.

[0072] In some embodiments, x is about 40 to about 100 in total. In some embodiments, x is about 50 to about 100 in total. In some embodiments, x is about 40 to about 70 in total. In some embodiments, x is about 60 to about 80 in total. In some embodiments, x is about 70 in total.

[0073] In some embodiments, R¹ and R² together have from 5 to 14 carbons. In some embodiments, R 1 and R2 together have from 5 to 12 carbons. In some embodiments, R 1 and

R 2 together have from 5 to 10 carbons. In some embodiments, R 1 and R2 together have from

5 to 8 carbons. In some embodiments, R 1 and R 2 together have from 6 to 12 carbons. In some embodiments, R 1 and R2 together have from 6 to 10 carbons. In some embodiments, R 1 and

R 2 together have from 6 to 8 carbons. In various embodiments, R 1 and R2 together have 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbons. In some embodiments, R 1 and R 2 each have 3 to

8 carbons. In some embodiments, R 1 and/or R2 comprise 3 carbons. In some embodiments, R 1 and/or R 2 comprise 4 carbons. In some embodiments, R 1 and/or R2 comprise 5 carbons. In some embodiments, R 1 and/or R2 comprise 6 carbons. In some embodiments, R 1 and/or R2 comprise 7 carbons. In some embodiments, R 1 and/or R 2 comprise 8 carbons. In some embodiments, R 1 and R2 are the same. In some embodiments, R 1 and R2 are different. In some embodiments, R 1 and R 2 are each independently straight or branched alkyl. In some embodiments, R 1 and R2 are each straight alkyl. In some embodiments, R 1 and R2 are each branched alkyl. Suitable alkyl groups for R 1 and R 2 include, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, and pentadecyl, including various possible skeletal isomers for each alkyl group such as n-, iso-, sec-, tert-, neo-, etc., provided the total number of carbons in R is from 5 to 16. In some embodiments, R 1 and R 2 are propyl. In some embodiments, propyl is iso-propyl. In some embodiments, propyl is n-propyl. In some embodiments, R 1 and R 2 are butyl. In some embodiments, butyl is n-butyl. In some embodiments, butyl is iso-butyl. In some

embodiments, butyl is sec -butyl. In some embodiments, butyl is t-butyl. In some

embodiments, R 1 and R 2 join to form a ring. The ring may optionally be substituted with one or more alkyl groups, provided the total number of carbons in R is from 5 to 16. In some embodiments, R 1 and R 2 together form a ring having 5 to 10 carbons. In some embodiments,

R 1 and R2 together form a ring having 5 to 8 carbons. In some embodiments, R 1 and R2 together form a ring having 5 to 7 carbons. In some embodiments, R 1 and R 2 together are -

(CH₂)₅-. In some embodiments, R 1 and R 2 together are -(CH₂)₆-.

[0074] The hydrophobic polymer segment may be synthesized according to, e.g. Atom Transfer Radical Polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT). Exemplary methods of preparation of the aforementioned polymers are known in the art and can be found, for example, in PCT/US2011/001418. In some embodiments, the polydispersity index (PDI) for the hydrophobic polymer segment is less than about 1.2. In some embodiments, the polydispersity index (PDI) for the hydrophobic polymer segment is less than about 1.1.

[0075] In some embodiments, the micelle polymer comprises a polymer of the compound C5A-MA:

[0076] In some embodiments, the micelle polymer comprises a polymer of the compound C6A-MA:

[0077] In some embodiments, the micelle polymer comprises a polymer of the compound C7A-MA:

[0078] In some embodiments, the micelle polymer comprises a compound of the formula DBA-MA:

[0079] Exemplary methods of synthesis of the aforementioned monomers can be found in PCT/US2011/001418, filed August 11, 2011. For example, C6A-MA can be synthesized by dissolving (pentamethyleneimino)ethanol, triethylamine, and inhibitor hydrophinone in THF and adding methacryloyl chloride dropwise. The solution is refluxed in THF for 2 hours, followed by filtration and removal of the solvent by rotovap. The product monomer is purified by distillation in vacuo.

[0080] Exemplary methods of synthesis of the block copolymers described herein can be found in PCT/US2011/001418, filed August 11, 2011, including the atom transfer radical polymerization method. For example, PEO-b-PDPA can be synthesized according to the following procedure: 2-(diisopropyl amino)ethyl methacrylate, N,N,N,N",N"- pentamethyldiethylenetriamine, and MeO-PEG-Br are dissolved in DMF. After three cycles of freeze-pump-thaw to remove oxygen, CuBr is added under nitrogen atmosphere.

Polymerization is carried out at 40°C for 8 hours, after which the reaction mixture is diluted with THF, passed through an alumina column to remove the catalyst, and rotovapped to remove the solvent. The residue is dialyzed in distilled water and lyophilized to obtain the product.

[0081] With regards to the compounds described herein, it is to be understood that polymerization reactions may result in a certain variability of polymer length, and that the numbers described herein indicating the number of monomer units within a particular polymer (e.g. x, y, z) may indicate an average number of monomer units. In some

embodiments, a polymer segment described herein (e.g. the hydrophobic polymer segment, the hydrophilic polymer segment) has a polydispersity index (PDI) less than about 1.2. In some embodiments, the polydispersity index (PDI) for the polymer segment is less than about 1.1. In some embodiments, the polydispersity index (PDI) for the block copolymer is less than about 1.2. In some embodiments, the polydispersity index (PDI) for the block copolymer is less than about 1.1. pH Sensitivity

[0082] In some embodiments, one or more block copolymers (e.g. 2, 3, 4, 5, or more) described herein may be used to form a pH-sensitive micelle. In some embodiments, a composition comprises a single type of micelle. In some embodiments, two or more (e.g. 2, 3, 4, 5, or more) different types of micelles may be combined to form a mixed-micelle composition.

[0083] The pH-sensitive micelle compositions of the invention may advantageously have a narrow pH transition range, in contrast to other pH sensitive compositions in which the pH response is very broad (i.e. 2 pH units). In some embodiments, the micelles have a pH transition range of less than about 1 pH unit. In various embodiments, the micelles have a pH transition range of less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1 pH unit. In some embodiments, the micelles have a pH transition range of less than about 0.5 pH unit. In some embodiments, the micelles have a pH transition range of less than about 0.25 pH unit.

[0084] The micelles may have different pH transition values within physiological range, in order to target specific cells or microenvironments. In some embodiments, the micelles have a pH transition value of about 5 to about 8. In some embodiments, the micelles have a pH transition value of about 5 to about 6. In some embodiments, the micelles have a pH transition value of about 6 to about 7. In some embodiments, the micelles have a pH transition value of about 7 to about 8. In some embodiments, the micelles have a pH transition value of about 6.3 to about 6.9 (e.g. tumor microenvironment). In some

embodiments, the micelles have a pH transition value of about 5.0 to about 6.2 (e.g.

intracellular organelles). In some embodiments, the micelles have a pH transition value of about 5.9 to about 6.2 (e.g. early endosomes). In some embodiments, the micelles have a pH transition value of about 5.0 to about 5.5 (e.g. late endosomes or lysosomes).

[0085] Without wishing to be bound by theory, the use of micelles in cancer therapy may enhance anti-tumor efficacy and reduce toxicity to healthy tissues, in part due to the size of the micelles. While small molecules such as certain chemotherapeutic agents (e.g.

doxorubicin) can enter both normal and tumor tissues, non-targeted micelle nanoparticles may preferentially cross leaky tumor vasculature. In some embodiments, the micelles have a size of about 10 to about 200 nm. In some embodiments, the micelles have a size of about 20 to about 100 nm. In some embodiments, the micelles have a size of about 30 to about 50 nm. β-Lapachone Compounds

[0086] In some aspects of the invention, the micelle composition further comprises a drug encapsulated within the micelle. Due to the hydrophobic interior of the micelle, hydrophobic drugs may be more readily encapsulated within the micelles. In some embodiments, the drug is hydrophobic and has low water solubility. In some embodiments, the drug has a log p of about 2 to about 8.

