WO2018172942A1 - Quercetin nanoparticles - Google Patents

Abstract

Disclosed herein are lipid nanoparticles comprising a flavonol molecule, such as quercetin phosphate, for use as a chemotherapeutic. Some embodiments disclosed herein pertain to methods of making and use of the lipid nanoparticles.

Description

QUERCETIN NANOPARTICLES

GOVERNMENT INTEREST

This invention was made with government support under Grant Numbers

CA149363, CA149387, CA151652, and DK100664 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The subject matter described herein relates to lipid nanoparticles comprising a flavonoid, such as quercetin phosphate. Additionally disclosed are methods of synthesis and the use of the same as a chemo therapeutic.

BACKGROUND OF THE INVENTION

Stroma cells, including tumor associated fibroblasts (TAFs), macrophage, and endothelial cells contribute to the resistance of nanochemotherapies. These cells form a physical barrier within tumors to inhibit penetration of the therapeutic nanoparticles (NP), as well as secrete growth-inducing cytokines and growth factors to facilitate the survival of tumor cells. Furthermore, during the chemotherapy processes, chronic damage to stroma cells elicit the secretion of damage response program (DRP) molecules to promote the survival and growth of neighboring cells, thus causing the acquired resistance to the chemotherapies. One of these DRP molecules was reported by Sun et al., who observed that treatment-induced DNA damage in the neighboring benign stroma cells promotes chemotherapy resistance through paracrine secretion of Wntl6. (Sun, Y. et al., "Treatment-Induced Damage to the Tumor Microenvironment Promotes Prostate Cancer Therapy Resistance through Wntl6b," Nat. Med., 2012, 18, pp. 1359-1368). Wntl6 is a member of the wingless-type MMTV integration site (Wnt) family and is considered one of the major mitogenic growth factors that constitute DRP molecules.

Natural chemicals have gained substantial attention in cancer therapy due to the safety profile (low toxicities). Unfortunately, the antitumor effect of natural products alone is usually far from satisfactory. There are numerous natural products with anti- fibrotic properties such as astragaloside IV, tetrandrine, salvianolic acid and quercetin. However, their effect on the DRP molecule Wnt 16 was unknown. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. Quercetin regulates multiple biological pathways eliciting induction of apoptosis as well as inhibiting angiogenesis and proliferation. It has also been reported to have a protective ability against oxidative stress and mutagenesisin normal cells. However, quercetin' s poor water solubility and bioavailability have limited its use as a pharmaceutical. Quercetin was in early-stage clinical trials as an anticancer agent decades ago. Yet, due to its poor solubility, the administration required the use of solvents such as dimethylsulfoxide or ethanol, which caused dose-dependent hemolysis as well as liver and kidneys impairments. Thus, alternative strategies are needed to improve the water solubility and/or the bioavailability as well as the tumor site delivery. The present disclosure addresses these and other needs within the field.

BRIEF SUMMARY OF THE INVENTION

In several embodiments, provided herein are lipid nanoparticles comprising a compound of Formula I. The lipid nanoparticles can be used to deliver compounds of Formula I to cells.

In several embodiments, the lipid nanoparticles comprising a compound of Formula I as provided for herein are advantageous in that they provide for one or more of improved bioavailability, improved metabolic stability, enhanced delivery to a target site, a high loading efficiency, and enhanced anti-tumor effects (e.g., modulation of the tumor microenvironment and/or Wntl6 levels).

Several embodiments pertain to a method of delivering a compound of Formula I to a subject (e.g., use of lipid nanoparticles). In several embodiments, the method comprises a step of administering an effective amount of a lipid nanoparticle comprising a compound of Formula I as disclosed herein to the subject.

In several embodiments are provided methods for the treatment of diseases or unwanted conditions in a subject, wherein the method comprises administering a lipid nanoparticle comprising a compound of Formula I. In additional embodiments, the methods further comprise administering an additional therapeutic agent.

In several embodiments, the lipid nanoparticles comprise a targeting ligand that can specifically target the bioactive compound to diseased cells, enhancing the effectiveness and minimizing the toxicity of the lipid nanoparticles. Several embodiments pertain to a method of treating a disease state. In several embodiments, the method comprises a step of administering an effective amount of a lipid nanoparticle comprising a compound of Formula I as described herein to a subject in need of treatment. In several embodiments, the disease state is a cancer.

Further provided herein are methods for making the lipid nanoparticles comprising a compound of Formula I.

These and other aspects of the subject matter disclosed herein are disclosed in more detail in the description of the invention given below. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show the effects of selected natural chemicals on Wntl6 expression in TGF-β activated NIH3T3 cells. (FIG. 1A) Western-blot bands and (FIG. IB) Quantification of Western-blot band intensities normalized to control. (1)

Tanshinone II A, (2) Astragaloside IV, (3) Notoginsenoside Rl, (4) Matrine, (5) Artemisinin, (6) Quercetin, (7) Rheinic acid, (8) Salvianolic acid B, (9) Ligustrazine, (10) Scutellarin, (11) Salvianolic acid A, (12) Tetrandrine, and (FIG. 1C) Western-blot bands and quantification showed effect of quercetin on Wntl6 expression in cisplatin treated activated NIH3T3 cells. *p<0.05, n=3.

FIGS. 2A-2D show the preparation and characterization of LCP-QP. FIG. 2A shows the preparation procedure for LCP-QP. FIG. 2B shows the TEM photograph of LCP-QP cores and final particles. FIG. 2C shows the dynamic light scattering measurements of particle size and distribution of LCP-QP. FIG. 2D shows a photograph of LCP-QP solution.

FIGS. 3A-3E depict the QP conversion to quercetin. Shown is the experimental procedure (FIG. 3 A) and HPLC spectrum (FIG. 3B) of the solution of free QP and alkaline phosphatase after 1 h incubation at 37°C. Also shown is the experimental procedure (FIG. 3C) and HPLC spectrum (FIG. 3D) of the cell medium after 4 h incubation of LCP-QP at 37 °C with NIH3T3 cells. FIG. 3E shows the tumor quercetin accumulation detected by UPLC-MS one hour after i.v. injection of free QP and LCP-QP respectively. *p<0.05, n=3.

FIGS. 4A-4F depict the tumor inhibition effects of LCP-QP, LPC, and LCP-QP plus LPC on a stroma-rich UMUC3 bladder cancer xenograft model after five intravenous injections (blue arrows, four mice per group). FIG. 4A shows the tumor volume change. FIG. 4B shows the tumor weight at the end of the experiment (day 19). FIG. 4C shows the western-blot bands and quantification of band intensities of Wntl6 expression in the tumor tissue after different treatments normalized to control (n=3). FIG. 4D shows the TUNEL staining of tumor sections after different treatments. FIG. 4E shows the quantification of TUNEL fluorescence signal expressed as the percentage of total cell number (DAPI signal). **p <0.01, *p<0.05, «=5. FIG. 4F shows the cisplatin concentration in the tumor tissue after LPC and LPC+LCP-QP treatment.

FIGS. 5 A and 5B depict: the effects (FIG. 5 A) of different treatments on the inhibition of fibroblast growth and Masson's trichrome stain for collagen and quantification results expressed as the percentage of total cell number; and (FIG. 5B) the effect of LCP-QP on the penetration of l'-dioctadecyl-3,3,3'3'- tetramethylindocarbocyanine (hereinafter "Dil") labeled nanoparticles and quantification of fluorescence signal (Dil labeled red) expressed as the percentage of cell number (DAPI signal) detected on frozen tumor sections. GFP positive fibroblasts (green), DAPI labeled nuclei (blue), and Dil labeled LCP-QP particles (red). **p <0.01, *p<0.05, «=5.

FIGS. 6A-6C depict: (FIG. 6A) Body weight change, (FIG. 6B) Serum ALT, AST, BUN and creatinine levels and, (FIG. 6C) HE staining of major drug

accumulating organs after five injections of different treatments.

FIGS. 7A-7C show the structure and ³¹P-NMR (FIG. 7A), Ή-NMR (FIG. 7B), and ¹³C-NMR (FIG. 7C) spectra of quercetin and phosphorylated quercetin.

FIG. 8 depicts the effect of LCP-QP on the tumor microenvironment after administrated with cisplatin nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

In some embodiments, the subject matter disclosed herein relates to lipid nanoparticles comprising a flavonoid, such as quercetin phosphate. As disclosed herein, flavonoids, such as the dietary flavonoid quercetin, were found to suppress Wntl6 expression in activated fibroblasts. In some embodiments, in order to facilitate drug delivery, a targeted Lipid/Calcium/Phosphate (LCP) nanoparticle formulation was prepared consisting of a prodrug of quercetin, i.e. quercetin phosphate, with a high loading efficiency (26.6% w/w). In some embodiments, a quercetin nanoparticle has a particle size of around 35 nm and was found to significantly improve the bioavailability and metabolic stability of parent quercetin. As disclosed herein, the quercetin phosphate may be released from the nanoparticles and converted back to the parent quercetin under physiological conditions. In some embodiments, following systemic administration of quercetin phosphate nanoparticles, a significant downregulation in Wntl6 expression was observed. In some embodiments, quercetin phosphate significantly remodeled the tumor microenvironment and increased the penetration of second-wave nanoparticles into the tumor nests.

In further embodiments, a synergistic antitumor effect with cisplatin nanoparticles in a stro ma-rich bladder carcinoma model was observed. Also disclosed herein is that the a-SMA-positive fibroblast levels and collagen content within the tumor decreased significantly after combination treatment. Without being bound by theory, the results disclosed herein suggest that the remodeling of the tumor microenvironment induced by quercetin plays a critical role in promoting the synergy. As such, quercetin phosphate nanoparticles are a safe and effective way to improve therapeutic treatment for desmoplastic tumors.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. The terminology used in the description of the subject matter described herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter described herein.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this subject matter pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Provided according to embodiments of the subject matter disclosed herein are lipid nanoparticles comprising a compound of Formula I:

wherein,

R¹ is selected from the group consisting of hydroxyl, -0-(O-C6 alkyl), and -0-P(0)(OR^x)₂, wherein R^x is independently hydrogen or Ci-Ce alkyl;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from the group consisting of hydrogen, hydroxyl, -0-(O-C6 alkyl), and -O- P(0)(OR^x)₂;

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹⁰ is -O- P(0)(OR^x)₂;

wherein the compound of Formula I is encapsulated by a lipid bilayer wherein;

the lipid bilayer comprises an inner leaflet and an outer leaflet, wherein the inner leaflet comprises a first lipid and the outer leaflet comprises a second lipid, wherein the lipid bilayer is asymmetric.

As used herein, the term "alkyl" refers to a straight-chained or branched hydrocarbon group containing 1 to 12 carbon atoms. The term "lower alkyl" refers to a Ci-C₆ alkyl chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl.

As in any embodiment above, a lipid nanoparticle wherein the lipid nanoparticle has a diameter of about 20 nm to about 90 nm.

As in any embodiment above, a lipid nanoparticle wherein the lipid nanoparticle has a diameter of about 25 nm to about 45 nm.

As in any embodiment above, a lipid nanoparticle wherein the compound of Formula I is present in an amount of at least 25 % wt. of the lipid nanoparticle.

As in any embodiment above, a lipid nanoparticle wherein the inner leaflet comprises a neutral or anionic lipid. As in any embodiment above, a lipid nanoparticle wherein the lipid is DOPA.

As in any embodiment above, a lipid nanoparticle wherein the outer leaflet comprises one or more of cholesterol, a cationic lipid, or a neutral lipid.

As in any embodiment above, a lipid nanoparticle wherein the outer leaflet comprises a lipid selected from the group consisting of cholesterol, DOTAP, DSPE- PEG, and DSPE-PEG-AA, and combinations thereof.

As in any embodiment above, a lipid nanoparticle wherein the outer leaflet further comprises a targeting ligand.

As in any embodiment above, a lipid nanoparticle wherein at least two of the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ groups are each independently selected from the group consisting of hydro xyl, -0-(O-C6 alkyl), and -0-P(0)(OR^x)₂, wherein R^x is independently hydrogen or Ci-Ce alkyl.

As in any embodiment above, a lipid nanoparticle wherein R¹, R², R⁴, R⁷, and R⁸ are independently selected from the group consisting of hydro xyl and -O- P(0)(OH)₂.

As in any embodiment above, a lipid nanoparticle wherein R¹ is hydroxyl.

As in any embodiment above, a lipid nanoparticle wherein R¹ is -0-P(0)(OH)₂. As in any embodiment above, a lipid nanoparticle wherein the compound of Formula I is

As in any embodiment above, a lipid nanoparticle wherein lipid nanoparticle comprises a compound of Formula I which is:

cr I "OH

OH the inner leaflet comprises DOPA; and the outer leaflet comprises a lipid selected from the group consisting of DOTAP, cholesterol, DSPE-PEG, and DSPE-PEG- AA, and mixtures thereof.

As in any embodiment above, a pharmaceutical composition comprising a lipid nanoparticle of any embodiment above and a pharmaceutically acceptable excipient.

In embodiments, a method of preparing a lipid nanoparticle as in any embodiment above, the method comprising:

a. contacting a first reverse emulsion comprising a compound of Formula I with a second reverse emulsion comprising a reagent that is capable of forming a species that can combine with the compound of Formula I to form a precipitated LCP-QP core, wherein at least one of the first and second reverse emulsions further comprises a neutral or anionic lipid and;

b. allowing the precipitated LCP-QP core to form; and

c. contacting the precipitated LCP-QP core from (b) with one or more lipids to prepare a lipid nanoparticle comprising a compound of Formula I.

As in any embodiment above, a method wherein the neutral or anionic lipid is

DOPA.

