WO2012138379A2 - Multifunctional chelator-free radioactive nanoparticles for imaging and therapy - Google Patents

Abstract

Methods and composition for improving therapeutic and imaging potential of radioactive nanoparticles are provided. For example, in certain aspects methods for preparing a composition containing a nanoparticle having an integrated metal radioisotope and uses thereof are described. Furthermore, the invention provides such a composition.

Description

DESCRIPTION

MULTIFUNCTIONAL CHELATOR-FREE RADIOACTIVE NANOPARTICLES

FOR IMAGING AND THERAPY

[0001] This application claims the benefit of United States Provisional Patent Application No. 61/394,654, filed October 19, 2010, the entirety of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] This invention was made with government support under grant nos. R01 CAI 19387 and CAI 19387— 05S awarded by the National Institute of Health and grant no. W81XWH-07-2-0101 awarded by the U.S. Department of Army Telemedicine and Advanced Technology Research Center. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the field of diagnosis and therapy, especially for cancer patients. More particularly, it concerns radiolabeled nanoparticles with a plurality of diagnostic and therapeutic functions.

2. Description of Related Art

[0004] Radioisotopes have been introduced to various nanoparticles (NPs), including quantum dots (Schipper et al., 2007; Cai et al., 2007), gold NPs (Lu et al., 2010; Melancon et al., 2008; Zhang et al., 2009), carbon nanomaterials (McDevitt et al., 2007; Liu et al., 2007), and polymeric NPs (Pressly et al., 2007; Schluep et al., 2009; Yang et al., 2007; Yang et al., 2009), through radionuclide labeling to allow noninvasive in vivo nuclear imaging of NPs' pharmacokinetics, tissue distribution, and clearance. In several studies, PET has been used to investigate the pharmacokinetics and biodistribution of ⁶⁴Cu-labeled NPs (Lu et al., 2010; Schluep et al., 2009). In all of these studies, the radioisotopes are linked to NPs through chelators such as diethylene triamine pentaacetic acid or 1, 4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid to form stable complexes.

[0005] However, there are two inherent limitations associated with the use of radiometal-chelator complexes for nuclear imaging and in vivo study of NPs (Sanhai et al., 2008). First, the physico chemical properties of NPs attached to radiometal-chelator complex are not exactly the same as those of NPs without radiotracers. It is well known that the biodistribution and pharmacokinetic properties of NPs are influenced by their surface properties (Schipper et ah, 2009). Therefore, the data obtained using NPs conjugated with radiometal-chelator complexes may not reflect the pharmacological properties of unlabeled NPs. Second, the radiometal-chelator complexes may be detached from the surface of the NPs, or the radiometal ions may be displaced in vivo from the radiometal-chelator complexes owing to transchelation in the presence of high plasma protein concentrations, which again could lead to in vivo data not accurately reflecting the pharmacokinetics and biodistribution of NPs (Bass et al, 2000). [0006] Therefore, there is a continued need in the medical arts for developing chelator-free radiolabeling techniques with independent analytical tools.

SUMMARY OF THE INVENTION

[0007] Nanotechnology is an applied science that creates and studies molecules or aggregates that have an overall size in the 1-1000 nm range (<1 μιη). In the last few years, nanoparticles have been used in biomedical studies investigating new and improved diagnosis and therapy agents. Oncology is one of the disciplines that has benefited most from nanotechnology. Radioisotopes linked to nanoparticles through a chelating agent have been investigated in non-invasive nuclear imaging. However, chelator-based radio-labeling techniques have problems in several aspects as described above, particularly, chelated radioisotopes in in vivo administered radiolabeled nanoparticles are inherently prone to displacement by serum proteins with metal-binding activity, reducing the available radioactivity available for tumor uptake.

[0008] Therefore, aspects of the present invention overcome a major deficiency in the art by providing a novel inorganic nanoparticle, wherein the nanoparticle comprises a metal radioisotope as an integral component of the nanoparticle. One advantage of the nanoparticle is to obviate conjugation of radioisotopes to the nanoparticle by a chelating agent, and thus the issues associated with chelate instability, as such stability is the primary reason for accumulation of high radioactivity in non-target organs. In particular aspects, the nanoparticle has small size, strong absorption in near infrared absorption and integration of radioisotope into the nanoparticle, making it ideally suited for a plethora of functions in diagnosis and therapy. For example, the nanoparticle can be used for the "see and treat" strategy in which nuclear imaging is used to noninvasive ly monitor nanoparticles' in vivo distribution and near-infrared light is applied to tumor sites where accumulation of nanoparticles in the tumor is confirmed. Surprisingly, the use of such nanoparticles for administering both radiotherapy and photothermal ablation therapy for tumor has a synergistic effect than either treatment alone. [0009] Nanoparticles of the embodiments may have any transition metal as an integral component, such as the core of nanoparticles. Transition metals in the nanoparticles are virtually insoluble in aqueous solutions due to partially covalent character of the crystal lattice. Non-limiting examples of transition metals in the nanoparticles include one or more of zinc, molybdenum, cadmium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, yttrium, zirconium, niobium, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, rohrium, hassium, meitnerium, darmstadtium, roentgenium, and copernicium. In a particular example, the nanoparticle is a copper nanoparticle. [0010] Nanoparticles may be made from metal chalcogenides, such as metal oxides or metal sulfides. These chalcogenides all have an element from Group VI of the periodic table, including oxides, sulfides, selenides, and tellurides, and compounds of polonium. In particular aspects, the nanoparticle may be a copper chalcogenides, such as copper sulfides or copper oxides. Copper sulfides that may be used include a family of chemical compounds and minerals with the formula CuxS where x = 1-2, such as CuS (covellite), Cui.₅S, Cu₂S or a combination thereof. Thus, in some aspects, metal sulfide nanoparticles such as copper sulfide nanoparticles can be formulated by varying the molar ratio of metal to sulfide during

2+ 2- particle formation. For example, a [metal (e.g., Cu )] to [S ^"] ratio of between about 1 and about 2, such as about 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.6, 1.9 or 2.0 can be used. As further detailed herein by increasing the metal to sulfide ratio the absorbance peak of the resulting nanoparticles can be increased in wavelength.