[0087] In some embodiments, the drug is a chemotherapeutic agent (such as an anti-cancer drug). In some embodiments, the drug is a β-lapachone compound or a pharmaceutically acceptable salt thereof. In some embodiments, the β-lapachone compound is β-lapachone, as shown in Formula I:

[0088] The β-lapachone compound described herein also includes analogs and derivatives of β-lapachone having similar anti-cancer activity as β-lapachone. In some embodiments, the β-lapachone compound is a prodrug of β-lapachone. In some embodiments, the β-lapachone compound is a polymer conjugated with a pH-sensitive prodrug of beta-lapachone, wherein the compound is capable of forming a micelle, for example, as described in

PCT/US2011/047497.

[0089] In some embodiments, the β-lapachone compound is a β-lapachone derivative. In some embodiments the β-lapachone derivative is a compound of Formula II:

wherein Ri is H, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkoxycarbonyl, -

(CH₂)_n-amino, -(CH₂)_n-aryl, -(CH₂)_n-heteroaryl, -(CH₂)_n-heterocycle, -or -(CH₂)_n-phenyl, wherein n is an integer from 0 to 10; and R₂ and R₃ are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkoxycarbonyl, -(CH₂)_n-aryl, -(CH₂)_n-heteroaryl, -(CH₂)_n- cycloalkyl, (CH₂)_n-heterocycloalkyl, hydroxyl, substituted or unsubstituted thiol, halogen, nitro or cyano, for example, as described in U.S. 6,875,745.

[0090] In some embodiments, the beta-lapachone compound is menadione, 2,2-dimethyl- (Z)-6-phenylimino-3,4,5,6-tetrahydro-2H-naphtho[l,2-b]oxin-5-one [phenylimine

lapachone], 2,2-dimethyl-(Z)-6-(4-methyl-phenylimino)-3,4,5,6-tetrahydro-2H-naphtho[l,2- b]oxin-5-one [p-methylphenylimine lapachone], 2,2-dimethyl-(Z)-6-(4- methoxyphenylimino)-3,4,5,6-tetrahydro-2H-naphtho[l,2-b]oxin-5-one [p- methoxyphenylimine lapachone] , 2,2-dimethyl-(Z)-6-(4-nitrophenylimino)-3,4,5,6- tetrahydro-2H-naphtho[l,2-b]oxin-5-one [p-nitrophenylimine lapachone], or 2,2-dimethyl- (Z)-6-(4-bromophenylimino)-3,4,5,6-tetrahydro-2H-naphtho[l,2-b]oxin-5-one [p- bromophenylimine lapachone] (see K.E. Reinicke et al, Clin. Cancer Res., 2005, 11(8), 3055- 3064).

[0091] In some embodiments, the beta-lapachone compound is a prodrug of beta-lap. In some embodiments, the beta-lapachone compound comprises a polymer conjugated with a pH-sensitive prodrug of beta-lapachone, wherein the compound is capable of forming a micelle, and wherein the pH-sensitive prodrug comprises a pH-sensitive linker selected from the group consisting of: an aryl imine and an aliphatic imine. In some embodiments, the pH- sensitive linker is an aryl imine. In some embodiments, the aryl imine is a phenyl imine. In some embodiments, the phenyl comprises a substitutent. In some embodiments, the substituent is at the para position. In some embodiments, the substituent is -OH, -NH₂, -SH,

or maleimide

. In some embodiments, the substituent is maleimide

.

In some embodiments, the pH-sensitive linker is an aliphatic imine. In some embodiments, the Ca of the aliphatic imine comprises at least one substitutent. In some embodiments, the Ca of the aliphatic imine comprises two substitutents. In some embodiments, the substitutents are both methyl. In some embodiments, the prodrug is selected from the group consisting of:

and

wherein R₈ is a side chain of a D or L amino acid other than -H; R₃ is -NH₂, -OH, -SH, or

; each of R₄, R5, R₆, and R₇ is independently -H, -X, -OCH₃, or -CH₃; X is a halogen; and p is an integer between 0 and 20. In some embodiments, R₈ is -CH₃. In some

embodiments, R₃ is

. In some embodiments, R₃ is -OH. In some embodiments, each of R₄, R5, R₆, and R₇ is H. In some embodiments, X is CI, Br, I, or F. In some embodiments, p is 0-6. In some embodiments, the prodrug is linked to the polymer by a bond selected from the group consisting of: an ester bond, an amide bond, a disulfide bond, or a thioether bond.

[0092] In some embodiments, the micelle formulation is stable at a neutral pH (e.g. a physiologically neutral pH) and releases beta-lapachone at an acidic pH (e.g. a

physiologically acidic pH). In some embodiments, the therapeutic agent is a β-lapachone prodrug with a linkage of: ketal, acyl hydrazone, aliphatic imine, aromatic imine bond, or a combination thereof. In some embodiments, the ketal, acyl hydrazone, aliphatic imine, or

wherein Ri is a side chain of D or L amino acids; R₂ is an alkyl group or an aromatic group; R₃ is NH₂, OH, or SH; each of R₄, R₅, R₆, and R₇ is independently H, X, OCH₃, or CH₃; X is a halogen; and n is an integer between 1 and 20. In some embodiments, X is CI, Br, I, or F. In some embodiments, R₂ is CH₃, CH₂CH₃, or Bzl. Non-limiting examples of prodrugs of the invention include the following:

[0093] In some embodiments, the biocompatible polymeric prodrug micelle has the formula:

wherein R1 and R2 are each independently selected from:

In some embodiments, the micelle is stable at a neutral pH and releases beta-lapachone at a physiologically acidic pH.

[0094] In some embodiments, the drug is an NQOl bioactivable drug, such as DNQ or strep tonigrin.

[0095] The drug may be incorporated into the micelles using methods known in the art, such as solvent evaporation. Briefly, for example, drug may be encapsulated in micelles by first dissolving the drug and the block co-polymer in organic solution. Addition of this solution to an aqueous solution, optionally under sonication, may result in micelle- encapsulated drug.

[0096] In some embodiments, the drug loading in the micelle may be from about 1% and about 20%. For example, the drug loading is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the drug loading is between about 1% and about 15%. In some embodiments, the drug loading is between about 5% and about 10%. In some embodiments, the drug loading is between about 3% and about 8%.

Superparamagnetic Iron Oxide Nanoparticles

[0097] In some aspects of the invention, the micelle composition further comprises magnetic nanoparticles encapsulated within the micelles. In some aspects, the micelles further comprise magnetic nanoparticles and a drug (such as an NQOl bioactivable drug described herein) encapsulated within the micelles.

[0098] In some embodiments, the magnetic nanoparticles are superparamagnetic iron oxide nanoparticles (SPION). In some embodiments, the superparamagnetic iron oxide

nanoparticles have the average particle size between about 3 nm and about 20 nm. In some embodiments, the surface of superparamagnetic iron oxide nanoparticles is coated by oleic acid. In some embodiments, the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles. In some embodiments, the single or multiple superparamagnetic iron oxide nanoparticles are encapsulated within the micellar cores. In some embodiments, the transverse relaxivity of solution ranges from 50 Fe mM^"1 s^"1 to 600.0 Fe mM^"1 s In some embodiments, the superparamagnetic iron oxide nanoparticles increase of cytotoxicity of beta-lapachone. In some embodiments, the cytotoxicity of the composition is increased by at least about 2 times (e.g., about 2-8 times) induced by the increase of reactive oxygen species induced by superparamagnetic iron oxide nanoparticles, when compared with the micelles without superparamagnetic iron oxide nanoparticles.

[0099] In some embodiments, the SPION and the drug are formulated within the same micelles. In some embodiments, the SPION and the drug are formulated within different micelles. In some embodiments, the SPION is formulated within micelles and the drug is formulated in solution. In some embodiments, the loading of the SPION in the micelles is between about 1% and 20%. In some embodiments, the loading of the SPION is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20%. In some embodiments, the SPION increases the loading of the drug in the micelles by about any of 2, 3, 5, 10, or 20-fold compared to SPION-free micelles.