As in any embodiment above, a method wherein the one or more lipids from step (c) are selected from the group consisting of DOTAP, cholesterol, DSPE-PEG, and DSPE-PEG- AA, and mixtures thereof.

As in any embodiment above, a method of treating a cancer comprising administering a lipid nanoparticle of any embodiment above to a subject. As in any embodiment above, a method wherein the cancer is a carcinoma. As in any embodiment above, a method wherein the carcinoma is bladder carcinoma.

As in any embodiment above, a method further comprising administering an additional bioactive compound.

As in any embodiment above, a method wherein the additional bioactive compound is a cisplatin nanoparticle.

As in any embodiment above, a method of reducing the size of a tumor, comprising contacting the tumor with the lipid nanoparticle of any embodiment above, wherein the tumor is reduced.

As in any embodiment above, a method of remodeling the tumor

microenvironment in a subject, comprising contacting the subject with the lipid nanoparticle of any embodiment above.

As in any embodiment above, a method wherein the Wntl6 expression in the tumor microenvironment is downregulated. In embodiments, the Wntl6 expression is downregulated by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%. In embodiments, the Wntl6 expression is reduced by an amount in a range of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 95%. In an embodiment, the reduction is about 50% when compared to a control.

As in any embodiment above, a method wherein the level of oc-SMA-positive fibrolast is decreased. In embodiments, the decreased level of oc-SMA-positive fibrolasts is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

As in any embodiment above, a method further comprising administering an additional bioactive compound.

As in any embodiment above, a method wherein the additional bioactive compound is a cisplatin nanoparticle.

As in any embodiment above, a method wherein the administering can be simultaneous, sequential or separate.

As in any embodiment above, a method wherein the administration of the lipid nanoparticle of any embodiment above with the cisplatin nanoparticle has a synergistic effect on tumor reduction. In embodiments, a method wherein the administration of the lipid nanoparticle of any embodiment above with the cisplatin nanoparticle results in a greater amount of cisplatin in the tumor cell as compared to administration of the cisplatin nanoparticle without administration of the lipid nanoparticle of any

embodiment above. In embodiments, the increase in amount of cisplatin may be in a range of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 100%, or in an increase of about 100% or more.

As in any embodiment above, a method wherein the lipid nanoparticle further comprises a targeting ligand.

As in any embodiment above, a method wherein the body weight of the subject remains unchanged during treatment. In some embodiments, the body weight of the subject changes in the range from about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, or from about 20% to about 50% when compared to a control.

As in any embodiment above, a method wherein the amount of quercetin in the tumor is increased upon administration of the LCP-QP when compared to

administration of just QP. In some embodiments, the increase in quercetin is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more. In some embodiments, the increase in the amount of quercetin may be in a range of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 100%, or in an increase of about 100% or more.

As in any embodiment above, a method wherein the level of collagen is decreased. In embodiments, the decreased level of collagen is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% when compared to a control. In some embodiments, the reduction in collagen level is in the range from about 5% to about 25%, about 25% to about 50%, or about 50% to about 75% when compared to a control.

As used herein, a "chemotherapeutic agent" is a chemical compound or biologic useful in the treatment of cancer. In embodiments, non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and

cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;

ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin;

podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et ah, Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzino statin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and

deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluoro uracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane;

rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;

mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara- C"); thiotepa; taxoids, e.g. , paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®);

chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin;

vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin. In an embodiment, the chemotherapeutic agent is cisplatin or a cisplatin containing nanoparticle.

As in any embodiment above, a method of reducing the size (i.e., volume or weight) of a tumor, comprising contacting the tumor with a nanoparticle as in any embodiment above, wherein the tumor is reduced. In an embodiment, the reduction can be at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% in tumor size (i.e., volume or weight). In some embodiments, the tumor size (i.e., volume or weight) is reduced in a range of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100% when compared to a control.

Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.

The term "consists essentially of (and grammatical variants), as applied to the compositions of the subject matter described herein, means the composition can contain additional components as long as the additional components do not materially alter the composition.

As used herein, the terms "increase," "increases," "increased," "increasing",

"improve", "enhance", and similar terms indicate an elevation in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more.

As used herein, the terms "reduce," "reduces," "reduced," "reduction",

"inhibit", and similar terms refer to a decrease in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

Combined therapy against both tumor and supporting microenvironment can potentially be more effective due to synergistic enhancement of drug action. Tumor microenvironment plays an important role in angiogenesis, tumor progression, invasion and metastasis. Remodeling the tumor microenvironment may be a powerful strategy to sensitize tumor cells to chemotherapy. Therefore, as set forth herein, a potential therapeutic strategy can be based, in part, on treatment- induced change in the tumor microenvironment. As used herein, the term "modulating the tumor

microenvironment" refers to a change in characteristics of the tumor, including, but not limited to, the Wntl6 expression level, oc-SMA-positive fibroblast level, amount of collagen disposition, or level of nanoparticle penetration.

As used herein, the term "synergistic" includes a synergistic effect of a combination of therapies which permits the use of lower dosages of one or more of the therapies and/or less frequent administration of the therapies to a subject. The ability to utilize lower dosages of a therapy and/or to administer the therapy less frequently reduces the toxicity associated with the administration of the therapy to a subject without reducing the efficacy of the therapy in the prevention or treatment of the disease or disorder to be treated. In addition, a synergistic effect can result in improved efficacy of agents in the treatment of the disease or disorder to be treated. Finally, a synergistic effect of a combination of therapies may avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.

As used herein "enhancing the therapeutic efficacy" means that the combination therapy improves the response as compared to a single therapy alone. The level of enhancement can be measured by any known method and, in embodiments, is a 1, 2, 5, 10, 15, 25, 50, 75% or more improvement.

As used herein, "less toxicity," "reducing toxicity," and similar phrases means less or non-toxic in comparison to alternate treatments for the disease or disorder.

As used herein, "improved efficacy" means efficacy of the lipid nanoparticle that is better in kind or degree in comparison to alternate treatment methods.

/. Particles The presently disclosed lipid nanoparticles comprise a lipid nanoparticle that encapsulates a bioactive compound, such as a flavonol. In other words, the bioactive compound(s) is precipitated and is encapsulated or coated on at least a portion of its surface by a lipid to form the precipitate. Methods of preparing a single lipid bilayer are disclosed in WO2011/017297, herein incorporated by reference in its entirety.

Liposomes are self-assembling, substantially spherical vesicles comprising a lipid bilayer that encircles a core, which can be aqueous, wherein the lipid bilayer comprises amphipathic lipids having hydrophilic headgroups and hydrophobic tails, in which the hydrophilic headgroups of the amphipathic lipid molecules are oriented toward the core or surrounding solution, while the hydrophobic tails orient toward the interior of the bilayer.

The lipid bilayer structure thereby comprises two opposing monolayers that are referred to as the "inner leaflet" and the "outer leaflet," wherein the hydrophobic tails are shielded from contact with the surrounding medium. The "inner leaflet" is the monolayer wherein the hydrophilic head groups are oriented toward the core of the liposome. The "outer leaflet" is the monolayer comprising amphipathic lipids, wherein the hydrophilic head groups are oriented towards the outer surface of the liposome. Liposomes typically have a diameter ranging from about 25 nm to about 1 μιη. (see, e.g., Shah (ed.) (1998) Micelles, Microemulsions, and Monolayers: Science and Technology, Marcel Dekker; Janoff (ed.) (1998) Liposomes: Rational Design, Marcel Dekker). The term "liposome" encompasses both multilamellar liposomes comprised of anywhere from two to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase and unilamellar vesicles that are comprised of a single lipid bilayer. Methods for making liposomes are well known in the art and are described elsewhere herein.

As used herein, the term "lipid" refers to a member of a group of organic compounds that has lipophilic or amphipathic properties, including, but not limited to, fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes), and fatty acids,. The term "lipid" encompasses both naturally occurring and synthetically produced lipids.

"Lipophilic" refers to those organic compounds that dissolve in fats, oils, lipids, and non-polar solvents, such as organic solvents. Lipophilic compounds are sparingly soluble or insoluble in water. Thus, lipophilic compounds are hydrophobic.

Amphipathic lipids, also referred to herein as "amphiphilic lipids" refer to a lipid molecule having both hydrophilic and hydrophobic characteristics. The hydrophobic group of an amphipathic lipid, as described in more detail immediately herein below, can be a long chain hydrocarbon group. The hydrophilic group of an amphipathic lipid can include a charged group, e.g., an anionic or a cationic group, or a polar, uncharged group. Amphipathic lipids can have multiple hydrophobic groups, multiple hydrophilic groups, and combinations thereof. Because of the presence of both a hydrophobic group and a hydrophilic group, amphipathic lipids can be soluble in water, and to some extent, in organic solvents.

As used herein, "hydrophilic" is a physical property of a molecule that is capable of hydrogen bonding with a water (H₂0) molecule and is soluble in water and other polar solvents. The terms "hydrophilic" and "polar" can be used interchangeably. Hydrophilic characteristics derive from the presence of polar or charged groups, such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.

Conversely, the term "hydrophobic" is a physical property of a molecule that is repelled from a mass of water and can be referred to as "nonpolar," or "apolar," all of which are terms that can be used interchangeably with "hydrophobic." Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids. Representative examples of

phospholipids include, but are not limited to, phosphatidylcholine,

phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,

lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,

dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidic acid, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and β-acyloxyacids, also are within the group designated as amphipathic lipids.

In some embodiments, the liposome or lipid bilayer comprises cationic lipids. As used herein, the term "cationic lipid" encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. Such lipids include, but are not limited to, the cationic lipids of formula (I) disclosed in International Application No.

PCT/US2009/042476, entitled "Methods and Compositions Comprising Novel Cationic Lipids," which was filed on May 1, 2009, and is herein incorporated by reference in its entirety. These include, but are not limited to, N-methyl-N-(2-(arginoylamino) ethyl)- N, N- Di octadecyl aminium chloride or di stearoyl arginyl ammonium chloride]

(DSAA), N,N-di-myristoyl-N-methyl-N-2[N'-(N⁶-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N-2[N²-guanidino-L- lysinyl] aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2[N'-(N2, N6- di-guanidino-L-lysinyl)] aminoethyl ammonium chloride, and

N,N-di-stearoyl-N-methyl-N-2[N'-(N6-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DSGLA). Other non-limiting examples of cationic lipids that can be present in the liposome or lipid bilayer of the presently disclosed lipid nanoparticles include N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3- dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); N-(2,3- dioleyloxy) propyl)-N,N,N-trimethylammonium chloride ("DOTMA") or other N- (N,N-l-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; N,N-distearyl- Ν,Ν-dimethylammonium bromide ("DDAB"); 3-(N-(N',N'-dimethylaminoethane)- carbamoyl) cholesterol ("DC-Choi") and N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"); l,3-dioleoyl-3- trimethylammonium-propane, N-(l-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethy- 1 ammonium trifluoro-acetate (DOSPA); GAP-DLRIE; DMDHP; 3-p[⁴N-(H⁸N-diguanidino spermidine)-carbamoyl] cholesterol (BGSC); 3-P[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N\N²,N³ Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N'- tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin) ; dimethyldioctadecyl ammonium bromide (DDAB); l,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)- 1-methyl- lH-imidazole (DPIM)

N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3 dioleoyloxy- 1 ,4- butanediammonium iodide) (Tfx-50); 1,2 dioleoyl-3-(4'-trimethylammonio) butanol-sn- glycerol (DOBT) or cholesteryl (4'trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DL-l,2-dioleoyl-3- dimethylaminopropyl-P-hydroxyethylammonium (DORI) or DL-l,2-0-dioleoyl-3- dimethylaminopropyl-P-hydroxyethylammonium (DORIE) or analogs thereof as disclosed in International Application Publication No. WO 93/03709, which is herein incorporated by reference in its entirety; l,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl

phosphatidylethanolamylspermine (DPPES) or the cationic lipids disclosed in U.S. Pat. No. 5,283,185, which is herein incorporated by reference in its entirety; cholesteryl-3P- carboxyl-amido-ethylenetrimethylammonium iodide; l-dimethylamino-3- trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide; cholesteryl-3-β- carboxyamidoethyleneamine; cholesteryl-3-P-oxysuccinamido- ethylenetrimethylammonium iodide; l-dimethylamino-3-trimethylammonio-DL-2- propyl-cholesteryl-3-P-oxysuccinate iodide; 2-(2-trimethylammonio)- ethylmethylamino ethyl-cholesteryl-3-P-oxysuccinate iodide; and 3-β-Ν- (polyethyleneimine)-carbamoylcholesterol.

In some embodiments, the liposomes or lipid bilayers can contain co-lipids that are negatively charged or neutral. As used herein, a "co-lipid" refers to a non-cationic lipid, which includes neutral (uncharged) or anionic lipids. The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. The term "anionic lipid" encompasses any of a number of lipid species that carry a net negative charge at physiological pH. Co-lipids can include, but are not limited to, diacylphosphatidylcholine,

diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols, phospholipid-related materials, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,

phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid,

dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleyolphosphatidylglycerol (POPG),

dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylchol- ine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dioleoyl phosphatidic acid (DOPA), stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,

glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, lysophosphatidylcholine, and dioctadecyldimethyl ammonium bromide and the like. Co-lipids also include polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to phospholipids or to ceramides, as described in U.S. Pat. No. 5,820,873, herein incorporated by reference in its entirety.