[0011] The nanoparticle can be made radioactive by utilizing radioactive starting material or by external activation (such as exposure to neutrons) after the nanoparticles are synthesized. In some aspects, the nanoparticle can have one metal composition throughout the particle (impurities may be present but not desired). In some other aspects, the nanoparticle can have more than one metal compositions. [0012] For example, the metal integral to the nanoparticle may be a mixture of a metal radioisotope (e.g. , a copper radioisotope) and a stable metal isotope (e.g., a stable

1 2 3 4 5 6 7 copper isotope) at ratio of about, at least or at most 1 , 10^" , 10^" , 10^" , 10^" , 10^" , 10^" , 10^" , 10^"

8 -9

, 10^" or any range derivable therein. The metal radioisotope and stable metal isotope may be of the same metal type or different metal type. The metal radioisotope or stable isotope may be, for example, a radioisotope or a stable isotope of gold, silver, copper, iron, zinc, or any other transition metal. In certain aspects, the radioisotope is a gamma emitter, a positron emitter or a beta-emitter. For instance, the radioisotope can be Cu-64, In-I l l , Tc-99m, Ga- 67, Ga-68, Y-90, Lu-177 or a mixture thereof. In a particular aspect, the radioisotope may be a copper radioisotope. For example, the copper radioisotope is Cu-64, Cu-67, Cu-62, Cu-63, Cu-61 , or any other copper radioisotopes. Depending on the desired application, the radioisotopes may be chosen which are alpha, beta, or gamma emitters.

[0013] In certain aspects, the nanoparticle may further comprise a surface stabilizer and/or targeting agent. Non-limiting examples of surface stabilizer include citrate, polyacetylene glycol, cysteine, folic acid, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol, polylysine, polyvinyl alcohol, human serum albumin, bovine serum albumin, hyaluranic acid, polyethyleimine (PEI), polyvinylprrolidone (PVP) or any chemical compound that passivates the surface of nanoparticle and protects the nanoparticle from further growth. For example, the polyacetylene glycol is polyethylene glycol (PEG). The polyethylene glycol may have a molecular weight ranging from 500- 20,000 dalton, or more particularly, from 1000-5000 dalton. For example, the PEG may have at least, at most or about 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000 dalton, or any range derivable therefrom. In some aspects, the surface stabilizer can also act as a targeting agent (e.g., hyaluranic acid and folic acid can be used to target particular classes of tumor cells).

[0014] The surface stabilizer may be virtually composed of a monomolecular layer. The surface property of nanoparticles, particularly property of the surface stabilizer layer, could determine the solubility, chemical reactivity, biodistribution, or pharmacokinetic properties of nanoparticles. Very small size, uniformity of particles, and critical role of the surface distinguishes nanoparticles from both colloidal and molecular systems.

[0015] The term nanoparticle may be used to describe objects of from 1 to 300 nm in diameter represented by vesicles, polymers or colloids. These species are used for drug delivery and for diagnostic purposes. The nanoparticle may have a particle diameter of less than 1000 nm, about 10 nm to about 500 nm, about 10 to 200 nm, about 20 to about 200 nm, about 20 to about 100 nm, about 20 to about 30 nm, or any range derivable therein. The particle diameter may be a mean or an average diameter. [0016] The term nanoparticles in certain aspects of the present embodiments includes particles that have a diameter of at least, about, or at most, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 150 nm, or any intermediate ranges.

[0017] Photothermal ablation (PTA) therapy has been used in minimally invasive treatments for cancer. Nanoparticles with optical properties such as infrared absorption may be used as photothermal coupling agents for PTA therapy to convert optical energy to thermal energy, enabling optical energy into tumors for thermal ablation. In certain aspects, the nanoparticle has a near-infrared absorption. For example, the nanoparticle has a maximum absorption at about, at least or at most 700, 750, 800, 850, 900, 950, 980, 1000, 1064, 1100 nm or any intermediate range.

[0018] In a further aspect, the nanoparticle may be coupled with a tumor targeting moiety, a targeting ligand, a therapeutic, an imaging agent, a peptide, an antibody, a nucleic acid, a small molecule, or a polymer. For example, the targeting ligand may be a CD44 targeting ligand, such as hyaluronic acid (HA), for selective targeting of nanoparticles to CD44 positive tumor cells. In a further aspect, the targeting ligand may bind to folate receptors, such as a targeting ligand comprising folic acid (FA). For example, NPs comprising FA can be used to target head and neck tumors or ovarian tumors.

[0019] In certain aspects, the nanoparticle may be formulated in a pharmaceutically acceptable nanoparticle composition. The nanoparticle formulation can be a liquid formulation or a solid formulation, such as a powder. Particularly, the composition may be dehydrated or lyophilized for long term storage with improved stability. Alternatively, the composition may be present in a substantially aqueous solution. The composition may be rehydrated or re-suspended in a solution or liquid from the previously lyophilized composition. The composition used in the methods may be previously dehydrated, lyophilized or in some other aspects, an aqueous solution or liquid formulation of previously lyophilized or dehydrated composition, an effective amount of which are administered to the subject. The present invention also provides, in certain aspects, previously lyophilized or dried composition after being stored at 4 degree for at least 1 week, for at least 3 weeks, for up to 4 weeks, or any period derivable therein, for treating the disease with retained activity after resuspension or rehydration. [0020] Application of nanoparticles having integral radioisotopes may significantly improve the detection limit of cancer, increase effectiveness of radiotherapy and photothermal ablation therapy, and reduce the overall side effects. In certain aspects, there may be provided a method of treating an angiogenic or a malignant tissue in a subject. The method may comprise administering to a subject or an angiogenic or a malignant tissue in a subject an effective amount of such inorganic nanoparticles. In certain aspects, the nanoparticle has a metal radioisotope as an integral component of the nanoparticle. The method may be further defined as a radiotherapy or brachytherapy due to the radiotherapeutic effect of the integrated radioactive isotope in the nanoparticle, such as Cu-64 or Cu-67.