Targeting Moiety

[0100] In some embodiments, the micelle composition further comprises one or more targeting moiety. For example, a targeting moiety can target a cancer cell surface marker, such as an angiogenesis biomarker. [0101] In some embodiments, the targeting moiety binds to an angiogenesis biomarker. In some embodiments, the angiogenesis biomarker is VEGF-VEGFR complex or endoglin. In some embodiments, the targeting moiety binds to VEGFR2. In some embodiments, the targeting moiety is a Fab' fragment of RAFL-1 mAb. In some embodiments, the targeting moiety binds to α_νβ₃ integrin. In some embodiments, the targeting moiety is cRGDfK (SEQ ID NO. 1).

[0102] The targeting moiety may be conjugated to the block copolymer (e.g., the hydrophilic polymer segment) by methods known in the art.

[0103] In some embodiments, targeting moiety is conjugated with poly (lactide)- poly(ethylene glycol) maleimide. In some embodiments, the molecular weight of

poly(ethylene glycol) moiety is between 2000-10000 and the molecular weight of poly (lactide) moiety is between 2000-5000. In some embodiments, the targeting moiety including cyclic (RGDfK(SEQ ID NO. 1)) peptide, and lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL(SEQ ID NO. 2)).

Methods of Making pH-Sensitive Micelle Formulations

[0104] Exemplary methods of generating micelles from block copolymers may be found, for example, in PCT/US2011/001418, filed August 11, 2011. For example, block copolymer is first dissolved in organic solvent (e.g. THF) and may be added to an aqueous solution, optionally under sonication, wherein the copolymer self-assembles to form micelles in the solution.

THERAPEUTIC METHODS

[0105] The invention also provides pharmaceutical compositions comprising a micelle formulation described herein and a pharmaceutically acceptable carrier for therapeutic use.

[0106] Micelle compositions described herein (including pharmaceutical compositions) may be used to treat or delay progression of cancer wherein the drug(s) encapsulated in the micelle may be delivered to the appropriate location due to localized pH differences (e.g. a pH different from physiological pH (7.4)). Micelles for therapeutic methods may optionally further comprise a labeling moiety (e.g. to assist in the imaging of the treatment) and/or a targeting moiety (e.g. to target a specific cell surface marker or to target the micelles for endocytic delivery). In some embodiments, the cancer treated is a solid tumor. In some embodiments, the cancer is selected from the group consisting of lung (such as non-small cell lung cancer) cancer, prostate cancer, breast cancer, pancreatic cancer, colon cancer (including colorectal cancer), and melanoma. In embodiments wherein the micelle comprises a targeting moiety, non- solid cancers may be treated.

[0107] An effective amount of the micelle composition described herein may be administered to an individual for treating cancer by any suitable methods, for example, by injection or infusion. In some embodiments, the composition is administered locally or systemically. In some embodiments, the composition is administered by intraperitoneal, intravenous, subcutaneous, and intramuscular injections, and other forms of administration such as oral, mucosal, via inhalation, sublingually, etc. The dosage required for the treatment depends on the choice of the route of administration, the nature of the formulation, the nature of the individual's illness, the individual's size, weight, surface area, age and sex; other drugs being administered, and the judgment of the attending physician. In some subjects, more than one dose may be required. Frequency of administration may be determined and adjusted over the course of therapy. For example, frequency of administration may be determined or adjusted based on the type and stage of the cancer to be treated, whether the agent is administered for therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. Administration of the composition in accordance with the method in the present invention can be continuous or intermittent. In some embodiments, the micelle composition comprising SPION and a β- lapachone compound may be administered once a day, once in two days, or once in three days. In some embodiments, a micelle composition comprising SPION is administered prior to administration of an anti-cancer drug (such as a β-lapachone compound) composition. In some embodiments, the period between the administration of the micelle composition comprising SPION and the anti-cancer drug is about 6 hours to about 48 hours, such as between about 6 hours to about 24 hours, about 24 hours to about 48 hours.

[0108] In some embodiments, an "effective amount" of drug, compound, or composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibiting, to some extent, tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more anti-cancer drug, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0109] The composition described herein may be used in conjunction with another anticancer treatment. As used herein, "in conjunction with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0110] In some embodiments, "treatment" or "treating" is an approach for obtaining beneficial or desired results including and preferably clinical results. For example, beneficial or desired clinical results include, but are not limited to, one or more of the following:

reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.

[0111] In some embodiments, "delaying development of a disease" includes defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed. [0112] In some embodiments, an "individual" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets (such as cats, dogs, horses), primates, mice and rats.

[0113] In some embodiments, the micelles have a pH transition value of about 6.3 to about 7.2 (e.g. for delivery to the tumor microenvironment). In some embodiments, the micelles have a pH transition value of about 5.0 to about 6.5 (e.g. for delivery to intracellular organelles). In some embodiments, the micelles have a pH transition value of about 6.2 or above 6.2 (e.g. for delivery to early endosomes). In some embodiments, the micelles have a pH transition value of about 5.5 (e.g. for delivery to late endosomes or lysosomes). In some embodiments, the micelles have a pH transition value of about 6.3 to about 6.9. In some embodiments, the micelles have a pH transition value of about 5.0 to about 6.2. In some embodiments, the micelles have a pH transition value of about 5.9 to about 6.2. In some embodiments, the micelles have a pH transition value of about 5.0 to about 5.5. In some embodiments, non-targeted pHAM with higher pH_t (e.g. 7.2, 6.8) may be used to delivery drug to tumors. In some embodiments, targeted pHAM with lower pH_t (e.g. 5.4, 6.3) may be used to delivery drug to endocytic compartments.

[0114] The invention also provides kits for use in the instant methods. Kits of the invention include a micelle formulation described herein. The micelle formulation may be in one or more containers. In some embodiments, the kits further comprise instructions for use in accordance with any of the methods described herein. In some embodiments, these instructions comprise a description of administering the composition (including

pharmaceutical composition) to an individual for treating cancer.

[0115] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly indicates otherwise.

[0116] Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X."

[0117] It is understood that aspect and variations of the invention described herein include "consisting" and/or "consisting essentially of aspects and variations.

[0118] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any manner. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

Example I: Synthesis

[0119] Superparamagnetic iron oxide nanoparticles as a multifunctional synergist was describe as follows: iron(III) acetylacetonate (2 mmol) was mixed with 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), and oleylamine (6 mmol) in benzyl ether (20 ml) under nitrogen. The mixture was then heated to reflux (200 °C) for 2 h, followed by refluxing for another 1 h at 300°C. After cooling to room temperature, the solution was treated with ethanol under air to yield a dark-brown precipitate. The product was dispersed in hexane in the presence of oleic acid and oleylamine, and then re-precipitated with ethanol to give 5-10 nm SPION.

Example 2: The encapsulation of beta lapachone into the nanoscale micelles

[0120] Poly(lactide)5000-poly(ethylene glycol)5000 (20 mg), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (25 mg), benzyl alcohol (100 mg), phosphatidyl choline (200 mg) and beta-lapachone (20 mg) were mixed to form viscous liquid film. The aqueous micelle was prepared by dissolving the film into purified water (5.0 ml) under ultrasonication. The yield of beta lapachone in the micelles is evaluated by measuring the content of beta lapachone in the micelles. The yield of micelles is 90%. Beta lapachone was also incorporated into poly(lactide)5000-poly(ethylene glycol)5000 (PEG- PLA) to form beta lapachone-loaded PEG-PLA micelles as control using same procedure. The yield of PEG-PLA micelles is only 20%. The micelles had an average particle size of 50.0 nm ranging from 10 nm to 100 nm according to the measurement of dynamic light scattering instrument.