In some embodiments, the liposome of the lipid nanoparticle is a cationic liposome and in other embodiments, the liposome is anionic. The term "cationic liposome" as used herein is intended to encompass any liposome as defined above which has a net positive charge or has a zeta potential of greater than 0 mV at physiological pH. Alternatively, the term "anionic liposome" refers to a liposome as defined above which has a net negative charge or a zeta potential of less than 0 mV at physiological pH. The zeta potential or charge of the liposome can be measured using any method known to one of skill in the art. It should be noted that the liposome itself is the entity that is being determined as cationic or anionic, meaning that the liposome that has a measurable positive charge or negative charge at physiological pH, respectively, can, within an in vivo environment, become attached to other substances or may be associated with other charged components within the aqueous core of the liposome, which can thereby result in the formation of a structure that does not have a net charge. After a lipid nanoparticle comprising a cationic or anionic liposome is produced, molecules such as lipid-PEG conjugates can be post-inserted into the bilayer of the liposome as described elsewhere herein, thus shielding the surface charge of the lipid nanoparticle.

In those embodiments in which the liposome of the lipid nanoparticle is a cationic liposome, the cationic liposome need not be comprised completely of cationic lipids, however, but must be comprised of a sufficient amount of cationic lipids such that the liposome has a positive charge at physiological pH. The cationic liposomes also can contain co-lipids that are negatively charged or neutral, so long as the net charge of the liposome is positive and/or the surface of the liposome is positively charged at physiological pH. In these embodiments, the ratio of cationic lipids to co- lipids is such that the overall charge of the resulting liposome is positive at

physiological pH. For example, cationic lipids are present in the cationic liposome at from about 10 mole % to about 100 mole % of total liposomal lipid, in some embodiments, from about 20 mole % to about 80 mole % and, in other embodiments, from about 20 mole % to about 60 mole %. Neutral lipids, when included in the cationic liposome, can be present at a concentration of from about 0 mole % to about 90 mole % of the total liposomal lipid, in some embodiments from about 20 mole % to about 80 mole %, and in other embodiments, from about 40 mole % to about 80 mole %. Anionic lipids, when included in the cationic liposome, can be present at a concentration ranging from about 0 mole % to about 49 mole % of the total liposomal lipid, and in certain embodiments, from about 0 mole % to about 40 mole %.

Likewise, in those embodiments in which the liposome of the lipid nanoparticle is an anionic liposome, the anionic liposome need not be comprised completely of anionic lipids, however, but must be comprised of a sufficient amount of anionic lipids such that the liposome has a negative charge at physiological pH. The anionic liposomes also can contain neutral co-lipids or cationic lipids, so long as the net charge of the liposome is negative and/or the surface of the liposome is negatively charged at physiological pH. In these embodiments, the ratio of anionic lipids to neutral co-lipids or cationic lipids is such that the overall charge of the resulting liposome is negative at physiological pH. For example, the anionic lipid is present in the anionic liposome at from about 10 mole % to about 100 mole % of total liposomal lipid, in some embodiments, from about 20 mole % to about 80 mole % and, in other embodiments, from about 20 mole % to about 60 mole %. The neutral lipid, when included in the anionic liposome, can be present at a concentration of from about 0 mole % to about 90 mole % of the total liposomal lipid, in some embodiments from about 20 mole % to about 80 mole %, and in other embodiments, from about 40 mole % to about 80 mole %. The positively charged lipid, when included in the anionic liposome, can be present at a concentration ranging from about 0 mole % to about 49 mole % of the total liposomal lipid, and in certain embodiments, from about 0 mole % to about 40 mole %.

In some embodiments in which the lipid vehicle is a cationic liposome or an anionic liposome, the lipid nanoparticle as a whole has a net positive charge. By "net positive charge" is meant that the positive charges of the components of the lipid nanoparticle (e.g., cationic lipid of liposome, cation of precipitate, cationic bioactive compound) exceed the negative charges of the components of the lipid nanoparticle (e.g., anionic lipid of liposome, anion of precipitate, anionic bioactive compound). It is to be understood, however, that the presently disclosed subject matter also encompasses lipid nanoparticles having a positively charged surface irrespective of whether the net charge of the complex is positive, neutral or even negative. The charge of the surface of a lipid nanoparticle can be measured by the migration of the complex in an electric field by methods known to those in the art, such as by measuring zeta potential (Martin, Swarick, and Cammarata (1983) Physical Pharmacy & Physical Chemical Principles in the Pharmaceutical Sciences, 3rd ed. Lea and Febiger) or by the binding affinity of the lipid nanoparticle to cell surfaces. Complexes exhibiting a positively charged surface have a greater binding affinity to cell surfaces than complexes having a neutral or negatively charged surface. Further, it is to be understood that the positively charged surface can be sterically shielded by the addition of non-ionic polar compounds, for example, polyethylene glycol, as described elsewhere herein.

In particular non- limiting embodiments, the lipid nanoparticle has a charge ratio of positive to negative charge (+ : -) of between about 0.5: 1 and about 100: 1, including but not limited to about 0.5: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 15: 1, about 20: 1, about 40: 1, or about 100: 1. In a specific non- limiting embodiment, the + : - charge ratio is about 1: 1.

The presently disclosed lipid nanoparticles comprise liposomes that

encapsulate, or coat at least a portion of, a bioactive compound.

While not being bound by any particular theory or mechanism of action, it is believed the presently disclosed lipid nanoparticles enter cells through endocytosis and are found in endosomes, which exhibit a relatively low pH (e.g., pH 5.0). Thus, in some embodiments, the bioactive compound is released at endosomal pH. In certain embodiments, the pH level is less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, or less than about 4.0, including but not limited to, about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6, about 5.5, about 5.4, about 5.3, about 5.2, about 5.1, about 5.0, about 4.9, about 4.8, about 4.7, about 4.6, about 4.5, about 4.4, about 4.3, about 4.2, about 4.1, about 4.0, or less.

The lipid nanoparticles can be of any size, so long as the complex is capable of delivering the incorporated bioactive compound to a cell (e.g., in vitro, in vivo), physiological site, or tissue. In some embodiments, the lipid nanoparticle comprises a liposome encapsulating the bioactive compound. As used herein, the term

"nanoparticle" refers to particles of any shape having at least one dimension that is less than about 1000 nm. In some embodiments, nanoparticles have at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, and 1000). In certain embodiments, the nanoparticles have at least one dimension that is about 150 nm. Particle size can be determined using any method known in the art, including, but not limited to, sedimentation field flow fractionation, photon correlation spectroscopy, disk centrifugation, and dynamic light scattering (using, for example, a submicron particle sizer such as the NICOMP particle sizing system from

AutodilutePAT Model 370; Santa Barbara, CA).

//. Flavonoid Compounds

Flavonoids are polyphenolic compounds that occur in foods of plant origin and are well known for their antioxidant capacities. Major dietary sources of flavonoids are vegetables, fruits, and beverages such as tea and red wine. Among the dietary flavonoids, quercetin-glycosides are amongst the most abundant. Flavonoids in general have been reported to confer a number of health benefits and are believed to act by intervention in various metabolic pathways such as by inhibition of 5-cyclooxygenase. Included within the general term flavonoid are flavonols, flavones, flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols and stilbenes, which are characterized by variations in core structures. One of the flavonoids, known as flavonols, is distinguished by the core structure pictured below:

Flavonols represent a large class of molecules all based on the core structure above and natural variation is achieved by attachment of other molecular entities e.g. hydroxyls, sugar, methyl groups etc, at different positions of the flavonol core-ring structure. Glycosylated forms are very abundantly found in nature, although the un- glycosylated form (aglycon) can occur as well. The main types of flavonols found in plants are based on quercetin, kaempferol and myrecetin. The structures of kaempferol,

Quercetin Kaempferol Myrecetin

Additional flavonols include, but are not limited to 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, isorhamnetin, morin, natsudaidain, pachpodol, rhamnazin, and rhamnetin.

Quercetin is a natural protective bioflavonoid which possesses many diverse pharmacologic activities including antioxidant, anti- inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenic activities. Quercetin has been shown to trigger multiple signal transduction pathways involving MEK/ERK, β-catenin, STAT3, EGFR/PI3K/Akt/mTOR and Nrf2/keapl, which are associated with inflammation and carcinogenesis. Downregulation of Wnt/ β-catenin can be induced by quercetin in various types of cells, such as 4T1 mammary cancer cells and SW480 colon cancer cells. Although quercetin has been reported to be a potent β-catenin inhibitor, its effect on the production of Wntl6 has not been previously observed. As disclosed herein, in some embodiments, quercetin downregulates Wntl6 expression in addition to reducing the ability of tumor cells to gain resistance and restructure the stroma. In addition to the effect on Wntl6, quercetin also has a modulating effect on multiple other pathways, such as mTOR, which may also contribute to the antitumor effect of LCP-QP.

Quercetin has also shown the ability to reverse the oxidative stress environment, decreasing inflammation, as well as inducing rearrangement of extracellular matrix (ECM) in aortic fibroblast disorders.

However, quercetin' s poor water solubility and bioavailability have limited its use as a pharmaceutical. Due to the poor physiochemical properties of quercetin, much effort has been put forth to develop nano formulations in an effort to increase the bioavailability of this phytochemical. Polymeric nanocapsules, nanomicelles, liposomes, nanodiamondsas well as various other nanoformulations have been explored to increase the bioavailability, protective, or anticancer properties of quercetin.

In contrast to previously reported methods, as disclosed herein, to improve the bioavailability and stability of quercetin, quercetin phosphate (QP) was synthesized and precipitated with calcium to be entrapped into the targeted LCP nanoparticles. The prepared LCP-QP protects the QP from degradation and facilitates increased

accumulation at the tumor site through the tumor's enhanced permeability and retention (EPR) effect.

///. Nanoparticle Preparation

Methods for preparing lipid nanoparticles (i.e., liposomes) are known in the art. For example, a review of methodologies of liposome preparation may be found in Liposome Technology (CFC Press NY 1984); Liposomes by Ostro (Marcel Dekker,

1987); Lichtenberg and Barenholz (1988) Methods Biochem Anal. 33:337-462 and U.S. Pat. No. 5,283,185, each of which is herein incorporated by reference in its entirety. For example, cationic lipids and optionally co-lipids can be emulsified by the use of a homogenizer, lyophilized, and melted to obtain multilamellar liposomes. Alternatively, unilamellar liposomes can be produced by the reverse phase evaporation method

(Szoka and Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198, which is herein incorporated by reference in its entirety). In some embodiments, the liposomes are produced using thin film hydration (Bangham et al. (1965) J. Mol. Biol. 13:238- 252, which is herein incorporated by reference in its entirety). In certain embodiments, the liposome formulation can be briefly sonicated and incubated at 50°C for a short period of time (e.g., about 10 minutes) prior to sizing (see Templeton et al. (1997) Nature Biotechnology 15:647-652, which is herein incorporated by reference in its entirety).

In some embodiments, the prepared liposome can be sized wherein the liposomes are selected from a population of liposomes based on the size (e.g., diameter) of the liposomes. The liposomes can be sized using techniques such as ultrasonication, high-speed homogenization, and pressure filtration (Hope et al. (1985) Biochimica et Biophysica Acta 812:55; U.S. Pat. Nos. 4,529,561 and 4,737,323, each of which is herein incorporated by reference in its entirety). Sonicating a liposome either by bath or probe sonication produces a progressive size reduction down to small vesicles less than about 0.05 microns in size. Vesicles can be recirculated through a standard emulsion homogenizer to the desired size, typically between about 0.1 microns and about 0.5 microns. The size of the liposomes can be determined by quasi-elastic light scattering (QELS) (Bloomfield (1981) Ann. Rev. Biophys. Bioeng. 10:421-450). The average diameter can be reduced by sonication of the liposomes. Intermittent sonication cycles can be alternated with QELS assessment to guide efficient liposome synthesis. Alternatively, liposomes can be extruded through a small-pore

polycarbonate membrane or an asymmetric ceramic membrane to yield a well-defined size distribution. Typically, a suspension is cycled through the membrane one or more times until the desired size distribution is achieved. The complexes can be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size. In particular embodiments, the liposomes are extruded through a membrane having a pore size of about 100 nm.

An emulsion is a dispersion of one liquid in a second immiscible liquid. The term "immiscible" when referring to two liquids refers to the inability of these liquids to be mixed or blended into a homogeneous solution. Two immiscible liquids when added together will always form two separate phases. The organic solvent used in the presently disclosed methods is essentially immiscible with water. Emulsions are essentially swollen micelles, although not all micellar solutions can be swollen to form an emulsion. Micelles are colloidal aggregates of amphipathic molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the hydrophobic portions of the lipid molecules at the interior of the micelle and the hydrophilic portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) has a range from about 50 to about 100. The term "micelles" also refers to inverse or reverse micelles, which are formed in an organic solvent, wherein the hydrophobic portions are at the exterior surface, exposed to the organic solvent and the hydrophilic portion is oriented towards the interior of the micelle.

An oil-in-water (O/W) emulsion consists of droplets of an organic compound (e.g., oil) dispersed in water and a water-in-oil (W/O) emulsion is one in which the phases are reversed and is comprised of droplets of water dispersed in an organic compound (e.g., oil). A water-in-oil emulsion is also referred to herein as a reverse emulsion. Thermodynamically stable emulsions are those that comprise a surfactant (e.g, an amphipathic molecule) and are formed spontaneously. The term "emulsion" can refer to microemulsions or macroemulsions, depending on the size of the particles. Droplet diameters in microemulsions typically range from about 10 to about 100 nm. In contrast, the term macroemulsions refers to droplets having diameters greater than about 100 nm.

It will be evident to one of skill in the art that sufficient amounts of the aqueous solutions, organic solvent, and surfactants are added to the reaction solution to form the water-in-oil emulsion.