[0021] For detection of angiogenic or malignant tissues, the nanoparticle may enter an angiogenic or a malignant tissue in the subject. In particular, the nanoparticle can be a metal chalcogenide comprising a metal radioisotope as an integral component. For example, the metal radioisotope comprises copper radioisotope.

[0022] For imaging an angiogenic or a malignant tissue, the method may comprise imaging the nanoparticles in the subject after a period of time that is sufficient for the nanoparticles to enter an angiogenic or a malignant tissue. The imaging method may be any optical or nuclear imaging method, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), or photoacoustic tomography. In certain aspects, a combined imaging method can be used. For example, PET imaging can be used in conjunction with photoacoustic imaging (e.g., for lymph node mapping). [0023] The imaging may further comprise imaging of lymph nodes in the subject. For example, [⁶⁴Cu]CuS NP can be used for lymph node imaging with PET/CT and/or photoacoustic imaging. This application can be potentially used in noninvasive detection of lymph node metastasis. The ability to detect lymph nodes metastasis is extremely important in cancer staging, determining prognosis, and monitoring treatment outcome. [0024] The method may further comprise administering a photothermal ablation therapy to the tissue having the nanoparticles. The photothermal ablation therapy may comprise administering to the tissue a near-infrared light, for example, from a near-infrared laser at about 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 W/cm , or any intermediate range for about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes or any intermediate range. The near-infrared light may have a wavelength of about 700, 750, 800, 850, 900, 950, 980, 1000, 1064, 1 100 nm or any intermediate range. In certain aspects, a photothermal ablation therapy comprises use of a laser having a wavelength of about 900 nm to 1 100 nm (e.g., about 980 nm or 1064 nm). A laser for use in the methods of the embodiments can be a continuous wave laser or a pulsed laser (e.g., a nanosecond, microsecond or millisecond pulsed laser).

[0025] The subject may be any animal, like a mammal. In particular, the subject may be a human or a mouse. The subject may have cancer. The cancer may include melanoma, leukemia, ovarian cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, breast cancer, gastric cancer, colon cancer, head and neck cancer, esophagus cancer, synovium cancer, brain cancer, bronchus cancer or any known cancer. [0026] For a safe and effective dosage, the nanoparticle or nanoparticle formulation may be administered at a dose of at least, at most or about lxlO⁴, lxlO⁵, lxlO⁶, lxlO⁷, lxlO⁸, lxlO⁹, lxlO¹⁰, lxlO¹¹, lxlO¹², lxlO¹³, 2xl0¹⁴, 3xl0¹⁴, 4xl0¹⁴, 5xl0¹⁴, 8xl0¹⁴, lxlO¹⁵, lxlO¹⁶,

17 18 19 20

1x 10 , 1x10 , 1x10 , 1x10 particles or any intermediate range per kg body weight or per tumor. The nanoparticle or nanoparticle formulation administered to the subject may have a radioactivity of at least, at most or about 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 250, 300, 350, 400 μα per kg body weight or per tumor. The nanoparticle administered may have a radioactivity of at least, about or at most 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 35, 30, 35, 40, 45, 50 mCi (or any range derivable therein) per subject. In certain aspects, the nanoparticle or nanoparticle formulation may be administered about 500 mg/m (body

2 2

surface)/day, about 10 to about 300 mg/m /day, 20 to about 200 mg/m /day, about 30 to

2 2 2 about 200 mg/m /day, about 40 to about 100 mg/m /day, about 50 to about 100 mg/m /day or any range derivable therein to a subject such as a human.

[0027] The nanoparticle or nanoparticle formulation may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, intrathecally, locally, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage. For example, the composition may be administered by injection or infusion. [0028] To have a better therapeutic benefit, the nanoparticle or nanoparticle formulation may be administered in combination with at least an additional agent such as a radiotherapeutic agent, a hormonal therapy agent, an immunotherapeutic agent, a chemotherapeutic agent, a cryotherapeutic agent and/or a gene therapy agent.

[0029] In certain aspects, there may be provided a method of preparing such a nanoparticle. The method may comprise providing a mixture comprising a copper radioisotope, a stable copper salt and a non-copper chalcogenide. The mixture may be incubated at a reaction condition having a temperature of at least, about or at most 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130°C, or any intermediate ranges. The reaction condition may have a pH of at least, about or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or any intermediate ranges. The reaction condition may last at least, at most or about 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1440, 1500, 1600 minutes or any intermediate ranges. The mixture may be aqueous or may be non-aqueous. The mixture may further comprise a source of a surface stabilizer for the nanoparticle, such as citrate or PEG. The ratio of copper radioisotope and a stable copper salt may be about, at least or at most 1, 10^"1, 10^"2, 10^"3, 10^"4, 10^"5, 10^"6, 10^"7, 10^"8, 10^"9 or any range derivable therein. The mixture may further comprise an adjuvant such as a tumor targeting moiety, a targeting ligand, a therapeutic, a peptide, an antibody, a nucleic acid, a small molecule, or a polymer. The reaction condition may be also suitable for coupling the nanoparticle with any adjuvant.

[0030] In certain aspects, there is provided a method of preparing a nanoparticle coated with a homing ligand. The method comprising providing a composition comprising a copper radioisotope, a stable copper salt and a non-copper chalcogenide at the ratio specified above. The reaction may be carried out under the reaction conditions (temperature, pH, solvent, and time) specified in the foregoing paragraph. The mixture may further comprise a source of a surface stabilizer for the nanoparticle, such as citrate or PEG in the presence of the homing ligands. Alternatively, nanoparticle may be coated with homing ligands after nanoparticles are formed using standard bioconjugation chemistry, click chemistry, and other methods of introducing homing ligands.

[0031] The skilled artisan will understand that there may also be provided methods for preparing a composition comprising admixing the nanoparticle into a pharmaceutically acceptable nanoparticle formulation. The method may further comprise homogenization and/or sonication for homogenous dispersion. In order for long term storage, the method may further comprise dehydrating or lyophilizating the formulation. For in vivo administration, the method may further comprise rehydrating or resuspending in a solution or liquid.

[0032] Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

[0033] As used herein the terms "encode" or "encoding" with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan however these terms may be used interchangeably with "comprise" or "comprising" respectively.