Example 3: The encapsulation of beta lapachone into the nanoscale micelles

[0121] l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (20 mg), and polyoxyethylene(660) esters of 12-hydroxy stearic acid (200 mg) and beta-lapachone (10 mg) were mixed to form viscous liquid film. The aqueous micelle was prepared by dissolving the film into purified water (1.0 ml) under ultrasonication. The yield of beta lapachone in the micelles is evaluated by measuring the content of beta lapachone in the micelles. The yield of micelles is 95%. The micelles had an average particle size of 40.0 nm ranging from 10 nm to 150 nm according to the measurement of dynamic light scattering instrument

Example 4: The encapsulation of beta lapachone into the nanoscale micelles

[0122] l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)5000] (15 mg), benzyl alcohol (40 mg) and polyoxyethylene(660) esters of 12- hydroxystearic acid (450 mg) and beta-lapachone (6 mg) were dissolved in 15 ml tetrahydrofuran. A drug-polymer film was obtained using rotary evaporation of the solvent at 60 °C for 10 min. The aqueous micelle was prepared by dissolving the film into purified water (3.0 ml) under ultrasonication. The yield of beta lapachone in the micelles is evaluated by measuring the content of beta lapachone in the micelles. The yield of micelles is 95%. The average particle size is about 45 nm.

Example 5: The encapsulation of beta lapachone into the nanoscale micelles

[0123] l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)5000] (60 mg), polyoxyethylene(660) esters of 12-hydroxystearic acid (500 mg) , superparamagnetic iron oxide nanoparticles (15 mg) and beta-lapachone (15 mg) were dissolved in 5 ml tetrahydrofuran. A film was obtained using rotary evaporation of the solvent at 60 °C for 10 min. The aqueous micelle was prepared by dissolving the film using purified water (5.0 ml) under ultrasonication. The micelle yield of the micelles is evaluated by measuring the content of beta lapachone in the micelles. The yield of the micelles is 90.0%. The average particle size is about 55.0 nm.

Example 6: The encapsulation of beta lapachone into the nanoscale micelles

[0124] The poly(lactide)5000-poly(ethylene glycol)10000 (30 mg), polyoxyethylene(660) esters of 12-hydroxystearic acid (300 mg), superparamagnetic iron oxide nanoparticles (30 mg), and beta-lapachone (15 mg) were dissolved in 5 ml tetrahydrofuran. A film was obtained using rotary evaporation of the solvent at 60 °C for 10 min. The aqueous micelle was prepared by dissolving the film into purified water 1.5 ml) under ultrasonication. The yield of the micelles is evaluated by measuring the content of beta lapachone in the micelles. The yield of micelles in micelles is 88%. The average particle size is about 75.0 nm. Example 7: The post modification of ligand peptides to micelles

[0125] l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (10 mg), phosphatidyl choline (400 mg), benzyl alcohol(40 mg) and beta-lapachone (15 mg) were dissolved in 8 ml tetrahydrofuran. A viscous film was obtained using rotary evaporation of the solvent at 60 °C for 10 min. The aqueous micelle was prepared by dissolving the film using purified water (5 ml) under ultrasonication. Then, cRGD peptide and 0.05 M hydroxy amine in HEPES/EDTA aqueous solution were added into solutions of micelles. The conjugation was allowed to occur for 4 h followed by filtration through a Millipore centrifugal filter (pore size 0.45 mm). The cRGD-micelles were dialyzed with Spectra/Por dialysis membrane (molecular weight cutoff = 50,000 Da) until free cRGD was completely removed. The yield of the micelles is 73%. The micelles had an average particle size of 61.0 nm.

Example 8: The post modification of ligand peptides to micelles

[0126] Poly(lactide)2000-poly(ethylene glycol)5000-maleimide (10 mg), polyoxyethylene (660) esters of 12-hydroxystearic acid (400 mg), superparamagnetic iron oxide nanoparticles (15 mg) and beta-lapachone (15 mg) were dissolved in 12 ml tetrahydrofuran. A drug- polymer film was obtained using rotary evaporation of the solvent at 60 °C for 10 min. The aqueous micelle was prepared by dissolving the film into purified water (8.0 ml) under ultrasonication. Lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL (SEQ ID NO.2)) was added into the micelle solution. Then the peptide and 0.05 M hydroxy amine in HEPES/EDTA aqueous solution were added into solutions of micelles. The conjugation was allowed to occur for 4 h followed by filtration through a Millipore centrifugal filter (pore size 0.45 mm). The micelles were further dialyzed with Spectra/Por dialysis membrane (molecular weight cutoff = 50,000 Da) until free peptide was completely removed. The drug yield in micelles is 75.0%. The average particle size of the micelles is 77.0 nm.

Example 9

[0127] The cytotoxicity of the compositions from example 2, example 3, example 4, example 5, example 6, example 7 and example 8 was evaluated. Relative survival assays based on DNA content were performed in A549. A549 cells were seeded (10,000 cells/well) into each well of 48-well plates. On the following day, media were removed, and media containing predetermined doses of free β-lap drug (dissolved in DMSO) or the compositions were added for a duration of 2 h. Beta lapachone-loaded Poly(lactide)5000-poly(ethylene glycol)5000 (PEG5k-PLA5k) micelles and free beta lapachone were also evaluated as a control. After 4 h exposures, media were then removed, control growth media added, and cells were allowed to grow for an additional 7 days. DNA content was determined by DNA fluorescence Hoescht dye 33258. Samples were read in a Perkin Elmer HTS 7000 Bio Assay Reader (Waltham, MA). The IC50 of the compositions are shown in Table 1. The

compositions showed significantly increase of cytotoxicity compared to the control groups.

Example 10: Transverse relaxivity

[0128] The transverse relaxivities of the micelles of the compositions prepared in example 6 and example 8 were measured at 1.41 T using a standard Carr-Purcell-Meiboom-Gill (CPMG) sequence on a Bruker desktop relaxometer (MQ60 model, Ettlingen, Germany). The Fe concentration in micelle samples was determined on a Varian SpectrAA 50 spectrometer (air/acetylene flame). The samples were diluted to 1.0 ml purified water, followed by vortexing. After incubation for 30 min, the T2 values were measured. The relaxation rates (1/T2, s-1) were plotted as a function of Fe concentrations and the slopes were measured as the r2 value for all the samples. The transverse relaxivities of the formulations from example 5 and example 7 were 250 mM-1 s-1, and 300 mM-1 s-1, which are significantly higher than that of commercial product (-100 mM-1 s-1). Example 11

[0129] The half-life time (tmp) in pharmacokinetic studies determining blood concentration over time of compositions from example 2 and example 8, were evaluated in 6- to 8-week-old randomized tumor-bearing female athymic nude mice (-25 g each). Log-phase A549 cells (5 x 106) were injected s.c. into the flanks of mice. Pharmacokinetic studies were initiated with randomized mice containing average tumor volumes of -300 mm3. The micelle composition containing 1% 3H-labeled micelles from Example 2 and example 7 were injected into mice via the tail vein. Blood was collected from the ocular vein at various times (1 min to 24 h) after injection. Plasma was isolated and mixed with a tissue solubilizer (1 mL, BTS-450; Beckman) at room temperature for 5 hours followed by addition of liquid scintillation mixture (10 mL), and the mixture was incubated for 12 hours. All experiments were performed in triplicate, and data were analyzed using a two-compartment pharmacokinetic model. The results showed all the composition had long circulation effects, the half-life time of both compositions are above 20 h.

Example 12: Synergistic effect of superparamagnetic iron oxide nanoparticles with β- lapachone

[0130] Preparation of micelle-coated superparamagentic iron oxide nanoparticles (mSPION). mSPION were prepared as described in the following representative example: 8.0 mg PEG₁₁₄-PDPA₁₂₀ and 2.0 mg SPION were first dissolved in 1.0 mL tetrahydrofuran (THF) in a glass vial. Then the solution was added to purified water (3.0 mL) under vigorous ultrasonic agitation using a Type 60 Sonic Dismembrator (Fisher Scientific). The vial was then open to air overnight, allowing slow evaporation of THF and the formation of micelles. The residual THF was completely removed using centrifugation dialysis (MW cutoff 100 kD, Millipore, MA) (3,000 rpm, 30 min, 3 times). Finally, 0.5 mL mSPION were filtered through a 0.22 μπι filter and stored at 4 °C.