Surfactants are added to the reaction solution in order to facilitate the development of and stabilize the water-in-oil microemulsion. Surfactants are molecules that can reduce the surface tension of a liquid. Surfactants have both hydrophilic and hydrophobic properties, and thus, can be solubilized to some extent in either water or organic solvents. Surfactants are classified into four primary groups: cationic, anionic, non-ionic, and zwitterionic. Preferably, the surfactants are non-ionic surfactants. Non-ionic surfactants are those surfactants that have no charge when dissolved or dispersed in aqueous solutions. Thus, the hydrophilic moieties of non- ionic surfactants are uncharged, polar groups. Representative non-limiting examples of non-ionic surfactants suitable for use for the presently disclosed methods and compositions include polyethylene glycol, polysorbates, including but not limited to, polyethoxylated sorbitan fatty acid esters (e.g., Tween® compounds) and sorbitan derivatives (e,g., Span® compounds); ethylene oxide/propylene oxide copolymers (e.g., Pluronic® compounds, which are also known as poloxamers); polyoxy ethylene ether compounds, such as those of the Brij® family, including but not limited to polyoxyethylene stearyl ether (also known as polyoxyethylene (100) stearyl ether and by the trade name Brij® 700); ethers of fatty alcohols. In particular embodiments, the non- ionic surfactant comprises octyl phenol ethoxylate (i.e., Triton X-100), which is commercially available from multiple suppliers (e.g., Sigma- Aldrich, St. Louis, MO).

Polyethoxylated sorbitan fatty acid esters (polysorbates) are commercially available from multiple suppliers (e.g., Sigma- Aldrich, St Louis, MO) under the trade name Tween®, and include, but are not limited to, polyoxyethylene (POE) sorbitan monooleate (Tween® 80), POE sorbitan monostearate (Tween® 60), POE sorbitan monolaurate (Tween® 20), and POE sorbitan monopalmitate (Tween® 40).

Ethylene oxide/propylene oxide copolymers include the block copolymers known as poloxamers, which are also known by the trade name Pluronic® and can be purchased from BASF Corporation (Florham Park, New Jersey). Poloxamers are composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) and are represented by the following chemical structure: HO(C2H40)_a(C3H60)b(C2H40)_aH; wherein the C2H4O subunits are ethylene oxide monomers and the C3H6O subunits are propylene oxide monomers, and wherein a and b can be any integer ranging from 20 to 150.

Organic solvents that can be used in the presently disclosed methods include those that are immiscible or essentially immiscible with water. Non-limiting examples of organic solvents that can be used in the presently disclosed methods include chloroform, methanol, ether, ethyl acetate, hexanol, cyclohexane, and dichloromethane. In particular embodiments, the organic solvent is nonpolar or essentially nonpolar.

In some embodiments, mixtures of more than one organic solvent can be used in the presently disclosed methods. In some of these embodiments, the organic solvent comprises a mixture of cyclohexane and hexanol. In particular embodiments, the organic solvent comprises cyclohexane and hexanol at a volume/volume ratio of about 7.5: 1.7. As noted elsewhere herein, the non- ionic surfactant can be added to the reaction solution (comprising aqueous solutions of cation, anion, bioactive compound, and organic solvent) separately, or it can first be mixed with the organic solvent and the organic solvent/surfactant mixture can be added to the aqueous solutions of the anion, cation, and bioactive compound. In some of these embodiments, a mixture of cyclohexane, hexanol, and Triton X-100 is added to the reaction solution. In particular embodiments, the volume/volume/volume ratio of the cyclohexane:hexanol:Triton X- 100 of the mixture that is added to the reaction solution is about 7.5: 1.7: 1.8.

It should be noted that the volume/volume ratio of the nonionic surfactant to the organic solvent regulates the size of the water-in-oil microemulsion and therefore, the bioactive compound contained therein and the resultant lipid nanoparticle, with a greater surfactant:organic solvent ratio resulting in lipid nanoparticles with larger diameters and smaller surfactant:organic solvent ratios resulting in lipid nanoparticles with smaller diameters.

The reaction solution may be mixed to form the water-in-oil microemulsion and the solution may also be incubated for a period of time. This incubation step can be performed at room temperature. In some embodiments, the reaction solution is mixed at room temperature for a period of time of between about 5 minutes and about 60 minutes, including but not limited to about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, and about 60 minutes. In particular embodiments, the reaction solution is mixed at room temperature for about 15 minutes.

In order to complex a bioactive compound with a liposome, the bioactive compound may be a precipitate. In other embodiments, the precipitate may be a nano- precipitate. Further, the surface of the precipitated bioactive compound can be charged, either positively or negatively. In some embodiments, the precipitate will have a charged surface following its formation. Those precipitates with positively charged surfaces can be mixed with anionic liposomes, whereas those precipitates with negatively charged surfaces can be mixed with cationic liposomes.

In certain embodiments, the surface charge of the nano-precipitate can be enhanced or reversed using any method known in the art. For example, a nano- precipitate having a positively charged surface can be modified to create a negatively charged surface. Alternatively, a nano-precipitate having a negatively charged surface can be modified to create a positively charged surface.

In those embodiments wherein a nano-precipitate is created having a positive surface charge, the surface charge can be made negative through the addition of sodium citrate to the water-in-oil microemulsion. In some embodiments, sodium citrate is added at a concentration of about 15 mM to the microemulsion. In some of these embodiments, the total volume of the 15 mM sodium citrate added to the microemulsion is about 125 ul. Sodium citrate is especially useful for imparting a negative surface charge to the nano-precipitates because it is non-toxic.

In some embodiments, the precipitate has or is modified to have a zeta potential of less than -10 mV and in certain embodiments, the zeta potential is between about -14 mV and about -20 mV, including but not limited to about -14 mV, about -15 mV, about -16 mV, about -17 mV, about -18 mV, about -19 mV, and about -20 mV.

In those embodiments wherein the nano-precipitate has a negatively charged surface, a cationic liposome is complexed with the nano-precipitate. The ratio of the cationic liposome to the nano-precipitate, and/or the bioactive compound can regulate the size and charge of the resultant lipid nanoparticle. In preferred embodiments, the zeta potential of a nanoparticle comprising a liposome is different than the zeta potential of a pure liposome containing the pure lipid, whether the zeta potential is a positive or negative value.

In embodiments, the lipid nanoparticles comprise asymmetric bilayers. As used herein, the term "asymmetric bilayer" or "asymmetric" refers to the distinct lipid composition that makes up the inner leaflet as compared to the lipid composition that makes up the outer leaflet. That is, within an asymmetric bilayer, the lipid composition of the inner and outer leaflets is not the same. As discussed further herein, the inner leaflet may be enriched in one type of lipid while the outer leaflet is enriched in another, different type of lipid. In some embodiments, the asymmetric lipid membrane can shield the charges that would be present on a pure liposome. In some embodiments the asymmetric bilayer has the inner leaflet of the bilayer enriched with the negatively charged lipid DOPA, but the outer leaflet is enriched with a different lipid. While not being bound by any theory or mechanism of action, it is believed that this energetically unfavorable state is stabilized because DOPA is ionically bonded to the surface of the CaP precipitate shell.

Following the production of the emulsion, nano-precipitated bioactive having a lipid coating is purified from the non- ionic surfactant and organic solvent. The nano- precipitate can be purified using any method known in the art, including but not limited to gel filtration chromatography. A nano-precipitate that has been purified from the non-ionic surfactants and organic solvent is a nano-precipitate that is essentially free of non-ionic surfactants or organic solvents (e.g, the nano-precipitate comprises less than 10%, less than 1%, less than 0.1% by weight of the non-ionic surfactant or organic solvent). In some of those embodiments wherein gel filtration is used to purify the nano-precipitate, the precipitate is adsorbed to a silica gel or to a similar type of a stationary phase, the silica gel or similar stationary phase is washed with a polar organic solvent (e.g., ethanol, methanol, acetone, DMSO, DMF) to remove the non- ionic surfactant and organic solvent, and the nano-precipitate is eluted from the silica gel or other solid surface with an aqueous solution comprising a polar organic solvent.

In some of these embodiments, the silica gel is washed with ethanol and the nano-precipitate is eluted with a mixture of water and ethanol. In particular

embodiments, the nano-precipitate is eluted with a mixture of water and ethanol, wherein the mixture comprises a volume/volume ratio of between about 1:9 and about 1: 1, including but not limited to, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, and about 1: 1. In particular embodiments, the volume/volume ratio of water to ethanol is about 1:3. In some of these embodiments, a mixture comprising 25 ml water and 75 ml ethanol is used for the elution step.

Following removal of the ethanol using, for example, rotary evaporation, the nano- precipitate can be dispersed in an aqueous solution (e.g., water) prior to mixing with the prepared liposomes.

In certain embodiments, the methods of making the lipid nanoparticles can further comprise an additional purification step following the production of the lipid nanoparticles, wherein the lipid nanoparticles are purified from excess free liposomes and unencapsulated nano-precipitates. Purification can be accomplished through any method known in the art, including, but not limited to, centrifugation through a sucrose density gradient or other media which is suitable to form a density gradient. It is understood, however, that other methods of purification such as chromatography, filtration, phase partition, precipitation or absorption can also be utilized. In one method, purification via centrifugation through a sucrose density gradient is utilized. The sucrose gradient can range from about 0% sucrose to about 60% sucrose or from about 5% sucrose to about 30% sucrose. The buffer in which the sucrose gradient is made can be any aqueous buffer suitable for storage of the fraction containing the complexes and in some embodiments, a buffer suitable for administration of the complex to cells and tissues.

In some embodiments, a targeted delivery system or a PEGylated delivery system is made as described elsewhere herein, wherein the methods further comprise a post-insertion step following the preparation of the liposome or following the production of the lipid nanoparticle, wherein a lipid-targeting ligand conjugate or a PEGylated lipid is post-inserted into the liposome. Liposomes or lipid nanoparticles comprising a lipid-targeting ligand conjugate or a lipid-PEG conjugate can be prepared following techniques known in the art, including but not limited to those presented herein (see Experimental section; Ishida et al. (1999) FEBS Lett. 460: 129-133;

Perouzel et al. (2003) Bioconjug. Chem. 14:884-898, which is herein incorporated by reference in its entirety). The post-insertion step can comprise mixing the liposomes or the lipid nanoparticles with the lipid-targeting ligand conjugate or a lipid-PEG conjugate and incubating the particles at about 50°C to about 60°C for a brief period of time (e.g., about 5 minutes, about 10 minutes). In some embodiments, the lipid nanoparticles or liposomes are incubated with a lipid-PEG conjugate or a lipid-PEG- targeting ligand conjugate at a concentration of about 5 to about 20 mol%, including but not limited to about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, and about 20 mol%, to form a stealth delivery system. In some of these embodiments, the concentration of the lipid-PEG conjugate is about 10 mol%. The polyethylene glycol moiety of the lipid-PEG conjugate can have a molecular weight ranging from about 100 to about 20,000 g/mol, including but not limited to about 100 g/mol, about 200 g/mol, about 300 g/mol, about 400 g/mol, about 500 g/mol, about 600 g/mol, about 700 g/mol, about 800 g/mol, about 900 g/mol, about 1000 g/mol, about 5000 g/mol, about 10,000 g/mol, about 15,000 g/mol, and about 20,000 g/mol. In certain embodiments, the lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In some embodiments, the lipid-PEG conjugate comprises DSPE-PEG2000. Lipid-PEG-targeting ligand conjugates can also be post- inserted into liposomes or lipid nanoparticles using the above described post-insertion methods.

The lipid nanoparticle comprising a nano-precipitated bioactive compound surrounded by a lipid bilayer comprising an inner and an outer leaflet can have a diameter of less than about 100 nm, including but not limited to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nm. In particular embodiments, the lipid nanoparticle has a diameter of about 25 to about 30 nm. The lipid bilayer surrounding the nano-precipitated bioactive compound has an inner and an outer leaflet. In some embodiments, the inner leaflet comprises an amphiphilic lipid having a free phosphate group. In embodiments, the amphiphilic lipid having a free phosphate group is dioleoyl phosphatidic acid (DOPA).

The outer leaflet of the lipid bilayer can comprise any type of lipid, but in some embodiments, it comprises a cationic lipid. In particular embodiments, the cationic lipid is DOTAP.

Useful neutral, anionic and cationic lipids include those listed elsewhere herein. In some embodiments, the neutral or anionic lipid is DOPA. Useful one or more lipids include co-lipids and cationic lipids listed elsewhere herein. In some embodiments, the one or more lipids are selected from the group consisting of DOTAP, cholesterol, DSPE-PEG and DSPE-PEG-AA.

Useful precursors are bioactive compounds that can be combined with an ion species to form a nano-precipitate in salt form. Such useful bioactive compounds are listed elsewhere herein. Precursors can combine with a cation, such as In⁺³, Gd⁺³, Mg⁺², Zn⁺² and Ba⁺² or an anion, such as a halide, to form a nano-precipitate in situ, i.e., during mixing of the reverse micro-emulsions.

The methods for preparing the lipid nanoparticles can include purifying and washing steps as disclosed herein. These steps employ solvents, washes and purification procedures described herein. In particular, the method further comprises a washing and/or purifying step after (b) and before (c). Generally, the methods can comprise mixing a first reverse microemulsion and a second reverse microemulsion to form a salt of a bioactive compound that itself is a nano-precipitate having a lipid coating, this nano-precipitate will have an outer leaflet lipid layer added in subsequent steps to form a nano-precipitate having a lipid bi- layer coat; washing the nano- precipitate; mixing the nano-precipitate in a volatile, organic solvent to form a nano- precipitate/solvent mixture; adding a lipid to the nano-precipitate/solvent mixture; and evaporating the volatile, organic solvent to produce the lipid nanoparticle.

In some embodiments, the first reverse microemulsion has the same or different pH as the second reverse microemulsion.