[0034] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0035] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0036] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0037] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0038] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0039] FIGS. 1A-B: Characterization of CuS NPs. (FIG. 1A) TEM image of Cit- CuS NPs. Inset: particle size distribution. (FIG. IB) Absorption spectra of Cit-CuS NPs and PEG-CuS NPs. Cit, citric acid; PEG = polyethylene glycol.

[0040] FIGS. 2A-C: Pharmacokinetic profiles of Cit-[⁶⁴Cu]-CuS NPs (n = 3) and PEG-[⁶⁴Cu]-CuS NPs (n = 5) following intravenous administration in mice (FIG. 2A); and biodistribution (FIG. 2B) of Cit-[⁶⁴Cu]-CuS NPs (n = 5) and PEG-[⁶⁴Cu]-CuS NPs (n = 5) at 24 h after intravenous injection in mice bearing subcutaneous U87 glioma xenografts. Data represent mean ± standard deviation. FIG. 2C: Biodistribution of PVP-and PEG-coated CuSNPs at 24 h after iv injection in mice bearing subcutaneous U87. PVP - polyvinylpyrrolidone .

[0041] FIG. 3: Micro -PET/CT images of nude mice-bearing s.c. U87 glioma xenografts acquired at 1 , 6, and 24 h after i.v. injection of PEG-[⁶⁴Cu]-CuS NPs. Yellow arrow: tumor; orange arrow: bladder; Red arrow: standard.

[0042] FIGS. 4A-B: PEG-CuS NPs acted as an efficient photothermal coupling agent. (FIG. 4A) Temperature elevation over a period of 9 min of exposure to NIR light (808 nm, 16 W/cm") at various PEG-CuS NP concentrations. Water was used as control. (FIG. 4B) Temperature change (ΔΤ) over a period of 9 min as a function of PEG-CuS NP concentration expressed as CuS molecular units.

[0043] FIGS. 5A-C: PEG-CuS NPs induced photothermal destruction of U87 tumors in vivo. (FIG. 5A). Photographs of tumor-bearing mice before and at 24 h after NIR laser irradiation (12 W/cm at 808 nm for 5 min). (FIG. 5B). Representative microphotograph of tumors removed at 24 h after NIR laser treatment. The tissues were cryosectioned into 5 μιη slices and stained with H&E. Bar, top, 200 μηι; Bar, bottle, 20 μηι. (FIG. 5C). Quantitative analysis of percentage of necrosis zone induced by various treatments. The data were measured as a percentage of the whole tumor area. Asterisk indicates statistic significance compared to the no-treatment control (p = 0.006). Error bars, standard deviation (n = 5). NPs, CuS nanoparticles; NIR, near-infrared laser; T, tumor.

[0044] FIG. 6: Nuclear magnetic resonance (NMR) study of Cit-CuS NPs and PEG- CuS NPs. To confirm the occurrence of ligand exchange reaction, both CuS NPs as prepared and CuS NPs after ligand exchange were characterized using 1H NMR with the dry sample dispersed in D₂0. For CuS NPs as prepared by using sodium citrate, two singlet peaks were observed (2.689 and 2.687 ppm), corresponding to the proton of methylene of sodium citrate. In the spectra for CuS NPs after ligand exchange, the two citrate peaks were thoroughly substituted by a peak at 3.65 ppm characteristic of PEG, suggesting that CuS NPs were coated by PEG after ligand exchange reaction with SH-PEG.

[0045] FIG. 7: X-ray diffraction pattern (XRD) of CuS NPs. All diffraction peaks can be indexed as covellite-phase copper sulfide with lattice parameters similar to those of Joint Committee on Powder Diffraction Standards card 79-2321. The relatively broad diffraction peaks reflect the small size of copper sulfide crystals. No obvious impurity peaks were detected, indicating the acquirement of covellite CuS with high quality.

[0046] FIG. 8: Hydrodynamic size of CuS NPs by DLS. The particle size of Cit-CuS NPs and PEG-CuS NPs are 11.7 nm and 31.6 nm, respectively.

[0047] FIG. 9: High resolution transmission electron microscope (HRTEM) image of PEG-CuS NPs.

[0048] FIG. 10: Stability of Cit-CuS NPs and PEG-CuS NPs in different buffer solutions at 37°C for 7 days: photograph. Solutions were as follows: 1, water; 2, citrate solution (0.4 mM); 3, acetate buffer solution (100 mM); 4, NaCl solution(100 mM); 5, 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid buffer (HEPES); 6, Phosphate buffered saline (PBS); 7, bovine serum albumin solution (BSA) (50 mM); 8, PBS containing 10% fetal bovine serum (FBS); and 9, 100% FBS. [0049] FIG. 11: Radiolabeling efficiency and stability of radioactive CuS NPs. Representative radio-ITLC chromatogram of [⁶⁴Cu]-CuS NP solution in PBS and fetal bovine serum (FBS) at 37°C at 24 h.

[0050] FIG. 12: Instant thin layer chromatography (ITLC) of PEG-[⁶⁴Cu]-CuS NPs from urine 24 h after i.v. injection. ITLC revealed that PEG-[⁶⁴Cu]-CuS NPs was cleared from the renal system in the form of CuS NPs, indicating that a fraction of smaller CuS NPs were capable of clearance by the kidney in vivo.

[0051] FIG. 13: Cell viability after NIR laser irradiation. Fluorescence photomicrographs of U87 cells after PTA therapy in vitro. U87 cells were treated with PEG- CuS NPs for 2 h followed by NIR laser (808 nm) irradiation. Treatments with combined PEG-CuS NPs at 500 μΜ and NIR laser at power density greater than 16 W/cm caused significant depletion of viable tumor cells. No apparent damage to cells was observed when cells were treated with PEG-CuS NPs alone or NIR laser alone. Viable cells were stained green with calcein AM. Bar, 20 μιη. [0052] FIG. 14: Cytotoxicity of CuS nanoparticles. Cell viability test show with varying CuS NPs concentration of 0-1 mM at 48 h.