[0131] Morphology and Particle Size. Transmission electron microscopy (TEM) imaging was performed using a JEOL 1200 EX II instrument (Tokyo, Japan). Each sample was first diluted using water and then placed on a glow-discharged copper grid and allowed to adsorb for 45 seconds. The excess solution was removed by blotting the grid against filter paper, and then the grid was stained for 30 seconds using 2% phosphotungstic acid. After the excess solution was removed, the grid was directly dried in the air. The TEM image of SPION was obtained without staining. Dynamic light scattering (DLS) was used to measure particle size. The hydrodynamic diameters of mSPION were characterized by Zeta sizer (Malvern

NanoZS).

[0132] Release of Iron Ions from mSPION. The release of iron ions from mSPION was performed using dialysis (MW cut-off 3000) in an Oscillator shaker. 1.0 ml mSPION (1.0 mM) was placed in dialysis and 20 ml buffers at pH 7.4, 6.8, 6.2 and 5.0 were respectively used as the release mediums at (37 + 0.5) °C. Samples (1.0 mL each) were taken at 12, 24, 36, 48 and 72 h with the replacement of an equal volume of new release medium. The analysis of iron ions was performed using an atom absorption spectrometer.

[0133] Relative Survival Assay. A549 cells were grown in Dulbecco's minimal essential medium (DMEM) with 5% fetal bovine serum (FBS). Cells were cultured at 37 °C in a 5% C0₂-95% air humidified atmosphere and were free from mycoplasma contamination. DNA content assessments were done as described by J.J. Pink et al., /. Biological Chemistry, 2000, 275(8), 5416-5422. Briefly, cells were co-treated with mSPION and β-lap at indicated concentrations with or without 48-h pretreatment of mSPION. When indicated, deferoxamine (5 mM) or catalase (1000 U) pre- and co-treatments were also used. Drug-free media were then added and cells allowed to grow for an additional ~7 days or until control cells reached -95% confluence. DNA content of samples was determined by Hoeschst 33258 dye staining and fluorescence detection using a plate reader (Perkin-Elmer, Boston, MA). Results were reported as means, +SE from sextuplate repeats.

[0134] Confocal Laser Scanning Microscopy. Fluorescent mSPION (0.14 mM iron) were incubated with A549 cells for 4 h. Subsequently, the cells were treated with

LysoTracker Green DND26 (50 nM) for 5 minutes, followed by subsequent examination using confocal microscopy. The excitation/emission wavelengths were 488 nm/510 nm for LysoTracker Green DND26.

[0135] ROS Formation Analysis. ROS formation was monitored by microscopy using the superoxide indicator DHE staining. Briefly, cells were seeded on 35-mm glass cover slips and allowed to attach overnight followed by 48 h pre-incubation of mSPION (0.14 mM) if needed. Cells were then loaded with 10 μΜ of DHE in 1 x PBS for 30 min at 37 °C, washed twice with PBS, and co-treated with 3 μΜ β-lap and 0.14 mM mSPION. At indicated times, cells were fixed in 10% formalin for -30 min at room temperature, then washed twice in PBS and mounted in Vectashield (with DAPI, Vector Laboratories, Inc., Burlingame, CA). Images were taken using a Leica DM5500 Upright Microscope (Buffalo grove, IL). Quantitative data were analyzed using NIH Image J software, where each datum point represented the mean, +SE of 50 cells counted and data were representative of experiments performed in triplicate. [0136] This example demonstrates that micelle coated SPION (mSPION) can act as an intracellular iron donor to enhance free radical generation of anticancer drug, β-lap. The combination of β-lap and mSPION results in a synergistic effect on cancer cytotoxicity. mSPION may release iron ions (Fe²⁺ and Fe³⁺) in acidic organelles including early endosomes (pH -6.0), late endosomes (5.0-6.0), and lysosomes (5.0-5.5), which may act as the catalyst in the Fenton reaction, after SPION are internalized into acidic organelles (Figure 1). The catalysis is believed to improve the decomposition of peroxide hydrogen into hydroxyl radicals, and subsequently improve the cytotoxicity of anticancer drug due to the enhanced ROS formation.

[0137] Oleic acid-stabilized SPION with average size of 5.8 nm were synthesized as reported previously by S. Sun et al. (/. Amer. Chem. Soc, 2004, 126, 273-279). The particle size of SPION was distributed between 5 nm and 7 nm as confirmed by transmission electron microscopy (Fig. 2a). Amphiphilic poly(ethylene glycol)-b-poly(2-(2-diisopropylamino) ethyl methacrylate (PEGn₄-b-PDPAi₂₀) was synthesized for constructing micelle-coated SPION (mSPION) via micellization (Critical micelle concentration was 1.0 μg/ml). mSPION had an average diameter of 65.0 nm with narrow size distribution, which is significantly higher than that of SPION-free micelles (32.0 nm) because of the formation of SPION clusters in micellar cores (Fig. 2B and Fig. 2C). mSPION had a transverse relaxivity of 125.3 Fe mM^"1 s^"1 at pH 7.4.

[0138] In order to evaluate the release of iron ions (Fe²⁺ and Fe³⁺) from mSPION in acidic environment, the leakage of iron ions from mSPION was measured in the buffers at different pH values, where the iron concentration was determined by atomic absorption spectrometer. Fig. 2D showed that mSPION in the buffers at pH 5.0 and 6.2 had iron release of 60.0% and 11.2% after 72 h dialysis, respectively. The iron ions from mSPION through the membranes displayed a steady release flux, which could be explained by Fick's First Law of diffusion. The buffer at pH 5.0 could result in much higher release flux of iron ions when compared with that at pH 6.2. However, the release of iron ions from mSPION was not observed in the buffers at pH 7.4 and 6.8. The accumulative amounts of the released iron ions are related to pH, concentration of mSPION and release time. mSPION display a pH-dependent iron release responding to subtle pH change in the physiological pH range and are possibly act as the donor of iron ions.

[0139] To track the intracellular distribution of mSPION,rhodamine-conjugated PEG₁₁₄- PDPAi₂o (PEGii₄-PDPAi₂₀-Rh) was synthesized to construct fluorescent mSPION, in which rhodamine was entrapped into the micellar cores and could display highly fluorescent at acidic environment. Confocal microscopy was used to perform double fluorescence-labeling experiments and visualize red fluorescence from PEGn₄-PDPAi₂o-Rh in mSPION and green fluorescence from LysoTracker Green DND-26 that is selective for acidic lysosomes (or endosomes), respectively. Most (>80%) of mSPION were localized in the endosomes and lysosomes, as evidenced by co-localization of red fluorescence with that from LysoTracker Green (Fig. 3A). It shows that mSPION could be internalized into the endosomes and lysosomes and exposed in the acidic microenvironment for subsequent release of iron ions. No toxicity was observed with an exposure of up to 0.5 mM concentration of mSPION in the in vitro cell culture system.

[0140] The use of mSPION as a donor of iron ions to facilitate the generation of ROS from β-lap was demonstrated. This is expected to improve the cytotoxicity of β-lap. Here, cells were first pre-treated with mSPION, allowing mSPION to release iron ions before β-lap was used to treat cells. A549 cells were either pretreated in the medium containing mSPION at various concentrations of iron (up to 0.14 mM) or mSPION-free medium for 48 h, followed by the co-treatment with 3 μΜ β-lap and mSPION for another 4 h (Fig. 3B). It shows that 48 h pretreatment of mSPION led to significant increased β-lap cytotoxicity. Cell survival rate after 0.07 mM mSPION pretreatment showed about 6-fold decrease compared to 3 μΜ β-lap treatment only. In comparison, co-treatment only showed an average of about 60% cell survival rate, which was similar to the cells that were treated with 3 μΜ β-lap alone. It implies that the pretreatment of mSPION play a key role for releasing the iron ion from mSPION in the cells, thus improving cytotoxicity of β-lap. Additionally, a specific NQOl inhibitor, could completely block this synergistic effect, just as it rescued the cells from the toxicity of drug alone, indicating that the synergistic effects between mSPION and β-lap were also NQO-1 dependent and tumor selective. The synergistic effects were further evaluated using 0.14 mM of mSPION combined with different concentrations of β-lap (Fig. 3C). It showed that with 48 h pre-treatment of mSPION, synergistic effects could be achieved at as low as 1.0 μΜ β-lap. Without pretreatment but only co-treatment, cells were killed at the same level as treated with β-lap alone. It indicates that mSPION could induce the decrease of β-lap LD₅₀ at different concentrations and significantly improve the sensitivity of cells to β- lap within the therapeutic window.