The method can further comprise producing the first reverse microemulsion, which can include providing a solution comprising a bioactive compound or a precursor thereof, and mixing the solution with a non-ionic surfactant and an organic solvent. In particular, the first microemulsion can contain triton X-100, IGEPAL 520, which are both well-known in the art, and hexanol as co-surfactants in an organic solvent.

In some embodiments, the organic solvent is hexanol and/or cyclohexane. In particular embodiments, the organic solvent comprises cyclohexane and hexanol at a volume-to-volume ratio of about 78: 11.

The non-ionic surfactant can be any non-ionic surfactant, including those non- limiting examples provided elsewhere herein, but in certain embodiments, the non-ionic surfactant is Triton-X 100. In particular embodiments, the aqueous solution comprising calcium chloride is mixed with a solution of cyclohexane, hexanol, and Triton-X 100 at a volume/volume/volume ratio of about 78: 11: 11.

The method can further comprise providing a second reverse emulsion that contains the species that will combine with the bioactive compound or precursor of a bioactive compound to form a nano-precipitated bioactive compound. The species can be a cation or anion. In embodiments, the cation is a monovalent, divalent or a trivalent cation. The cations that used to form the salt nano-precipitates can be radioactive isotopes which will allow imaging of the lesion. An example is ^mIn which can be imaged by SPECT. Gd⁺³ can also be used as an MRI agent. Thus, the resulting liposomes will carry both a therapeutic and an imaging agent for theranostic

nanomedicines. In embodiments, the anion is a monovalent, divalent or a trivalent anion. In particular embodiments, the anion is a halide anion (fluoride (F^"), chloride (CI-), bromide (Br) and iodide (Γ)).

The second reverse microemulsion will comprise the ion species (by way of adding its precursor such as a halide salt) and a neutral and/or anionic lipid. Preferably, the lipid is DOPA. The second reverse emulsion will be an emulsion that can further comprise a non-ionic surfactant, and an organic solvent.

Again, the organic solvent can comprise hexanol and/or cyclohexane. In particular embodiments, the organic solvent comprises cyclohexane and hexanol at a volume-to-volume ratio of about 78: 11.

Likewise, the non- ionic surfactant used to produce the second reverse microemulsion can be any non-ionic surfactant, including those non-limiting examples provided elsewhere herein, but in certain embodiments, the non-ionic surfactant is Triton-X 100. In particular embodiments, the aqueous solution comprising sodium phosphate and the anionic lipid is mixed with a solution of cyclohexane, hexanol, and Triton-X 100 at a volume/volume/volume ratio of about 78: 11: 11. The volatile, organic solvent within which the nano-precipitate is mixed can be ethanol or chloroform. In some embodiments, the nano-precipitate is washed with ethanol, and the washing step can be performed about 1-5 times, including 1, 2, 3, 4, and 5.

The monolayer lipid nano-precipitate can be encapsulated with an outer leaflet comprising one or more of cholesterol, a cationic lipid such as DOTAP or a neutral lipid, such as dioleoyl phosphatidylcholine by combining one or more to the mixture containing the monolayer lipid nano-precipitate. In some embodiments, the outer leaflet comprises a lipid-polyethylene glycol (lipid-PEG) conjugate, a lipid-targeting ligand conjugate, or a combination thereof. In certain embodiments, a mixture of neutral lipids (e.g., DOPC) and a lipid-PEG conjugate, a lipid-targeting ligand conjugate, or a combination thereof is at a molar ratio of 10 neutral lipid (e.g., DOPC) to 1 lipid-PEG conjugate, lipid targeting ligand conjugate, or combination thereof (e.g., DSPE-PEG-AA). Alternatively, the lipid-PEG conjugate, lipid targeting ligand conjugate, or a combination thereof can be added to the outer leaflet of the lipid bilayer through post-insertion described elsewhere herein.

IV. PEGylated Delivery Systems and Targeted Delivery Systems

As described elsewhere herein, the lipid nanoparticles can have a surface charge (e.g., positive charge). In some embodiments, the surface charge of the liposome of the delivery system can be minimized by incorporating lipids comprising polyethylene glycol (PEG) moieties into the liposome. Reducing the surface charge of the liposome of the delivery system can reduce the amount of aggregation between the lipid nanoparticles and serum proteins and enhance the circulatory half- life of the complex (Yan, Scherphof, and Kamps (2005) J Liposome Res 15: 109-139). Thus, in some embodiments, the exterior surface of the liposome or the outer leaflet of the lipid bilayer of the delivery system comprises a PEG molecule. Such a complex is referred to herein as a PEGylated lipid nanoparticle. In these embodiments, the outer leaflet of the lipid bilayer of the liposome of the lipid nanoparticle comprises a lipid-PEG conjugate.

A PEGylated lipid nanoparticle can be generated through the post-insertion of a lipid-PEG conjugate into the lipid bilayer through the incubation of the lipid nanoparticle with micelles comprising lipid-PEG conjugates, as known in the art and described elsewhere herein (Ishida et al. (1999) FEBS Lett. 460: 129-133; Perouzel et al. (2003) Bioconjug. Chem. 14:884-898; see Experimental section). By "lipid- polyethylene glycol conjugate" or "lipid-PEG conjugate" is intended a lipid molecule that is covalently bound to at least one polyethylene glycol molecule. In some embodiments, the lipid-PEG conjugate comprises l,2-distearoyl-5n-glycero-3- phosphoethanolamine-N-carboxy-polyethylene glycol (DSPE-PEG). As described immediately below, these lipid-PEG conjugates can be further modified to include a targeting ligand, forming a lipid-PEG-targeting ligand conjugate (e.g., DSPE-PEG- AA). The term "lipid-PEG conjugate" also refers to these lipid-PEG-targeting ligand conjugates and a lipid nanoparticle comprising a liposome comprising a lipid-PEG targeting ligand conjugate are considered to be both a PEGylated lipid nanoparticle and a targeted lipid nanoparticle, as described immediately below.

Alternatively, the lipid nanoparticle can be PEGylated through the addition of a lipid-PEG conjugate during the formation of the outer leaflet of the lipid bilayer.

PEGylation of liposomes enhances the circulatory half-life of the liposome by reducing clearance of the complex by the reticuloendothelial (RES) system. While not being bound by any particular theory or mechanism of action, it is believed that a PEGylated lipid nanoparticle can evade the RES system by sterically blocking the opsonization of the complexes (Owens and Peppas (2006) Int J Pharm 307:93-102). In order to provide enough steric hindrance to avoid opsonization, the exterior surface of the liposome must be completely covered by PEG molecules in the "brush"

configuration. At low surface coverage, the PEG chains will typically have a

"mushroom" configuration, wherein the PEG molecules will be located closer to the surface of the liposome. In the "brush" configuration, the PEG molecules are extended further away from the liposome surface, enhancing the steric hindrance effect.

However, over-crowdedness of PEG on the surface may decrease the mobility of the polymer chains and thus decrease the steric hindrance effect (Owens and Peppas (2006) Int J Pharm 307:93-102).

The conformation of PEG depends upon the surface density and the molecular mass of the PEG on the surface of the liposome. The controlling factor is the distance between the PEG chains in the lipid bilayer (D) relative to their Flory dimension, R_F, which is defined as aN^3/5, wherein a is the persistence length of the monomer, and N is the number of monomer units in the PEG (see Nicholas et al. (2000) Biochim Biophys Acta 1463: 167-178, which is herein incorporated by reference). Three regimes can be defined: (1) when D>2 RF (interdigitated mushrooms); (2) when D<2 RF (mushrooms); and (3) when D< RF (brushes) (Nicholas et al.).

In certain embodiments, the PEGylated lipid nanoparticle comprises a stealth lipid nanoparticle. By "stealth lipid nanoparticle" is intended a lipid nanoparticle comprising a liposome wherein the outer leaflet of the lipid bilayer of the liposome comprises a sufficient number of lipid-PEG conjugates in a configuration that allows the lipid nanoparticle to exhibit a reduced uptake by the RES system in the liver when administered to a subject as compared to non PEGylated lipid nanoparticles. RES uptake can be measured using assays known in the art, including, but not limited to the liver perfusion assay described in International Application No. PCT/US2009/042485, filed on May 1, 2009. In some of these embodiments, the stealth lipid nanoparticle comprises a liposome, wherein the outer leaflet of the lipid bilayer of the liposome comprises PEG molecules, wherein the D<RF.

In some of those embodiments in which the PEGylated delivery system is a stealth system, the outer leaflet of the lipid bilayer of the cationic liposome comprises a lipid-PEG conjugate at a concentration of about 4 mol% to about 15 mol% of the outer leaflet lipids, including, but not limited to, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, and about 15 mol% PEG. In certain embodiments, the outer leaflet of the lipid bilayer of the cationic liposome of the stealth lipid nanoparticle comprises about 10.6 mol% PEG. Higher percentage values

(expressed in mol%) of PEG have also surprisingly been found to be useful. Useful mol% values include those from about 12 mol% to about 50 mol%. Preferably, the values are from about 15 mol% to about 40 mol%. Also preferred are values from about 15 mol% to about 35 mol%. Most preferred values are from about 20 mol% to about 25 mol%, for example 23 mol%.

The polyethylene glycol moiety of the lipid-PEG conjugate can have a molecular weight ranging from about 100 to about 20,000 g/mol, including but not limited to about 100 g/mol, about 200 g/mol, about 300 g/mol, about 400 g/mol, about 500 g/mol, about 600 g/mol, about 700 g/mol, about 800 g/mol, about 900 g/mol, about 1000 g/mol, about 5000 g/mol, about 10,000 g/mol, about 15,000 g/mol, and about 20,000 g/mol. In some embodiments, the lipid-PEG conjugate comprises a PEG molecule having a molecular weight of about 2000 g/mol. In certain embodiments, the lipid-PEG conjugate comprises DSPE-PEG2000. In some embodiments, the lipid nanoparticle comprises a liposome, wherein the exterior surface of the liposome, or the lipid nanoparticle comprises a lipid bilayer wherein the outer leaflet of the lipid bilayer, comprises a targeting ligand, thereby forming a targeted delivery system. In these embodiments, the outer leaflet of the liposome comprises a targeting ligand. By "targeting ligand" is intended a molecule that targets a physically associated molecule or complex to a targeted cell or tissue. As used herein, the term "physically associated" refers to either a covalent or non-covalent interaction between two molecules. A "conjugate" refers to the complex of molecules that are covalently bound to one another. For example, the complex of a lipid covalently bound to a targeting ligand can be referred to as a lipid-targeting ligand conjugate.

Alternatively, the targeting ligand can be non-covalently bound to a lipid. "Non- covalent bonds" or "non-covalent interactions" do not involve the sharing of pairs of electrons, but rather involve more dispersed variations of electromagnetic interactions, and can include hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bonds.

Targeting ligands can include, but are not limited to, small molecules, peptides, lipids, sugars, oligonucleotides, hormones, vitamins, antigens, antibodies or fragments thereof, specific membrane-receptor ligands, ligands capable of reacting with an anti- ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. Non-limiting examples of targeting ligands include asialoglycoprotein, insulin, low density lipoprotein (LDL), folate, benzamide derivatives, peptides comprising the arginine-glycine-aspartate (RGD) sequence, and monoclonal and polyclonal antibodies directed against cell surface molecules. In some embodiments, the small molecule comprises a benzamide derivative. In some of these embodiments, the benzamide derivative comprises anisamide.

The targeting ligand can be covalently bound to the lipids comprising the liposome or lipid bilayer of the delivery system, including a cationic lipid, or a co-lipid, forming a lipid-targeting ligand conjugate. As described above, a lipid-targeting ligand conjugate can be post-inserted into the lipid bilayer of a liposome using techniques known in the art and described elsewhere herein (Ishida et al. (1999) FEBS Lett.

460: 129-133; Perouzel et al. (2003) Bioconjug. Chem. 14:884-898; see Experimental section). Alternatively, the lipid-targeting ligand conjugate can be added during the formation of the outer leaflet of the lipid bilayer. Some lipid-targeting ligand conjugates comprise an intervening molecule in between the lipid and the targeting ligand, which is covalently bound to both the lipid and the targeting ligand. In some of these embodiments, the intervening molecule is polyethylene glycol (PEG), thus forming a lipid-PEG-targeting ligand conjugate. An example of such a lipid-targeting conjugate is DSPE-PEG-AA, in which the lipid 1,2- distearoyl-5n-glycero-3-phosphoethanolamine-N-carboxyl (DSPE) is bound to polyethylene glycol (PEG), which is bound to the targeting ligand anisamide (AA). Thus, in some embodiments, the cationic lipid vehicle of the delivery system comprises the lipid-targeting ligand conjugate DSPE-PEG-AA.

By "targeted cell" is intended the cell to which a targeting ligand recruits a physically associated molecule or complex. The targeting ligand can interact with one or more constituents of a target cell. The targeted cell can be any cell type or at any developmental stage, exhibiting various phenotypes, and can be in various pathological states (i.e., abnormal and normal states). For example, the targeting ligand can associate with normal, abnormal, and/or unique constituents on a microbe (i.e., a prokaryotic cell (bacteria), viruses, fungi, protozoa or parasites) or on a eukaryotic cell (e.g., epithelial cells, muscle cells, nerve cells, sensory cells, cancerous cells, secretory cells, malignant cells, erythroid and lymphoid cells, stem cells). Thus, the targeting ligand can associate with a constitutient on a target cell which is a disease-associated antigen including, for example, tumor-associated antigens and autoimmune disease- associated antigens. Such disease-associated antigens include, for example, growth factor receptors, cell cycle regulators, angiogenic factors, and signaling factors.