[0053] FIG. 15A-D: A, schematic of the experimental design for antitumor activity study in nude mice bearing s.c. BT474 tumors. B, tumor growth curves after treatments with radiotherapy (RT), photothermal therapy (PTT), and combined radio-photothermal therapy (RPTT). C, photographs of representative mouse from each treatment group on day 30 after treatment. D, Representative microphotograghs of tumors stained with hematoxylin and eosin. Tumors were removed on day 30 after the initiation of treatment. Bars: 200 μιη (top panel); 20 μιη (bottom panel).

[0054] FIG. 16: A, body weight changes as a function of time after treatments. B, histology of H&E stained tissues. No noticeable abnormality was observed in heart, liver, spleen, kidney, and lung.

[0055] FIG. 17: PTA images of chick breast tissues with imbedded CuS NP- containing gels containing, (a) Photograph of the cross section of chicken breast tissue with gelatin objects containing CuS NP (1 mM) and a steel needle, (b) Two dimensional photoacoustic image at the depth of ~ 5 cm from laser illuminated surface acquired with 800 nm laser.

[0056] FIG. 18: MicroPET/CT images of a rat 24 h after subcutanenous injection of the [64Cu]CuS NP in the right front paw. Arrows: lymph nodes. [0057] FIG. 19. Photothermal conducting effect of HA-CuS NP. HA-CuS NP showed high photothermal conducting efficiency. HA, hyaluronic acid.

[0058] FIG. 20. Selective binding of ⁶⁴Cu-labeled HA-CuS NP to human breast cancer MDA-MB231 cells that express CD44 receptors. Cells incubated with ⁶⁴Cu-labeled HA-CuS NP showed significantly higher uptake in the cancer cells than non-targeted 64Cu- labeled CuS NP, and this uptake could be completely blocked by co-incubation with cold HA.

[0059] FIG. 21. Selective binding of FITC-labeled HA-CuS NP to CD44-positive HeLa cells. Cells incubated with FITC-labeled HA-CuS NP showed selective uptake in HeLa cells. [0060] FIG. 22. Selective photothermal ablation of HeLa cells exposed to HA-CuS

NP and near-infrared (NIR) laser (808 nm). Cells were treated with EthD-1 for visualization of dead cells (red) and Calcein AM for visualization of viable cells (green).

[0061] FIG. 23. Physical properties of a CuS nanoparticles with incorporated Ga-68. ITLC results show that the Ga-68 is stably incorporated into CuS NP (FIG. 23A) and does not significantly alter the absorbance properties of the particles (FIG. 23B).

[0062] FIG. 24. Graph shows the optical absorbance peak of nanoparticles

2+ 2- 2+ 2- synthesized with different ratios of [Cu ] and [S ^"]. The molar ratio of [Cu ] to [S ^"] were 1.0, 1.2, and 1.5 for CuS NP that peaked at 930 nm, 984 nm, and 1046 nm, respectively.

[0063] FIG. 25A-B. CuS NPs efficiently convert laser energy to heat at 980 nm (A) or 808 nm (B). CuNPs were compared to hollow gold nanospheres (HAuNS), carbon nanotubes (CNTs), graphene and water alone (control). The concentration of all nanoparticles were 100 μg/ml. [0064] FIG. 26. CuS NPs can efficiently and repeatedly convert laser energy to heat. Particles were dispersed at a concentration of 50 mg/L and exposed to pulses of a 3W laser beam (1 cm diameter) having a wave length of 808 nm over the time periods shown.

[0065] FIG. 27. CuS NPs can efficiently convert pulsed laser energy to heat with superior spatial control. HeLa cells were treated as indicated with no therapy (Cell only); only laser application (Cell + laser) only NPs [CuS NP (2 OD)] or with laser in the presence of CuS NPs. Laser exposures in each case were for 10 seconds with nanosecond pulses using a 17 mJ, 1064 nm laser beam.

[0066] FIG. 28. Physical properties of FA coated CuS NPs. Particles were assessed for peak absorbance (A); photothermal conduction (B) particle size (C); and stability in a variety of buffers including FBS, NaCl, HEPES, BSA, PBS and water (D). FA, folic acid.

[0067] FIG. 29. Binding properties of FA coated CuS NPs on KB cells. KB cells were imaged to determine whether FA coated CuS NPs could specifically bind the cells. Cells were imaged to visualize DAPI staining, Cy5.5 staining or both in the presence of PEG- CuS NPs (1 mM), FA-CuS NPs (1 mM) or FA-CuS NPs (1 mM) with excess FA as indicated. FA, folic acid.

[0068] FIG. 30. FA-CuS NPs selectively target KB cells for photothermal ablation. A 3W, 2 min NIR laser exposure was applied to cells in the presence of FA-CuS NPs (1 mM); PEG-CuS NPs (1 mM); FA-CuS NPs plus blocking FA or without NPs (laser alone), as indicated. FA, folic acid.

[0069] FIG. 31. FA-CuS NPs selectively target KB tumors in vivo. Biodistribution of FA-coated CuS NPs after injection in mice KB tumors in the presence (Blocking) or absence of excess FA (FA-CuS). Tissue abbreviation are blood (Bl), heart (He), liver (Li), spleen (Sp), kidney (Ki), lung (Lu), stomach (St), intestinal (In), muscle (Mu), bone (Bo), brain (Br) and tumor (Tu). FA, folic acid.

[0070] FIG. 32. FA-CuS NPs are effective mediators of photothermal ablation in KB tumor tissue. Images show cells from KB tumors that were exposed to laser energy in the absence of NPs (Non-treatment, the presence of PEG-CuS NPs or FA-CuS NPs. FA, folic acid. [0071] FIG. 33. PEG-CuS NPs provide an excellent contrast for photoacoustic imaging. PEG-CuS NPs were imaged by photoacoustic tomography with a 1064 nm, 36.5 mJ laser at a depth of 1 cm. The samples in agarose gel were imbedded in chicken breast. The concentrations of PEG-CuS NP were 100 μ^πιΐ (1); 50 μg/ml (2); 25 μg/ml (3); 12.5 μ^πιΐ (4); 6.25 μg/ml (5) andr 3.125 μg/ml (6). (7) indicates agarose gel alone as a control.