[0141] To examine the effect of pre-incubation time of mSPION on iron ion release, the synergistic effects were compared after 4 h versus 48 h pretreatment at 0.07 mM and 0.14 mM of mSPION, respectively (Fig. 3A). Results showed that 4 h pre-treatment with subsequently 44 h incubation in mSPION-free medium has already significantly improved β- lap cytotoxicity at 3 μΜ. Furthermore, 48 hr pre-incubation further intensified the synergistic effects indicating that prolonged incubation of mSPION might facilitate more efficient iron ion release from mSPION rather than more uptake of mSPION.

[0142] To confirm that the released iron ions from mSPION are indeed the contributor for improved β-lap cytotoxicity, deferoxamine, a FDA-approved chelating agent that isused to bind free iron ions in the body, was used to block the synergistic killing effects. A549 cells were pretreated in the medium containing mSPION (0.07mM and 0.14 mM iron respectively) for 48 h with or without the presence of 5.0 μg/mL deferoxamine, a relatively non-toxic dose, followed by 4 h co-treatment with 3.0 μΜ β-lap (Fig. 4B). Data showed that the presence of deferoxamine in medium only resulted in a slight change in the viability of A549 cells suggesting that the synergistic effects between mSPION and β-lap were blocked by deferoxamine. On the contrary, pretreatment of mSPION still led to a significant decrease of the cell viability in the absence of deferoxamine. This indicates that deferoxamine could lead to the cleavage of the iron ions from mSPION in cells, which could not induce the generation of enough hydroxyl radicals and subsequently more ROS via the Fenton reaction. Thus, the presence of the iron ions with adequate concentration in cells is a prerequisite for achieving the synergistic effect. Additionally, it also implies that the synergistic effect was impossibly attributed to the intrinsic peroxidase-like activity on the surface of mSPION as suggested by Gao et al (Nat. Nanotechnol. 2007, 2, 577-583). If the intrinsic activity of mSPION could also act as the catalyst to potentiate the β-lap toxicity, the use of deferoxamine should not be able to completely block the synergistic effect.Thus, iron ions from mSPION play a key role in achieving the synergy between mSPION and β-lap.

[0143] To further explore the role of free radicals in mediating the synergistic effect, dihydroethidium (DHE) staining was used to detect 0₂ ^~ level within the first 60 min of co- treatment of mSPION and β-lap with or without pretreating the cells with mSPION (Fig. 5A- B). The oxidized form of DHE by 0₂ ^~ to 2-hydroxyethidium can intercalate DNA and emit bright red fluorescence inside cell nuclei. Fig. 5A showed that cells at 20 min post co- treatment of 3 μΜ β-lap after 48 h pretreatment of mSPION (0.14 mM) had more than 10- fold increase of ROS intensity compared to those cells without pretreatment of mSPION (Fig. 5B). This effect kept being intensified at 60 min post co-treatment indicating that the cellular accumulation of iron ions from mSPION pretreatment can escalate ROS formation in the cells, thereafter leading to cell death. Thus, the formation of a large amount of ROS is the key factor for the synergistic effect between mSPION and β-lap. [0144] As it is known that H₂0₂ might be one of the major executers of free radicals that can go into the nuclei and damage DNA via the Fenton reaction, whether H₂0₂ is essential for the synergistic effect caused by mSPION and β-lap was evaluated. Catalase as a H₂0₂ scavenger was exploited to interfere with the synergistic treatment. Fig. 5C shows that without catalase, pre-treatment of mSPION led to significant synergy. Exogenously added 1000 u catalase into the cultural media completely blocked the synergistic effects of mSPION at both 0.07 and 0.14 mM. It could possibly be attributed to the catalase-triggered

decomposition of hydrogen peroxide from β-lap in the futile cycle into water and oxygen. The decrease of hydrogen peroxide level resulted in an alleviation of the cytotoxicity of pre- and co-treatment regimen even though iron ions were still available in the cells.

[0145] In summary, the synergistic effect of mSPION on improving anticancer drug efficiency was demonstrated. mSPION are highly internalized into acidic organelles and subsequently release iron ions due to their pH-dependent release behavior. The released iron ions from mSPION in cells possibly catalyze the ROS formation primarily via Fenton reaction, which could result in the decrease of drug LD₅₀ and significantly improve the sensitivity of cells to drug within the therapeutic window. The prolonged incubation of mSPION might induce stronger synergistic effect on drug cytotoxicity because more efficient iron ions are released from mSPION. The formation of more ROS via a series of

sophisticated reactions is responsible for the synergistic effect between mSPION and anticancer drugs holding a mechanism of action through ROS. mSPION as a biocompatible agent display an intriguing biological potential for cancer therapy and opens up a new route for their application in nanomedicine.

Example 13: Improved Loading of β-Lapachone in Micelles

[0146] β-lap micelles with various SPIO loadings were synthesized using a solvent evaporation method. First, a mixture of PEG-PDPA polymers, β-lap (10% theoretical loading) and SPIO were dissolved in 1 ml THF in a glass vial. Next, the mixture was added slowly into 3 ml of de-ionized water under sonication (60 Sonic Dismembrator, Fisher

Scientific). After overnight shaking at room temperature to evaporate THF, the micelle solution was filtered through a 0.45 μπι syringe filter to remove any large aggregates and further purified by centrifuge dialysis (MW cutoff 100 kDa) to remove any free drugs.

[0147] After micelle production, the hydrodynamic diameters of β-lap and SPIO

encapsulated micelles were measured using a Dynamic Light Scattering instrument (Malvern

Nanosizer). β-lap loading content (LC) was calculated as the percentage of loaded β-lap over the total weight of micelles, β-lap loading was determined by a UV-vis spectrometer

(Shimadzu UV-1800, Japan). First, micelle solutions were lyophilized and re-dissolved in a mixture of chloroform and DMSO (1:1) under bath sonication for 30 min. The suspending SPIO nanoparticles were removed by centrifugation, and the upper solution was collected to measure the β-lap amount by UV absorbance at 257.2 nm (ε =105 mL mg-1 cm-1).

[0148] For in vitro drug release study, 0.2 ml micelle solution was first added to 1.8 ml of appropriate buffer solution with different pH values and the solution mixed by vortex. This solution was then placed into a Spectrum Float- A-Lyzer dialysis tube (MW cutoff 100,000 Da). The tube was subsequently placed into 8 ml buffer solutions with different pH values. Release studies were performed at 37 °C in a New Brunswick Scientific C24 Incubator Shaker. At selected time points, buffer solution outside the dialysis bag was removed for UV-Vis analysis and replaced with fresh buffer solution. Release studies were performed in triplicate with the error bars representing the standard deviation between trials. The release of iron ions from micelles was performed using the same procedure as the β-lap release study. The concentration of iron ions released from micelles was measured by atomic absorption spectrometer.

[0149] Shown in Figure 6, by raising the loading amounts of SPIO in the β-lap and SPIO encapsulated micelles, the hydrodynamic size of micelles increased dramatically. The diameters of micelles changed from 24.2+1.6 to 83.1±12.6 nm when the SPIO loading was increased from 5% to 15% (Figure 6a). In addition, the introduction of SPIO inside the micelle hydrophobic core can improve the β-lap loading significantly. The optimized β-lap loading is only 0.8% for the SPIO free micelles, while the loading density can increase to 5.5% when 15% of SPIO was encapsulated in the micelles (Figure lb).