In some embodiments, the targeting ligand interacts with a cell surface protein on the targeted cell. In some of these embodiments, the expression level of the cell surface protein that is capable of binding to the targeting ligand is higher in the targeted cell relative to other cells. For example, cancer cells overexpress certain cell surface molecules, such as the HER2 receptor (breast cancer) or the sigma receptor. In certain embodiments wherein the targeting ligand comprises a benzamide derivative, such as anisamide, the targeting ligand targets the associated lipid nanoparticle to sigma- receptor overexpressing cells, which can include, but are not limited to, cancer cells such as small- and no n- small-cell lung carcinoma, renal carcinoma, colon carcinoma, sarcoma, breast cancer, melanoma, glioblastoma, neuroblastoma, and prostate cancer (Aydar, Palmer, and Djamgoz (2004) Cancer Res. 64:5029-5035). Thus, in some embodiments, the targeted cell comprises a cancer cell. As described elsewhere herein, the terms "cancer" or "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, "cancer cells" or "tumor cells" refer to the cells that are characterized by this unregulated cell growth.

V. Pharmaceutical Compositions

The lipid nanoparticles (i.e., liposomes) described herein are useful in mammalian tissue culture systems, in animal studies, and for therapeutic purposes. The lipid nanoparticles comprising a bioactive compound having therapeutic activity when expressed or introduced into a cell can be used in therapeutic applications. The lipid nanoparticles can be administered for therapeutic purposes or pharmaceutical compositions comprising the lipid nanoparticles along with additional pharmaceutical carriers can be formulated for delivery, i.e., administering to the subject, by any available route including, but not limited, to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. In some embodiments, the route of delivery is intravenous, parenteral, transmucosal, nasal, bronchial, vaginal, and oral.

As used herein the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Supplementary active compounds also can be incorporated into the compositions.

As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;

antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions or dispersions such as those described elsewhere herein and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. In some embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, or sodium chloride are included in the formulation. Prolonged absorption of the injectable formulation can be brought about by including in the formulation an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by filter sterilization. In certain embodiments, solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the lipid nanoparticles into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In those embodiments in which sterile powders are used for the preparation of sterile injectable solutions, the solutions can be prepared by vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The oral compositions can include a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the presently disclosed compositions can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used.

Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical or cosmetic carrier. The specification for the dosage unit forms of the presently disclosed subject matter are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Guidance regarding dosing is provided elsewhere herein.

The presently disclosed subject matter also includes an article of manufacture providing a lipid nanoparticle described herein. The article of manufacture can include a vial or other container that contains a composition suitable for the present method together with any carrier, either dried or in liquid form. The article of manufacture further includes instructions in the form of a label on the container and/or in the form of an insert included in a box in which the container is packaged, for carrying out the method of the presently disclosed subject matter. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow the subject or a worker in the field to administer the pharmaceutical composition. It is anticipated that a worker in the field encompasses any doctor, nurse, technician, spouse, or other caregiver that might administer the composition. The pharmaceutical composition can also be self- administered by the subject.

The presently disclosed subject matter provides methods for delivering a bioactive compound to a cell and for treating a disease or unwanted condition in a subject with a lipid nanoparticle comprising a bioactive compound that has therapeutic activity against the disease or unwanted condition. Further provided herein are methods for making the presently disclosed lipid nanoparticles.

The presently disclosed lipid nanoparticles can be used to deliver the bioactive compound to cells by contacting a cell with the lipid nanoparticles. As described elsewhere herein, the term "deliver" when referring to a bioactive compound refers to the process resulting in the placement of the composition within the intracellular space of the cell or the extracellular space surrounding the cell. The term "cell" encompasses cells that are in culture and cells within a subject.

In some embodiments, the exterior of the lipid nanoparticle comprises a lipid- PEG conjugate. In some of these embodiments, the lipid nanoparticle comprises a stealth lipid nanoparticle. In certain embodiments, the outer leaflet of the liposome of the delivery system comprises a targeting ligand, thereby forming a targeted lipid nanoparticle, wherein the targeting ligand targets the targeted lipid nanoparticle to a targeted cell.

The lipid nanoparticles described herein comprising a flavonol compound can be used for the treatment of a disease or unwanted condition in a subject, wherein the flavonol compound has therapeutic activity against the disease or unwanted condition when expressed or introduced into a cell. The flavonol compound is administered to the subject in a therapeutically effective amount. In those embodiments wherein the flavonol compound comprises QP, when QP is administered to a subject in

therapeutically effective amounts, the QP is capable of treating the disease or unwanted condition.

By "therapeutic activity" when referring to a bioactive compound is intended that the molecule is able to elicit a desired pharmacological or physiological effect when administered to a subject in need thereof.

As used herein, the terms "treatment" or "prevention" refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular infection or disease or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure of an infection or disease and/or adverse effect attributable to the infection or the disease. Accordingly, the method "prevents" (i.e., delays or inhibits) and/or "reduces" (i.e., decreases, slows, or ameliorates) the detrimental effects of a disease or disorder in the subject receiving the compositions of the subject matter described herein. For example, the disease may be cancer wherein a tumor is present. As such, treatment of cancer may result in reducing the size of a tumor associated with the cancer. Reducing the size of the tumor means that after administration the overall size of the tumor is less than it was before administration. Thus, as used herein, the "tumor reduction" can be at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or more.

The subject may be any animal, including a mammal, such as a human, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The disease or unwanted condition to be treated can encompass any type of condition or disease that can be treated therapeutically. In some embodiments, the disease or unwanted condition that is to be treated is a cancer. As described herein, the term "cancer" encompasses any type of unregulated cellular growth and includes all forms of cancer including, but not limited to, all forms of carcinomas, melanomas, sarcomas, lymphomas and leukemias, including without limitation, bladder carcinoma, brain tumors, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, endometrial cancer, hepatocellular carcinoma, laryngeal cancer, lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma and thyroid cancer. In some embodiments, the targeted cancer cell comprises a lung cancer cell. The term "lung cancer" refers to all types of lung cancers, including but not limited to, small cell lung cancer (SCLC), non-small-cell lung cancer (NSCLC, which includes large-cell lung cancer, squamous cell lung cancer, and adenocarcinoma of the lung), and mixed small-cell/large-cell lung cancer. In some embodiments, the cancer to be treated is a metastatic cancer. In particular, the cancer may be resistant to known therapies. Methods to detect the inhibition of cancer growth or progression are known in the art and include, but are not limited to, measuring the size of the primary tumor to detect a reduction in its size, delayed appearance of secondary tumors, slowed development of secondary tumors, decreased occurrence of secondary tumors, and slowed or decreased severity of secondary effects of disease. In embodiments, the type of cancer is a carcinoma, such as bladder carcinoma.

It will be understood by one of skill in the art that the lipid nanoparticles can be used alone or in conjunction with other therapeutic modalities, including, but not limited to, surgical therapy, radiotherapy, or treatment with any type of therapeutic agent, such as a drug. In those embodiments in which the subject is afflicted with cancer, the lipid nanoparticles can be delivered in combination with any

chemotherapeutic agent well known in the art.

As used herein, the term "in combination" includes the use of the nanoparticle with an additional treatment. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject with melanoma. A first therapy, either the additional treatment or the immunotherapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy.

When administered to a subject in need thereof, the lipid nanoparticles can further comprise a targeting ligand, as discussed elsewhere herein. In these

embodiments, the targeting ligand will target the physically associated complex to a targeted cell or tissue within the subject. In certain embodiments, the targeted cell or tissue comprises a diseased cell or tissue or a cell or tissue characterized by the unwanted condition. In some of these embodiments, the lipid nanoparticle is a stealth lipid nanoparticle wherein the surface charge is shielded through the association of PEG molecules and the liposome further comprises a targeting ligand to direct the lipid nanoparticle to targeted cells.

In some embodiments, particularly those in which the diameter of the lipid nanoparticle is less than 100 nm, the lipid nanoparticles can be used to deliver bioactive compounds across the blood-brain barrier (BBB) into the central nervous system or across the placental barrier. Non-limiting examples of targeting ligands that can be used to target the BBB include transferring and lactoferrin (Huang et al. (2008) Biomaterials

which is herein incorporated by reference in its entirety). Further, the lipid nanoparticles can be transcytosed across the endothelium into both skeletal and cardiac muscle cells. For example, exon-skipping oligonucleotides can be delivered to treat Duchene muscular dystrophy (Moulton et al. (2009) Ann N Y Acad Sci 1175:55-60, which is herein incorporated by reference in its entirety).

Delivery of a therapeutically effective amount of a lipid nanoparticle comprising a bioactive compound can be obtained via administration of a

pharmaceutical composition comprising a therapeutically effective dose of the bioactive compound or the lipid nanoparticle. By "therapeutically effective amount" or "dose" is meant the concentration of a delivery system or a bioactive compound comprised therein that is sufficient to elicit the desired therapeutic effect.

By "bioactive compound" is intended any agent that has a desired effect (e.g., therapeutic effect) on a living cell, tissue, or organism, or an agent that can desirably interact with a component (e.g., enzyme) of a living cell, tissue, or organism. Bioactive compounds can include, but are not limited to, polynucleotides, polypeptides, polysaccharides, organic and inorganic small molecules. The term "bioactive compound" encompasses both naturally occurring and synthetic bioactive compounds. The term "bioactive compound" can refer to a detection or diagnostic agent that interacts with a biological molecule to provide a detectable readout that reflects a particular physiological or pathological event. In some embodiments, the bioactive compound is quercetin or a phosphate derivative. In some embodiments, the bioactive compound is cisplatin or a cisplatin nanoparticle.

As used herein, "effective amount" is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times.

The effective amount of the lipid nanoparticle or bioactive compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound or complex, and, if desired, the adjuvant therapeutic agent being administered along with the lipid nanoparticle. Methods to determine efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996) Harrison 's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic (e.g., immuno toxic) and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the presently disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

The pharmaceutical formulation can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments. Further, treatment of a subject can include a single cosmetic application or, in some embodiments, can include a series of cosmetic applications.

The pharmaceutical formulation can be administered at various intervals and over different periods of time as required, e.g. , multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments. In an embodiment, the pharmaceutical formulation is administered preventatively, in one, two, or more doses. In another embodiment, the pharmaceutical formulation is administered after a tumor is established. In an embodiment, the pharmaceutical formulation is administered multiple times. In a further embodiment, the pharmaceutical formulation is administered in five doses. In a further embodiment, the second of the at least two administrations occurs about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first administration, for example at about 7 days after the first administration. In a further embodiment, the five doses are administered every other day. Further, treatment of a subject can include a single cosmetic application or, in some embodiments, can include a series of cosmetic applications.

It is understood that appropriate doses of a compound depend upon its potency and can optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject can depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

In some embodiments, the lipid nanoparticles are administered to the subject at a dose of between about 0.001 μg/kg and about 1000 mg/kg, including but not limited to about 0.001 μg/kg, 0.01 μg/kg, 0.05 μg/kg, 0.1 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 100 μg/kg, 250 μg/kg, 500 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg, and 200 mg/kg. In embodiments, the administered doses may be in the range from about 0.1 mg/kg to about 10 mg/kg, and in further embodiments from about 0.1 mg/kg to about 1 mg/kg, from about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 5 mg/kg, or about 5 mg/kg to about 10 mg/kg.

One of ordinary skill in the art upon review of the presently disclosed subject matter would appreciate that the presently disclosed compounds and pharmaceutical compositions thereof, can be administered directly to a cell, a cell culture, a cell culture medium, a tissue, a tissue culture, a tissue culture medium, and the like. When referring to the delivery systems of the presently disclosed subject matter, the term "administering," and derivations thereof, comprises any method that allows for the compound to contact a cell. The presently disclosed compounds or pharmaceutical compositions thereof, can be administered to (or contacted with) a cell or a tissue in vitro or ex vivo. The presently disclosed compounds or pharmaceutical compositions thereof, also can be administered to (or contacted with) a cell or a tissue in vivo by administration to an individual subject, e.g., a patient, for example, by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration) or topical application, as described elsewhere herein.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nanoparticle" is understood to represent one or more nanoparticles. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

Throughout this specification and the claims, the words "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments + 50%, in some embodiments + 20%, in some embodiments + 10%, in some embodiments + 5%, in some embodiments + 1%, in some embodiments + 0.5%, and in some embodiments + 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.

The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

Example 1

1.1 Materials and Methods

l,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP), 1,2-

Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (DSPE-PEG2000, referred herein as "DSPE-PEG"), and

dioleoylphosphatidic acid (DOPA) were purchased from Avanti Polar Lipids

(Alabaster, AL). Quercetin, cholesterol, hexanol, triton X-100 and cyclohexane were provided by Sigma-Aldrich (St. Louis, MO). Anisamide conjugated l,2-distearoyl-5n- glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol ("DSPE-PEG- AA") was synthesized based on the previous reported methods. Cisplatin was purchased from Acros Organics (Fair Lawn, NJ). All the other chemicals were obtained from Sigma-Aldrich unless otherwise mentioned.

The mouse embryonic fibroblast cell line NIH3T3 was purchased from UNC

Tissue Culture Facility. The human bladder transitional cell line UMUC3 was obtained from Dr. William Kim (University of North Carolina at Chapel Hill, NC). NIH3T3 and UMUC3 were cultured in Dulbecco's Modified Eagle's Media (Invitrogen, Carlsbad, CA), supplemented with 10% bovine calf serum (Hyclone, Logan, Utah) or 10% fetal bovine serum (Sigma, St. Louis MO) respectively, with penicillin (100 U/mL)

(Invitrogen) and streptomycin (100 μg/mL) (Invitrogen).

Female athymic Balb/C nude mice 6-8 weeks of age were obtained from the University of North Carolina animal facilities. All work performed on animals was approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.