[0072] FIG. 34. MicroPET/CT image of PEG-[⁶⁴Cu]CuS NPs and ⁶⁴CuCl₂ 24 h after intratumoral injection into B474 tumors in mice.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Introduction

[0073] A novel class of nanoparticles having radioactive isotope as an integral component, such as chelator-free [⁶⁴Cu]-CuS nanoparticles (NPs) suitable for both PET imaging, radiotherapy and as photothermal coupling agents for photothermal ablation, were synthesized and evaluated. For example, [⁶⁴Cu]-CuS NPs were simple to make, possessed excellent stability, and allowed robust noninvasive micro-PET imaging. Furthermore, radioactive CuS NPs displayed strong absorption in the near-infrared (NIR) region, passive targeting (long circulation in the blood and extravasation at tumor vasculature) prefers the tumor site, and mediated ablation of tumor cells upon exposure to NIR light both in vitro and in vivo after either intratumoral or intravenous injection. In specific examples, PEG-[⁶⁴Cu]- CuS NPs acted as an efficient radiotracer for pharmacokinetics, biodistribution, and PET imaging, and PEG-CuS NPs acted as an efficient photothermal coupling agent and photoacoustic contrast agent. PEG-[⁶⁴Cu]-CuS NPs showed high uptake in U87 human glioblastoma xenografts owing to enhanced permeability and retention effect.

[0074] Active targeting of such nanoparticles, such as radioactive CuS NPs, to solid tumors by conjugating receptor-specific or tumor-specific targeting moieties onto the NPs' surface may be contemplated in certain aspects. Improved tumor uptake of radioactive nanoparticles could increase the efficacy of photothermal ablation therapy, decrease the energy dose of the laser, and minimize the potential damage to surrounding normal tissues.

[0075] In addition to serving as a photothermal mediator, such radiolabeled NPs may also have an application for therapy, like radiotherapy or photothermal ablation therapy. Thus, the combination of small diameter, strong NIR absorption, and integration of a radioisotope as a structural component makes this novel nanopaticle ideally suited for combining its intrinsic nuclear and optical properties in multifunctional molecular imaging and therapy, for example, image-guided photothermal ablation therapy.

II. Metal Isotopes

[0076] In certain aspects, there may be provided nanoparticles having a metal radioisotope as an integral component. For example, the radio isotope can be astatine-211, chromium-51, cobalt-57, cobalt-58, copper-67, Eu-152, gallium-67, gallium-68, indium-I l l, iron-59, lutetium-177m, rhenium-186, rhenium-188, selenium-75, strontium-89, technicium- 99m, thorium-227 and/or yttrium-90. In particular, the radioisotope may be a copper radioisotope. Non-limiting examples of copper radioisotope include Copper-64, Copper- 70ml, Copper-70m2, Copper-68m, Copper-69m, Copper-71m, Copper-72m, Copper-76m, Copper-52, Copper-53, Copper-54, Copper-55, Copper-56, Copper-57, Copper-58, Copper- 59, Copper-60, Copper-61, Copper-62, Copper-66, Copper-67, Copper-73, Copper-74, Copper-75, Copper-77, Copper-78, Copper-79, and Copper-80. In particular, the copper radioisotope may be Copper-64 (with a half-life of about 12.70 hours) or Copper-67 (with a half-life of about 61.83 hours).

[0077] A skilled artisan will recognize that the type of radioisotope employed will depend on what the nanoparticle is used for. For example, a positron emitter such as Ga-68 can be employed for PET imaging. Examples of radioisotope for use in gamma imaging include, but are not limited to IN-11, Tc-99m and Ga-67. For therapeutic application a beta emitter, such as Y-90 or Lu- 177 may be preferred.

III. Nanoparticle Formulation

[0078] In certain aspects, the present invention affords compositions and methods involving a nanoparticle formulation comprising a nanoparticle having an integrated radioisotope. The disclosed formulations, e.g., stabilized radioactive nanoparticles, are designed to improve the stability as well as the pharmacokinetics of this nanoparticle so that a better in vivo therapeutic activity can be achieved. For example, the nanoparticle formulation may decrease the loss of radioactivity in blood circulation and increase the drug distribution to tumor tissues and uptake by cancer cells and thus enhance anticancer activity.

[0079] As used herein, the term "nanoparticle" refers to any particles having dimensions in the 1-1,000 nm range. In some embodiments, nanoparticles have dimensions in the 2-200 nm range, preferably in the 5-150 nm range, and even more preferably in the 10- 100 nm range. In certain aspects, the nanoparticles may be conjugated to a targeting moiety to provide structures with potential application for targeted delivery, controlled release, enhanced cellular uptake and intracellular trafficking in vitro and in vivo.

[0080] For example, as a p-type semiconductor material, CuS is of great interest for use in catalysis and photovoltaic. Several methods have been developed for the preparation of copper sulfide nanoparticles (Haram et ah, 1996; Xu et ah, 2009; Huang et ah, 2010). Semiconductor CuS NPs are a new class of promising photothermal coupling agents. Thioglycolic acid-stabilized CuS NPs have been synthesized and demonstrated for photothermal destruction of tumor cells in vitro using a NIR laser beam centered at 808 nm.

[0081] In certain exemplary aspects of the present invention, it has been developed a process for the rapid synthesis of radioactive [⁶⁴Cu]-CuS NPs in which ⁶⁴Cu is an integral building block of CuS rather than chelated to NPs. Both citrate and polyethylene glycol (PEG)-stabilized [⁶⁴Cu]-CuS NPs were evaluated for their pharmacokinetic and biodistribution. In vivo passive targeting of the nanoparticles to tumors was demonstrated. The photothermal killing effect of PEG-stabilized CuS NPs both in vitro and in vivo following intratumoral (i.t.) or intravenous (i.v.) injection was further disclosed. This could be the first disclosure on the use of chelator-free NPs for PET imaging and the first demonstration of chelator-free NPs for dual imaging and therapy in vivo.

A. Additional Therapeutic Agents

[0082] The compositions of the present invention may optionally include one or more additional therapeutic agents. For example, the therapeutic agent may be a chemotherapeutic agent.