[0150] These micelles provide stable drug and SPIO encapsulation at physicological pH, but can release β-lap or iron ions in acidic environments due to the protonation and swelling of micelles. For 10% SPIO loading micelles, the hydrodynamic size showed a significant increase from 51.4±4.3 to 155.8±8.8 nm when the pH value decreased from 7.4 to 5.0 (Figure 7a). In vitro β-lap release showed that at pH 7.4 and 6.8 the drug release is much slower, accounting for only 25% of drug release even after 6 days. When the micelles were incubated in acidic environment for 6 days, the drug release is much faster, with over 50% of drug released at pH 6.2 and 90% of drug released at pH 5.0 (Figure 7b). Moreover, the iron ions released from the micelles also showed a similar pH-dependent pattern. At pH 7.4 or 6.8, no iron ions were released from the micelles even after 6 days, while over 80% of irons were released at pH 5.0 after 6 days (Figure 7c). [0151] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A polymeric micelle composition comprising: a pH-sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle and a beta- lapachone compound encapsulated within the hydrophobic core of the micelle,

wherein the block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment,

wherein the hydrophilic polymer segment comprises a polymer selected from the group consisting of: poly(ethylene oxide) (PEO), poly(methacrylate phosphatidyl choline) (MPC), and polyvinylpyrrolidone (PVP),

wherein the hydrophobic polymer segment comprises

wherein R' is -H or -CH

wherein R is -NR 1 R2 , wherein R 1 and R2 are alkyl groups, wherein R 1 and R2 are the same or different, wherein R 1 and R2 together have from 5 to 16 carbons, wherein R 1 and R2 may optionally join to form a ring,

wherein n is 1 to about 10,

wherein x is about 20 to about 200 in total, and

wherein the block copolymer optionally comprises a labeling moiety.

2. The composition of claim 1, wherein the hydrophilic polymer segment comprises

PEO.

3. The composition of any one of claims 1-2, wherein n is 1 to 4.

4. The composition of any one of claims 1-3, wherein R' is -CH₃.

5. The composition of any one of claims 1-3, wherein R' is -H.

6. The composition of any one of claims 1-5, wherein x is about 40 to about 100 in total.

7. The composition of any one of claims 1-5, wherein x is about 50 to about 100 in total.

8. The composition of any one of claims 1-5, wherein x is about 40 to about 70 in total.

9. The composition of any one of claims 1-5, wherein x is about 60 to about 80 in total.

10. The composition of any one of claims 1-5, wherein x is about 70 in total.

11. The composition of any one of claims 1-10, wherein R 1 and R 2 are each straight or branched alkyl.

12. The composition of any one of claims 1-10, wherein R 1 and R 2 join to form a ring.

13. The composition of any one of claims 1-12, wherein R 1 and R 2 are the same.

14. The composition of any one of claims 1-12, wherein R 1 and R 2 are different.

15. The composition of any one of claims 1-14, wherein R 1 and R 2 each have 3 to 8 carbons.

16. The composition of claim 12, wherein R 1 and R 2 together form a ring having 5 to 10 carbons.

17. The composition of any one of claims 1-10, wherein R 1 and R 2 are propyl.

18. The composition of claim 17, wherein propyl is iso-propyl.

19. The composition of any one of claims 1-10, wherein R 1 and R 2 are butyl.

20. The composition of claim 19, wherein butyl is n-butyl.

21. The composition of any one of claims 1-10, wherein R 1 and R 2 together are -(CH₂)₅-.

22. The composition of any one of claims 1-10, wherein R 1 and R 2 together are -(CH₂)₆-.

23. The composition of any one of claims 1-22, wherein the micelle has a size of about 10 nm to about 200 nm.

24. The composition of any one of claims 1-23, wherein the micelle has a pH transition of less than about 1 pH unit.

25. The composition of any one of claims 1-24, wherein the micelle has a pH transition value of about 5 to about 8.

26. The composition of any one of claims 1-25, wherein the micelle further comprises a targeting moiety.

27. The composition of any one of claims 1-26, wherein the beta-lapachone compound is beta-lapachone, or a derivative thereof, or a pharmaceutically acceptable salt thereof.

28. The composition of any one of claims 1-27, wherein the loading of beta-lapachone compound is between about 3 and about 8%.

29. The composition of any one of claims 1-28, wherein the superparamagnetic iron oxide nanoparticle (SPION) has an average particle size of about 3 nm to about 20 nm.

30. The composition of any one of claims 1-29, wherein the micelle comprises about 1% to about 20% by weight of the superparamagnetic iron oxide nanoparticle.

31. The composition of any one of claims 1-30, wherein the block copolymer is poly(ethylene glycol)-P-poly(2-(2-diisopropylamino) ethyl methacrylate).

32. The composition of any one of claims 1-31, wherein the block copolymer is PEG₁₁₄- P-PDPA₁₂₀.

33. A polymeric micelle composition comprising: a pH-sensitive micelle comprising a block copolymer; and a hydrophobic superparamagnetic iron oxide nanoparticle encapsulated within the hydrophobic core of the micelle,

wherein the hydrophobic polymer segment comprises

wherein R' is -H or -CH₃,

wherein n is 1 to about 10,

wherein x is about 20 to about 200 in total, and

wherein the block copolymer optionally comprises a labeling moiety.

34. The composition of claim 33, wherein the hydrophilic polymer segment comprises PEO.

35. The composition of any one of claims 33-34, wherein n is 1 to 4.

36. The composition of any one of claims 33-35, wherein R' is -CH₃.

37. The composition of any one of claims 33-35, wherein R' is -H.

38. The composition of any one of claims 33-37, wherein x is about 40 to about 100 in total.

39. The composition of any one of claims 33-37, wherein x is about 50 to about 100 in total.

40. The composition of any one of claims 33-37, wherein x is about 40 to about 70 in total.

41. The composition of any one of claims 33-37, wherein x is about 60 to about 80 in total.

42. The composition of any one of claims 33-37, wherein x is about 70 in total.

43. The composition of any one of claims 33-42, wherein R 1 and R 2 are each straight or branched alkyl.

44. The composition of any one of claims 33-42, wherein R 1 and R 2 join to form a ring.

45. The composition of any one of claims 33-44, wherein R 1 and R 2 are the same.

46. The composition of any one of claims 33-44, wherein R 1 and R 2 are different.

47. The composition of any one of claims 33-46, wherein R 1 and R 2 each have 3 to 8 carbons.

48. The composition of claim 33-42, wherein R 1 and R 2 together form a ring having 5 to 10 carbons.

49. The composition of any one of claims 33-42, wherein R 1 and R 2 are propyl.

50. The composition of claim 49, wherein propyl is iso-propyl.

51. The composition of any one of claims 33-42, wherein R 1 and R 2 are butyl.

52. The composition of claim 51, wherein butyl is n-butyl.

53. The composition of any one of claims 33-42, wherein R 1 and R 2 together are -(CH₂)5.

54. The composition of any one of claims 33-42, wherein R 1 and R 2 together are -(CH₂)₆.

55. The composition of any one of claims 33-54, wherein the micelle has a size of about 10 nm to about 200 nm.

56. The composition of any one of claims 33-55, wherein the micelle has a pH transition of less than about 1 pH unit.

57. The composition of any one of claims 33-56, wherein the micelle has a pH transition value of about 5 to about 8.

58. The composition of any one of claims 33-57, wherein the micelle further comprises a targeting moiety.

59. The composition of any one of claims 33-58, wherein the block copolymer is poly(ethylene glycol)-P-poly(2-(2-diisopropylamino) ethyl methacrylate.