1.2 Statistical Analysis

Quantitative results were expressed as mean + SD. The analysis of variance was completed using student's t-test and one-way analysis of variance (ANOVA). A p value of p < 0.05 was considered statistically significant

1.3 Screening of TCMsfor Wntl6 Inhibition

Twelve anti-fibrotic natural chemicals selected according to the Chinese Pharmacopeia and literatures were tested for its effects on Wntl6 expression in TGF-β activated NIH3T3 cells. Specifically, NIH3T3 cells were pre-activated with 10 ng/mL TGF-β. Then, the next day, the cells were treated with different chemicals at a predetermined nontoxic concentration: (1) Tanshinone IIA, 4.7 μΜ (2) Astragaloside IV, 19 μΜ (3) Notoginsenoside Rl, 54 μΜ (4) Matrine, 0.53 μΜ (5) Artemisinin, 160 μΜ (6) Quercetin, 10 μΜ (7) Rheinic acid, 11.43 μΜ (8) Salvianolic acid B, 80 μΜ (9) Ligustrazine, 16 μΜ (10) Scutellarin, 80 μΜ (11) Salvianolic acid A, 20 μΜ (12) Tetrandrine, 13 μΜ. Twenty- four hours later, the cells were harvested, and western-blot assays were used to detect the expression levels of Wntl6. The chemical with the best Wntl6 inhibition effect was selected for further studies.

Among the candidates screened, quercetin showed superior Wntl6 knockdown efficiency in NIH3T3 murine fibroblasts in the presence or absence of cisplatin.

Example 2

2.1 - Synthesis and Characterization of Quercetin Phosphate (QP)

Quercetin phosphate (QP) was synthesized by phosphorylation of the hydroxyl groups of quercetin to improve the water solubility and facilitate the preparation of LCP-QP nanoparticles. The chemical structure of the synthesized QP was confirmed by MS and NMRs (FIGS. 7A-7C). The LC-MS results yielded an m/z of 702.88. The yellow colored QP has increased water solubility compared to quercetin, and its concentration can be determined using a UV spectrophotometer.

This phosphorylation of quercetin not only increased its water solubility, but also facilitated its precipitation with calcium to form an amorphous nanoparticle core used in the formulation of lipid calcium phosphate (LCP) nanoparticles. As disclosed herein, the phosphate groups of QP can be cleaved in vivo through interaction with phosphatases.

2.2 - Method

QP was synthesized based on the reported literature, with some modification.

Briefly, 3 mmol quercetin, DMAP (2.0 mmol per -OH group), and Et₃N (2.0 mmol per -OH group) were dissolved in 100 mL anhydrous THF. Then a solution of ClP(0)(OEt)2 (90 mmol) in anhydrous THF (50 mL) was added dropwise under stirring in an ice-water bath over 30 min. The reaction was continued at room temperature for 24 h under nitrogen. The reaction mixture was diluted with EtOAc, washed with 0.5 M HC1, 5% (w/v) NaOH, brine and water, and then dried over anhydrous Na₂S04. After removal of the solvent by rotary evaporation, the residue was purified using column chromatography on deactivated silica gel with petroleum ether/EtOAc (4: 1 - 2: 1) and dichloromethan/methanol (5: 1 - 2: 1) as eluent to give the ethyl protected QP, which was characterized by HPLC-MS.

The ethyl protected QP (1.70 mmol) was dissolved in 10 mL dry dichloromethane and 7.2 mmol trimethylsilyl bromide was added dropwise at 0° C. After stirring for 4 h, an excess of methanol was added and the mixture stirred for 30 min. The mixture was evaporated to dryness under vacuum overnight and the product was characterized by HPLC-MS, Ή-ΝΜΡν, and ³¹P-NMR. A QP standard curve was calculated using a UV absorbance spectrum and was used to calculated QP concentrations in the LCP.A synthesis of phosphorylated quercetin is depicted below in Scheme 1. The molecular weight of the ethyl protected QP intermediate was confirmed by MS spectrum (m/z=982.99). The presence of five phosphate groups of the final QP was confirmed by the appearance of five singlets in the ³¹P-NMR spectrum (FIG. 7 A). The five phosphate groups on QP were further confirmed by Ή-ΝΜΡ (FIG. 7B), ¹³C- NMR (FIG. 7C), and LC-MS (m/z=702.88). The QP has improved hydrophilicity compared to parent quercetin.

Scheme 1. Synthesis of phosphorylated quercetin. Example 3

Preparation and Characterization of LCP-QP

The LCP-QP cores were prepared by water-in-oil micro-emulsions in an oil phase containing cyclohexane/Igepal CO-520 solution (70/30, v/v) based on the reported literature. Briefly, three hundred microliters of 30 mg/mL QP was mixed with 600 μΐ_^ 2.5 M CaCb in 20 mL oil phase with continuous stirring. Six hundred microliters of 200 mM NH4HPO4 was added to a separate 20 mL oil phase. After 5 min, the two oil phases were mixed, 500 μΐ_^ of 20 mM DOPA in chloroform was added to the emulsion, and the mixture was stirred for 30 min. Then 40 mL of absolute ethanol was added slowly. The ethanol emulsion mixture was centrifuged at 10,000 g for 15 min and the precipitated LCP-QP core was collected. The precipitate was washed twice with absolute ethanol and dried under N₂. The LCP-QP cores were dissolved in 2 mL chloroform and stored in a glass vial at -20°C for future use.

To prepare the final LCP-QP nanoparticle, 11.5 mg LCP-QP core in chloroform was mixed with 0.6 mL of 20 mM cholesterol, 0.6 mL of 20 mM DOTAP, 0.24 mL of 20 mM DSPE-PEG and 0.06 mL of 20 mM DSPE-PEG-AA. After evaporating the chloroform, the residual lipids were suspended in water under brief sonication to form the final LCP-QP.

The Dil labeled LCP (LCP-Dil) were prepared by the same method without addition of quercetin but with 2% Dil added to the lipids.

The particle size and Zeta potential of LCP-QP was determined by a Malvern ZetaSizer Nano series (Westborough, MA). TEM images of LCP-QP cores and LCP- QP nanoparticles (negatively stained with 2% uranyl acetate) were acquired using a JEOL 100 CX II TEM (JEOL, Japan). The drug-loading capacity and encapsulation efficiency of QP were measured using ultraviolet spectrophotometer (D800, Beckman Coulter, Inc.). The LCPs were first lysed using a pH 4 acetic acid buffer and the concentration was determined using a standard curve. QP can be encapsulated into LCP nanoparticles with high encapsulation efficiency (60.8+5.2%) and loading (26.6+2.3%).

The LCP-QP core and LCP-QP final lipid nanoparticle were spherical and uniformly distributed under TEM (FIG. 2B). The final LCP-QP nanoparticle has a particle size of approximately 35 nm and appears opalescence with a yellow color (FIGS. 2C and 2D).

Example 4

4.1 - QP Conversion to Quercetin

To confirm that pharmacologically active quercetin is delivered to the tumor site, the conversion of QP to quercetin in vitro was validated using alkaline phosphatase. After incubating QP with alkaline phosphatase for lh at 37°C, quercetin was detected in the QP solution by HPLC analysis (FIGS. 3A and 3B). The retention time of the quercetin peak in the QP solution was identical to the quercetin standards, while the QP solution without alkaline phosphatase showed no quercetin peak. These results demonstrated that the QP was converted back to quercetin by phosphatase. Validation was also performed using intact NIH3T3 cells. LCP-QP was incubated with the cells for 2h and following incubation, free quercetin was detected by HPLC analysis (FIGS. 3C and 3D). To further confirm the conversion of QP to quercetin in vivo, QP and LCP-QP were i.v. injected to stroma rich UMUC3 bearing mice and the tumors were harvest one hour later to detect the quercetin concentration in tumor. Quercetin was detected in the tumor after both QP and LCP-QP injection with a higher accumulation by the LCP-QP (FIG. 3E). Together, these results suggested that the QP delivered by LCP-QP could be converted to the active parent, quercetin, by hydrolytic enzymes extensively distributed in vivo.

4.2 - QP Conversion to Quercetin by Alkaline Phosphatase

The conversion of QP into quercetin was first evaluated in vitro by alkaline phosphatase, a common hydrolytic enzyme. Two hundred micrograms of QP were mixed with 50 U of alkaline phosphatase in 1 mL OPTIZYME AP buffer and incubated at 37° C for 1 h. The mixture was then frozen using dry ice and lyophilized. Then 400iL acetonitrile was added to the extract and the resulting solution analyzed with HPLC (Waters 600 HPLC system/717 plus autosampler) with a dual absorbance UV detector. The separation of quercetin was achieved by using a Kromasil 100-5-C18 column with methanol/acetonitrile/water 40: 15:45 as the mobile phase with a flow rate of 1 mL/min at detection wavelength of 345 nm.

4.3 - QP Conversion to Quercetin by NIH3T3 Cells

The conversion of QP into quercetin was further validated in live NIH3T3 cells. LCP-QP containing 200 μg QP was added to NIH3T3 cells. After 2 h incubation, the medium and cells were collected and subjected to lyophilization in 1% triton X-100. Then 400 iL acetonitrile was added and the quercetin in the resulting solution was detected by HPLC analysis.

4.4 - QP Conversion to Quercetin in vivo

Free QP and LCP-QP were i.v. injected to stroma rich UMUC3 bearing mice at a QP dose of 30 mg/kg. One hour after injection, the mice were sacrificed and the tumors were harvested and analyzed for quercetin concentration by a UPLC-MS method. Baicalein was used as an internal standard. The homogenized tumor tissue was extracted with EtOAc and evaporated to dryness under nitrogen gas. After reconstituting with acetonitrile/0.1% formic acid and centrifugation, the supernatant was used for UPLC analysis. The mass spectrometer was operated in the positive ion mode with the Turbolonspray heater set at 450 °C (API3000 LC/MS/ MS system, Applied Biosystems, Foster City, CA, USA). The samples were analyzed using the transition of m/z 303→153 amu for quercetin and m/z 271→123 amu for baicalein.

Example 5

5.1 - Anti-Tumor Efficacy in Stroma-rich Xenografts

Antitumor efficacy of different treatments; LCP-QP, LPC (cisplatin

nanoparticle), or LCP-QP combined with LPC were investigated on a stroma-rich UMUC3 bladder cancer xenograft model (FIGS. 4A-4F). All groups were

administered five injections every other day (QOD). The intravenously injected treatments consisting of LCP-QP and LPC alone showed partial tumor inhibition effects resulting in a significantly decreased tumor volume compared to the untreated group (FIG. 4A). The anti-tumor efficacy of the LCP-QP + LPC combination treatment elicited a significantly greater response in tumor growth inhibition compared to either the LPC or LCP-QP treatment alone. This shows that the anti-tumor effect of LPC was greatly enhanced by the LCP-QP nanoparticles. The same trend was found in tumor weight results, which shows that LCP-QP combined with LPC treatment significantly reduced the tumor weight compared to the control, LPC-QP, and LPC groups (FIG. 4B). Furthermore, compared to the LPC group, the LCP-QP + LPC group showed a reduced Wntl6 level with an increased cisplatin level in the tumor tissue, suggesting that the normalization effect of LCP-QP on Wntl6 upregulation facilitated the penetration of LPC nanoparticles into the tumor (FIGS. 4C and 4F). After five doses of LPC treatment, the Wntl6 level in the tumors was increased 50% compared to the control group, suggesting there was a Wntl6 upregulation which correlated to tumor cell resistance. The TUNEL apoptosis assay results also illustrate the same pattern, in which the number of apoptotic cells in the LCP-QP+LPC treatment group was significantly higher than the other treatment groups, which is consistent with the tumor inhibition results (FIGS. 4D and 4E).

5.2 - Methods The stroma-rich xenograft model was established as previously reported in the literature. UMUC3 (5 x 10⁶) and NIH3T3 cells (2 x 10⁶) in 100 μΐ. of PBS were subcutaneously co-injected with Matrigel (BD Biosciences, CA) at a ratio of 1: 1 (v/v) into the right flank of the mice. On the ninth day, the mice were randomly divided into four groups and subjected to the following treatments every other day: (1) Control group, i.v. injection of 200 μΐ. PBS; (2) LCP-QP group, i.v. injection of LCP-QP corresponding to 5.5 mg/kg quercetin; (3) LPC group, i.v. injection of LPC corresponding to 1.7 mg/kg cisp latin; and (4) LCP-QP+LPC group, i.v. injection of both LCP-QP (5.5 mg/kg quercetin) and LPC (1.7 mg/kg cisp latin) on the same day. The LPC nanoparticles were prepared as described in the literature. These injections were given five times. Tumor volume and body weight of the mice were measured every day starting from the ninth day post inoculation. The formula: V = (L x W²)/2 was applied to calculate tumor volume, where V is the tumor volume, L the larger perpendicular diameter, and W is the smaller perpendicular diameter. Two days after final administration, the mice were sacrificed and the tumors were harvested and weighed. Western-blot was used to detect Wntl6 expression in the tumor tissue. ICP- MS was used to investigate the tumor accumulation of cisplatin. The tumor tissue was also washed with PBS and fixed by 4% paraformaldehyde for further studies.

TGFP activated NIH3T3 cells were treated withlO μΜ free cisplatin for 3 h before being treated with 10 μΜ quercetin. Two days later, the cells were harvested for a western-blot assay of Wntl6 expression.

5.3 - TUNEL Assay

Paraffin-embedded sections of the tumor were prepared by the UNC Tissue Procurement Core. Slides were deparaffinized and rehydrated then stained using a TUNEL assay kit (Pierce) according to the manufacturer's instruction.