[0083] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; 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); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; 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; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (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, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; 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; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethyl amine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0084] Also included in the compositions may be anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above. B. Administration of Nanoparticle Formulation

[0085] The composition of this invention may enable accumulation of nanoparticles to tissues adjacent to or distant from an administration site. The radioactive isotope integral to the nanoparticle may be capable of providing a local or systemic biological, physiological or therapeutic effect. For example, the nanoparticle may act to kill cancer cells, or to control or suppress tumor growth, among other functions.

[0086] The nanoparticles are administered in an amount effective to provide the desired level of biological, physiological, pharmacological and/or therapeutic effect. The nanoparticle may stimulate or inhibit a biological or physiological activity. The concentration of the nanoparticle should not be so high that the composition has a consistency that inhibits its delivery to the administration site by the desired method. The lower limit of the amount of the nanoparticle may depend on its activity and the period of time desired for treatment. C. Targeting of Nanoparticles

[0087] Targeted delivery is achieved by the addition of ligands without compromising the ability of nanoparticles to deliver their loads. It is contemplated that this may enable delivery to specific cells, tissues and organs. The targeting specificity of the ligand-based delivery systems is based on the distribution of the ligand receptors on different cell types. It is preferable that the ligand to be conjugated to the nanoparticles may bind to the receptors that specifically or predominantly express in tumor cells so that the nanoparticle may be preferentially delivered to the tumor cells. For example, specific antibodies such as anti- CD20 (Rituximab) may be conjugated to the nanoparticles to deliver nanoparticles to malignant B-cells such as those of chronic lymphocytic leukemia and B-cell lymphoma. [0088] The targeting ligand may either be non-covalently or covalently associated with a nanoparticle, and can be conjugated to the nanoparticles by a variety of methods as discussed herein. Examples of proteins or peptides that can be used to target nanoparticles include transferin, lactoferrin, TGF-a, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin, as well as others (Gupta et al., 2005; Ferrari, 2005). [0089] In further aspects, the nanoparticle may be coupled to a tumor targeting moeity. In certain embodiments the tumor targeting moiety is an antibody that binds an antigen selected from the group consisting of, a gastrointestinal cancer cell surface antigen, a lung cancer cell surface antigen, a brain tumor cell surface antigen, a glioma cell surface antigen, a breast cancer cell surface antigen, an esophageal cancer cell surface antigen, a common epithelial cancer cell surface antigen, a common sarcoma cell surface antigen, an osteosarcoma cell surface antigen, a fibrosarcoma cell surface antigen, a melanoma cell surface antigen, a gastric cancer cell surface antigen, a pancreatic cancer cell surface antigen, a colorectal cancer cell surface antigen, a urinary bladder cancer cell surface antigen, a prostatic cancer cell surface antigen, a renal cancer cell surface antigen, an ovarian cancer cell surface antigen, a testicular cancer cell surface antigen, an endometrial cancer cell surface antigen, a cervical cancer cell surface antigen, a Hodgkin's disease cell surface antigen, a lymphoma cell surface antigen, a leukemic cell surface antigen and a trophoblastic tumor cell surface antigen.

[0090] In particular embodiments, the tumor targeting moiety is an antibody or ligand that binds an antigen or a receptor selected from the group consisting of 5 alpha reductase, a- fetoprotein, AM-1, APC, APRIL, BAGE, β-catenin, Bcl2, bcr-abl (b3a2), CA-125, CASP- 8/FLICE, Cathepsins, CD19, CD20, CD21, CD23, CD22, CD38, CD33, CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59 (791Tgp72), CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, EGFR, EMBP, Ena78, FGF8b and FGF8a, FLK-l/KDR, Folic Acid Receptor, G250, GAGE-Family, gastrin 17, GD2/GD3/GM2, GnRH, GnTV, gplOO/Pmel 17, gp-100- in4, gpl5, gp75/TRP-l, hCG, Heparanase, Her2/neu, HERS, Her4, HMTV, HLA-DR10, Hsp70, hTERT, IGFR1, IL-13R, iNOS, Ki 67, KIAA0205, K-ras, H-ras, N-ras, KSA, (C017- 1A), LDLR-FUT, MAGE Family (MAGE1, MAGE3, etc.), Mammaglobin, MAP 17, Melan- AJ, MART-1, mesothelin, MIC A/B, MT-MMP's, such as MMP2, MMP3, MMPI, MMP9, Moxl, MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1, NY-ESO-1, Osteonectin, pl5, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PDGF, Plasminogen (uPA), PRAME, Probasin, Progenipoietin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX gene, family, STAT3, STn, TAG-72, TGF-a, TGF-β, and Thymosin β15, nucleolin, Cal5-3, astro Intestinal Tumor Antigen (Cal9-9), ovarian Tumor Antigen (Cal25), Tag72-4 Antigen (CA72-4) and carcinoembryonic antigen (CEA). In a particular example, the targeting ligand may be a CD44 binding ligand, such as hyaluronic acid (HA).

IV. Methods of Using Nanoparticless

[0091] Nanoparticles may be used in an imaging or detection method for diagnosis or localization of tumor or angiogenic tissues. Any optical or nuclear imaging method may be contemplated, such as PET, SPECT, CT, or photoacoustic tomography. The integrated radioactive isotope in the nanoparticle may exert a radiotherapy on the tissue incorporating such nanoparticle. In addition, a photothermal ablation therapy may be administered to the tissue having the nanoparticle to enhance the therapeutic effect.

[0092] Nanoparticles may be used in PET. Positron emission tomography (PET) is a powerful and widely used diagnostic tool that has the advantages of high sensitivity (down to the picomolar level) and ability to provide quantitative imaging analyses of in vivo abnormalities (Scheinin et al., 1999; Eckelman, 2003; Welch et al, 2009). ⁶⁴Cu (Ti_/2 = 12.7 h; β⁺, 0.653 MeV [17.8%]; β^", 0.579 MeV [38.4%]) has decay characteristics that allow for both PET imaging and targeted radiotherapy for cancer (Shokeen and Anderson, 2009). It has been investigated as a promising radiotracer for real-time PET monitoring of regional drug concentration, pharmacokinetics, and dosimetry during radiotherapy (Shokeen and Anderson, 2009; Lu et ah, 2010). PET may be used in certain aspects to trace nanoparticles in vivo. [0093] Nanoparticles may also be used in SPET. Single photon emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required.