60. The composition of any one of claims 33-59, wherein the block copolymer is PEG₁₁₄- P-PDPA₁₂₀.

61. A method of preparing the polymeric micelle composition of any one of claims 1-32, comprising mixing a mixture of the beta-lapachone compound, the superparamagnetic iron oxide nanoparticle, and the block copolymer with aqueous solution to form a stable micelle composition.

62. A method for treating cancer in an individual in need thereof, comprising

administering to the individual an effective amount of the composition of any one of claims 1-32.

63. The method of claim 62, wherein the cancer is a solid tumor with cancer cells expressing NQOl.

64. The method of claim 62, wherein the cancer is lung, prostate, breast, pancreatic, colon, or melanoma cancer.

65. The method of any one of claims 62-64, wherein the composition is administered once a day, once every two days, or once every three days.

66. The method of any one of claims 62-64, wherein the composition of any one of claim 33-60 is administered to the individual before the administration of the composition of any one of claims 1-32.

67. The method of claim 66, wherein the period between the two treatments is about 6 hours to about 48 hours.

68. A polymeric micelle formulation of comprising:

a. a hydrophobic core component, including polylactide;

b. an hydrophilic poly(ethylene glycol) segment as shell layers;

i. a injectable lipid solubilizer for enhancing drug loading of

hydrophobic drug, or ii. poly(lactide)-poly (ethylene glycol) maleimide or lipid-poly(ethylene glycol) maleimide with post-modification of cancer-targeting ligand such as cRGD; and

69. A polymeric micelle formulation comprising:

a. an amphiphilic polymer including monomethoxy poly (ethylene glycol)-b- polylactide or 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol);

b. an hydrophobic anti-cancer drug such as beta-lapachone encapsulated within hydrophobic cores; and

c. one of:

i. one or two injectable solubilizers for enhancing drug loading and

stability of hydrophobic drug in the micellar cores;

ii. a multifunctional hydrophobic synergist encapsulated within

70. The formulation of claim 68 or claim 69, wherein the therapeutic agent is a hydrophobic agent.

71. The formulation of claim 68 or claim 69, wherein the therapeutic agent is beta-lap.

72. The formulation of claim 68 or claim 69, wherein the hydrophilic monomethoxy poly (ethylene glycol) of amphiphilic polymers has an average molecular weight of between 1000 and 10000.

73. The formulation of claim 68 or claim 69, wherein the hydrophobic polylactide segments have an average molecular weight of between 2000 and 8000.

74. The formulation of claim 68 or claim 69, wherein the hydrophobic polylactide segments is selected from: l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol) include l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)2000] or l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)5000] .

75. The formulation of claim 68 or claim 69, wherein the biodegradable hydrophobic polylactide is synthesized from monomers selected from: D,L-lactide, D-lactide, and L- lactide.

76. The formulation of claim 68 or claim 69, wherein the injectable solubilizers are selected from: benzyl alcohol, polyoxyethylene (660) esters of 12-hydroxystearic acid, and phosphatidyl choline.

77. The formulation of claim 68 or claim 69, wherein the polymeric micelles comprise about 0.5-20% by weight of amphiphilic polymers and 60-95% by weight of solubilizers in total inactive ingredients and drug.

78. The formulation of claim 68 or claim 69, wherein the micelle has a size range between 10 and 200 nm.

79. The formulation of claim 68 or claim 69, wherein the drug dispersed in the total inactive ingredient and drug has a content of 0.5-20% weight percent.

80. The formulation of claim 68 or claim 69, wherein the formulation further comprises magnetic nanoparticles and/or targeting moieties.

81. The formulation of claim 80, wherein the magnetic nanoparticles are

superparamagnetic iron oxide nanoparticles.

82. The formulation of claim 80, wherein the superparamagnetic iron oxide nanoparticles have the average particle size between 3-20 nm.

83. The formulation of claim 80, wherein the surface of superparamagnetic iron oxide nanoparticles is coated by oleic acid.

84. The formulation of claim 80, wherein the polymeric micelles comprise about 1-20% by weight of superparamagnetic iron oxide nanoparticles.

85. The formulation of claim 80, wherein the single or multiple superparamagnetic iron oxide nanoparticles are encapsulated within the micellar cores.

86. The formulation of claim 80, wherein the transverse relaxivity of solution ranges from 50 Fe mM^"1 s^"1 to 600.0 Fe mM^"1 s

87. The formulation of claim 80, wherein the superparamagnetic iron oxide nanoparticles increase of cytotoxicity of beta-lapachone.

88. The formulation of claim 80, wherein the cytotoxicity of the composition is increased by up to 2-8 times induced by the increase of reactive oxygen species induced by

superparamagnetic iron oxide nanoparticles, when compared with the micelles without superparamagnetic iron oxide nanoparticles.

89. The formulation of claim 80, wherein the targeting moiety is conjugated with poly (lactide) -poly(ethylene glycol) maleimide.

90. The formulation of claim 80, wherein the molecular weight of poly(ethylene glycol) moiety is between 2000-10000 and the molecular weight of poly (lactide) moiety is between 2000-5000.

91. The formulation of claim 80, wherein the targeting moiety including cyclic (RGDfK (SEQ ID NO. 1)) peptide, and lung cancer peptide (aka H2009.1, with a sequence of RGDLATLRQL (SEQ ID NO. 2)).

92. The formulation of claim 68 or claim 69, wherein the formulations disclosed herein result in any of the following: (a) improvement in yield of micelles (e.g., a yield of about 60- 98%); (b) improvement in storage stability (e.g., storage stability of about 24 months); (c) improvement in infusion fluid stability (e.g., infusion stability of about 48 hours); (d) improvement in safety (e.g., reduction in hemolysis); (e) improvement in plasma

pharmacokinetics (e.g., a circulation time of >20 h); (f) improvement in tumor distribution; and (g) improvement in efficacy.

93. A method of treating a cancer, comprising administering to an individual in need thereof a composition disclosed herein.

94. The method of claim 93, wherein the composition is administered before, during, or simultaneously with the administration of a cytotoxic agent.

95. A method of preparing a formulation comprising micelles in aqueous solution, comprising: (a) preparing a mixture of drug, polymers and solubihzers; and (b) mixing the mixture with aqueous solution to form a stable micelle solution.

96. The method of claim 95, wherein preparing a mixture of drug, polymers and solubihzers comprises (a) mixing the amphiphilic polymers and solubihzers and a

hydrophobic drug, or (b) dissolving the amphiphilic polymers and solubihzers and a hydrophobic drug in an organic solvent followed by evaporation of solvent.

97. The method of claim 95, wherein mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing.

98. The method of claim 95, further comprising filtering the solution through filter paper with 0.22 μπι pore size.

99. A method of preparing the stable micelles with superparamagnetic iron oxide nanoparticles in aqueous solution, comprising: (a) preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles; and (b) mixing the film with aqueous solution.

100. The method of claim 99, wherein preparing a mixture of drug, polymers and superparamagnetic iron oxide nanoparticles comprises dissolving the amphiphilic

copolymers, solubihzers, superparamagnetic iron oxide nanoparticles and a hydrophobic drug in an organic solvent, followed by evaporating the micelle solution at room temperature to remove the organic solvent and form a film.

101. The method of claim 99, wherein mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing.

102. The method of claim 99, further comprising filtering the solution through a filter device with 0.22 μπι pore size.

103. A method of preparing stable micelles with targeting ligand in aqueous solution, comprising: (a) preparing a mixture of drug and polymers; (b) mixing the mixture with aqueous solution to form a stable micelle solution; and (c) conjugating cRGD peptide with micelle solution.

104. The method of claim 103, wherein preparing a mixture of drug and polymers comprises dissolving the amphiphilic polymers, solubihzers, or multifunctional synergist and poly(lactide)-poly(ethylene glycol) maleimide and hydrophobic dmg in an organic solvent, followed by evaporation of solvent.

105. The method of claim 104, wherein mixing the mixture with aqueous solution comprises ultrasonication or high speed mixing.

106. The method of claim 104, further comprising filtering the solution through a filter device with a 0.22 μm pore size.