Example 6

6.1 - NP Distribution and TME Remodeling

To observe the TME remodeling effect of LCP-QP, the influence of LCP-QP on ECM markers (a-SMA expressing fibroblast populations and collagen content) and LCP-Dil distribution was investigated. As shown in FIG. 5A, all treatment groups that included the LCP-QP significantly decreased the active fibroblasts growth and amount of collagen in the tumors. Alternately, the number of active fibroblasts and collagen increased significantly after LPC treatment, while the LCP-QP treatments resulted in 50% reduction in collagen compared to the untreated control group.

Furthermore, to understand the effect that decreased collagen content and activated fibroblast population play on subsequent nanoparticle tumor accumulation and penetration, LCP containing a fluorescent probe, l'-dioctadecyl-3,3,3'3'- tetramethylindocarbocyanine (hereinafter "Dil"), was employed in the LCP lipid bilayer. These LCP-Dil nanoparticles were administered following treatment of LCP-QP to test the effect of LCP-QP on penetration of nanoparticles to the tumor (FIG. 5B). The results showed that, after three successive injections of LCP-QP, not only was the total uptake of LCP-Dil by the tumor greatly enhanced, but importantly, the penetration of LCP-Dil into the tumor nest (TN) was also greatly enhanced. Free QP also resulted in a partial increase in the penetration of nanoparticles into tumor cells. Without being bound by theory, these findings, together with the TME remodeling results, suggest that the LCP- QP not only improves the QP delivery to the tumor site, but also improves the tumor penetration of subsequent nanoparticles administered after the LCP-QP. Such properties may play a crucial role in the prominent antitumor effects in vivo.

6.2 - Methods

Paraffin block sections of SRBC with different treatments were deparaffinized with xylene and a graded alcohol series. After antigen retrieval, sections were blocked with 10% goat serum and incubated with polyclonal rabbit anti-a-SMA antibody (Abeam, Cambridge, MA, USA) at 1: 100 dilution overnight at 4°C. The next day, the slides were incubated with Alexa Fluor 647 secondary antibody at a 1: 100 dilution for 1 h at room temperature in the dark. Slides were rinsed with PBS and cover-slipped with Vectashield containing DAPI (Vector laboratories, Burlingame, CA). Digital images were acquired via an Eclipse Ti-U inverted microscope (Nikon Corp., Tokyo, Japan) at 20x magnification and quantitatively analyzed on Image J (National Institutes of Health). Collagen content was visualized using Masson trichrome staining.

The effect of LCP-QP on LCP-Dil penetration was investigated on a GFP- 3T3/UMUC3 stroma-rich tumor model. When the tumor was around 500 mm³, the mice were i.v. administered three successive doses of LCP-blank, QP, and LCP-QP (corresponding to quercetin dose of 5.5 mg/kg). LCP-Dil was i.v. injected with the third dose of LCP-QP at a dose of 0.1 mg/kg Dil. The mice were sacrificed 24 h post LCP- Dil injection. In order to localize and visualize the LCP-Dil penetration, the tumor was frozen and sectioned. The sections were directly stained with DAPI and observed using a Nikon light microscope (NikonCorp., Tokyo).

Example 7

7.1 - In vivo toxicity ofLCP-QP

An important aspect of nano therapeutics is their safety. The body weight of the treated mice remained unchanged during the tumor inhibition experiments (FIG. 6A). The serum biochemical test results showed the BUN, creatinine, AST and ALT levels were all in the normal range, which suggests that there was no severe damage to renal and hepatic functions after LCP-QP and LCP-QP+LPC injections (FIG. 6B). Moreover, the H&E staining results also demonstrate that there was no tissue-specific toxicity to major organs (FIG. 6C). This suggests good biocompatibility and safety of the LCP-QP nanoparticles.

7.2 - Methods - Serum Biochemical Value Analysis and H&E Assay.

After five doses of LCP-QP, LPC, and LCP-QP+LPC injections, blood was collected and centrifuged at 4000 rpm for 5 min to obtain the serum. Blood urea nitrogen (BUN), creatinine, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were assayed as indicators of renal and hepatic function. Organs (heart, liver, spleen, lung, and kidney) were fixed and sectioned for H&E staining in order to evaluate the organ- specific toxicity.

Example 8

8.1 - LCP-QP Nanoparticles Downregulate Wntl6 Levels

Certain embodiments disclosed herein pertain to a LCP-QP nanoparticle which downregulates the Wntl6 levels in the TAFs and enhances the antitumor effect of cisplatin containing nanoparticles (LPC) in a stroma-rich bladder carcinoma model. In some embodiments, the phosphorylation of quercetin results in successful construction of LCP-QP with small particle size and high drug loading. Intravenously injected LCP- QP yielded significantly enhanced antitumor efficiency in combination with potent LPC. The effect of LCP-QP on decreasing active fibroblasts and collagen content in the TME contributes to the enhanced antitumor effect of LCP-QP. The preliminary toxicity studies disclosed herein show a promising safety profile of the LCP-QP. Thus, in embodiments, the LCP-QP is a TME remodeling nanoformulation that enhances the antitumor effects of nanotherapeutics. These results show that natural chemicals encapsulated into nanoparticles are a promising strategy to modulate TME and assist in traditional chemotherapy.

8.2 - Quercetin, QP, and LCP-QP

The effects of different natural chemicals on the expression of Wntl6 in activated fibroblasts NIH3T3 were detected by western-blot analysis (FIGS. 1A and IB). The expression of Wntl6 in activated fibroblasts was inhibited to different extents by selected chemicals. Among them, chemical No. 6 (quercetin) showed the most significant inhibition effect with only 44% of Wntl6 expressed compared to the control group.

Quercetin is has been postulated to reduce the toxicity and sensitization of some potent anticancer chemicals, such as cisplatin and gemcitabine. As a dietary

polyphenolic agent the safety profile of quercetin is well recognized.

Phosphorylation of quercetin into QP allows the precipitation of QP with calcium to form the particle core, which is further coated with asymmetric lipid bilayers decorated with a sigma receptor ligand aminoethylanisamide, AEAA, a tumor specific targeting molecule. The drug loading of LCP-QP is 26.6%, suggesting that one-quarter of the cargo consists of the drug. This high loading ability is attributed to the five phosphate groups on the QP as well as the supreme stability endowed by the core of the LCP particle.

8.3 - LCP-QP in Cells

The LCP-QP allows for increased tumor accumulation, cellular uptake, and intracellular release of the QP. Upon delivering into cells, QP is dephosphorylated by phosphatases to release the active drug quercetin. Phosphatase exists extensively on cell membranes, cytoplasm, and lysosomes in various organs. Interestingly, subtypes of phosphatases are elevated in tumor tissues. Such phosphatases include the prostatic acid phosphatase whose level is correlated to tumor grade, the seminoma marker Regan isoenzyme of alkaline phosphatase as well as the protein tyrosine phosphatase, PRL-3, which is upregulated in human myeloma cells and also considered as a metastasis- associated phosphatase. As such, in some embodiments, the upregulated phosphatase in the tumor microenvironment results in an enhanced conversion of QP to quercetin following the intratumoral delivery of LCP-QP.

As disclosed herein, the effect of the LCP-QP as an inhibitor to the DRP molecule Wntl6 was investigated in a stroma-rich bladder carcinoma model. The effects of LCP-QP on the tumor microenvironment (TME) including TAFs apoptosis, collagen deposition, and improved nanoparticle penetration were tested. Further, the in vivo toxicity of LCP-QP was inspected by biochemical indicator analysis and organ haematoxylin and eosin (HE) stain analysis.

Additionally, as disclosed herein, the quercetin nanoparticle LCP-QP can down regulate the a-SMA fibroblast populations and normalize the collagen content in the tumor tissue. This was consistent with the results found in human corneal fibroblasts in which quercetin is a key regulator of fibrotic markers and ECM assembly. The remodeling effect of quercetin on fibroblasts may normalize the fibroblasts and the ECM which likely plays a critical role in increasing the penetration of Dil nanoparticles into the tumor nest. Further, as disclosed herein, in an embodiment, the LCP-QP have better remodeling ability than free QP. Without being bound by theory, this may be attributed to the enhanced delivery and stability of QP after nanoparticle encapsulation.

Further, toxicity studies also demonstrated good biocompatibility of QP and LCP-QP. 8.4 - Combination of LCP-QP and Cisplatin Nanoparticles (LPC)

The Wntl6 inhibition effect of quercetin on cisplatin treated NIH3T3 cells was also examined. Cisplatin NP (LPC) induced a nearly 2- fold elevated secretion of Wntl6 in TAFs compared to untreated cells. Further, the cisplatin induced secretion of Wntl6 was abolished upon treatment with quercetin. (FIG. 1C). Many modifications and other embodiments of the subject matter described herein will come to mind to one skilled in the art to which the subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter described herein is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the foregoing list of embodiments and appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

THAT WHICH IS CLAIMED:

1. A lipid nanoparticle comprising a compound of Formula I:

wherein,

R¹ is selected from the group consisting of hydroxyl, -0-O-C6 alkyl, and -0-P(0)(OR^x)₂, wherein R^x is independently hydrogen or Ci-Ce alkyl;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from the group consisting of hydrogen, hydroxyl, -0-(O-C6 alkyl), and -O- P(0)(OR^x)₂;

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, or R¹⁰ is -O- P(0)(OR^x)₂;

wherein said compound of Formula I is encapsulated by a lipid bilayer wherein;

said lipid bilayer comprises an inner leaflet and an outer leaflet, wherein said inner leaflet comprises a first lipid and said outer leaflet comprises a second lipid, wherein said lipid bilayer is asymmetric.

2. The lipid nanoparticle of claim 1, wherein said lipid nanoparticle has diameter of about 20 nm to about 90 nm.

3. The lipid nanoparticle of claim 1, wherein said lipid nanoparticle has a diameter of about 25 nm to about 45 nm. 4. The lipid nanoparticle of claim 1, wherein said compound of Formula I is present in an amount of at least 25 % wt. of said lipid nanoparticle.

5. The lipid nanoparticle of claim 1, wherein said inner leaflet comprises a neutral or anionic lipid. 6. The lipid nanoparticle of claim 5, wherein said lipid is DOPA.

7. The lipid nanoparticle of claim 1, wherein said outer leaflet comprises one or more of cholesterol, a cationic lipid, or a neutral lipid. 8. The lipid nanoparticle of claim 7, wherein said outer leaflet comprises a lipid selected from the group consisting of cholesterol, DOTAP, DSPE-PEG, and DSPE-PEG- AA, and combinations thereof.

9. The lipid nanoparticle of claim 7, wherein said outer leaflet further comprises a targeting ligand.

10. The lipid nanoparticle of claim 1, wherein at least two of said R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from the group consisting of hydroxyl, -0-(O-C6 alkyl), and -0-P(0)(OR^x)₂, wherein R^x is independently hydrogen or Ci-C₆ alkyl.

11. The lipid nanoparticle of claim 10, wherein R¹, R², R⁴, R⁷, and R⁸ are independently selected from the group consisting of hydroxyl and -0-P(0)(OH)₂. 12. The lipid nanoparticle of claim 10, wherein R¹ is hydroxyl.

The lipid nanoparticle of claim 10, wherein R¹ is -0-P(0)(OH)₂

14. The lipid nanoparticle of claim 13, wherein the compound of Formula I is

15. The lipid nanoparticle of claim 1, wherein said nanoparticle comprises a compound of Formula I which is:

said inner leaflet comprises DOPA; and said outer leaflet comprises a lipid selected from the group consisting of DOTAP, cholesterol, DSPE-PEG, and DSPE-PEG-AA, and mixtures thereof.

A pharmaceutical composition comprising said lipid nanoparticle of a pharmaceutically acceptable excipient.

A method of preparing said lipid nanoparticle of claim 1, comprising: contacting a first reverse emulsion comprising a compound of Formula I with a second reverse emulsion comprising a reagent that is capable of forming a species that can combine with said compound of Formula I to form a precipitated LCP-QP core, wherein at least one of said first and second reverse emulsions further comprises a neutral or anionic lipid and;

b. allowing said precipitated LCP-QP core to form; and

c. contacting said precipitated LCP-QP core from (b) with one or more lipids to prepare a lipid nanoparticle comprising a compound of Formula I.

The method of claim 17, wherein said neutral or anionic lipid is DOPA.

19. The method of claim 18, wherein said one or more lipids from step (c) are selected from the group consisting of DOTAP, cholesterol, DSPE-PEG, and DSPE- PEG- AA, and mixtures thereof. 20. A method of treating a cancer comprising administering said lipid nanoparticle of claim 1 to a subject.

21. The method of claim 20, wherein said cancer is a carcinoma. 22. The method of claim 21, wherein said carcinoma is bladder carcinoma.

23. The method of claim 20, further comprising administering an additional bioactive compound. 24. The method of claim 23, wherein said additional bioactive compound is a cisplatin nanoparticle.

25. A method of reducing the size of a tumor, comprising contacting the tumor with the lipid nanoparticle of claim 1, wherein said tumor is reduced.

26. A method of remodeling the tumor microenvironment in a subject, comprising contacting the subject with the lipid nanoparticle of claim 1.

27. The method of claim 26, wherein the Wntl6 expression in the tumor microenvironment is downregulated.

28. The method of claim 26, wherein the level of a-SMA-positive fibrolast is decreased.

29. The method of claim 26, further comprising administering an additional bioactive compound. 30. The method of claim 29, wherein said additional bioactive compound is a cisplatin nanoparticle.

31. The method of claim 30, wherein said administering can be

simultaneous, sequential or separate.

32. The method of claim 30, wherein said administration of said lipid nanoparticle of claim 1 with said cisplatin nanoparticle has a synergistic effect on tumor reduction.

33. The method of claim 27, wherein said lipid nanoparticle further comprises a targeting ligand.