[0094] The SPET basic technique requires injection of a gamma-emitting radioisotope called radionuclide) into the bloodstream of the patient. In certain aspects the radioisotope is integrated into a nanoparticle, which has chemical properties which allow it to be concentrated in ways of medical interest for disease detection. In other aspects, a nanoparticle comprising a marker radioisotope, which is of interest for its radioactive properties, has been attached to a targeting ligand, which is of interest for its chemical binding properties to certain types of tissues. This marriage allows the combination of ligand and radioisotope (the radiopharmaceutical) to be carried and bound to a place of interest in the body, which then (due to the gamma-emission of the isotope) allows the ligand concentration to be seen by a gamma-camera.

[0095] Nanoparticles may also be used in CT. Computed tomography (CT) is a medical imaging method employing tomography created by computer processing. Digital geometry processing is used to generate a three-dimensional image of the inside of an object from a large series of two-dimensional X-ray images taken around a single axis of rotation. CT is used in medicine as a diagnostic tool and as a guide for interventional procedures. Sometimes contrast materials such as intravenous iodinated contrast are used. This is useful to highlight structures such as blood vessels that otherwise would be difficult to delineate from their surroundings. Using contrast material can also help to obtain functional information about tissues. [0096] Nanoparticles may also be used in photoacoustic tomography. Photoacoustic tomography (PAT), or photoacoustic computed tomography (PACT), is a materials analysis technique based on the reconstruction of an internal photoacoustic source distribution from measurements acquired by scanning ultrasound detectors over a surface that encloses the source under study. The PA source is produced inside the object by the thermal expansion that results from a small temperature rise, which is caused by the absorption of externally applied radiation of pulsed electromagnetic (EM) waves. This technique has great potential for applications in the biomedical field because of the advantages of ultrasonic resolution in combination with EM absorption contrast. PAT is also called optoacoustic tomography (OAT) or thermoacoustic tomography (TAT), with the term "thermoacoustic" emphasizing the thermal expansion mechanism in the PA generation. OAT refers particularly to light- induced PAT, while TAT is used to refer to rf-induced PAT.

[0097] In photoacoustic tomography (PAT), each temporal PA signal, measured at various detection positions, provides one-dimensional radial information about the PA source relative to the detector position; 2D surface scans offer other 2D lateral information about the PA source. Combining the temporal and spatial measurements affords sufficient information for a complete reconstruction of a 3D PA source. Because the PA signal received by each ultrasound detector is the integral of the ultrasound waves over the sensing aperture of the detector, the reconstruction algorithms depend on the detector apertures as well as the scanning geometries. Small-aperture detectors are often used to approximate point detectors, which receive PA signals originating from spherical shells, centered at each point detector, with radii determined by the acoustic times of flight. The three geometries commonly used are planar, cylindrical, and spherical surfaces. Both Fourier- and time-domain reconstruction formulas with point-detector measurements for these geometries have been well established. Besides, algorithms based on other detection methods, such as large-aperture (plane), line, or circle detectors have also been derived. [0098] Nanoparticles may also be used in photothermal ablation therapy.

Photothermal ablation (PTA) therapy has gained increasing attention in recent years as a minimally invasive alternative to conventional approaches to cancer treatment such as surgery and chemotherapy (Amin et ah, 1993;Nolsoe et ah, 1993; Fiedler et ah, 2001; Vogeland Venugopalan, 2003). NPs with unique optical properties— primarily gold nanostructures, such as gold nanoshells (Hirsch et ah, 2003; Loo e αί, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages (Chen et ah, 2007; Au et ah, 2008), and hollow gold nanospheres (Lu et ah, 2010; Melancon et al„ 2008; (Lu et αί, 2009), but also carbon nanotubes (Chakravarty et al., 2008 Burke et al., 2009)— have been investigated as photothermal coupling agents to enhance the efficacy of PTA therapy. These plasmonic nanomaterials exhibit strong absorption in the near-infrared (NIR) region (wavelength 700-1100 nm) and offer an opportunity to convert optical energy to thermal energy, enabling deposition of otherwise benign optical energy into tumors for thermal ablation of tumor cells.

V. Pharmaceutical Preparations

[0099] Where clinical application of the particles of the present invention is undertaken, it will generally be beneficial to prepare the particles as a pharmaceutical composition appropriate for the intended application. This may entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One may also employ appropriate buffers to render the complex stable and allow for uptake by target cells.

[00100] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal {e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. [00101] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. A pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable {e.g. , due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. [00102] The actual dosage amount of a composition of the present invention administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[00103] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound, such as the nanoparticle or the integrated metal radioisotope. In other embodiments, the active compound may comprise between about 1% to about 75% of the weight of the unit, or between about 5% to about 50%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 30 milligram/kg/body weight, about 40 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 microgram kg/body weight to about 5 milligram/kg/body weight, about 50 microgram kg/body weight to about 50 milligram/kg/body weight, etc., can be administered.

[00104] A nanoparticle may be administered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more mg of nanoparticle per dose. Each dose may be in a volume of 1, 10, 50, 100, 200, 500, 1000 or more μΐ or ml. [00105] Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. [00106] The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[00107] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.

[00108] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[00109] The therapeutic compositions of the present invention may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal, topical, or aerosol.

[00110] An effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired. [00111] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

A. Route of Administration

[00112] In pharmacology and toxicology, a route of administration is the path by which a drug, fluid, poison or other substance is brought into contact with the body. In certain aspects of the present invention, the composition comprising a nanoparticle formulation may be formulated in a topical formulation or an enteral formulation, and preferably, a parenteral formulation. In particular aspects, the compositions may be administered to a subject by injection or infusion for a systematic delivery.

[00113] Obviously, a substance must be transported from the site of entry to the part of the body where its action is desired to take place (even if this only means penetration through the stratum corneum into the skin). However, using the body's transport mechanisms for this purpose can be far from trivial. The pharmacokinetic properties of the nanoparticle (that is, those related to processes of uptake, distribution, and elimination) are critically influenced by the route of administration.

[00114] Routes of administration can broadly be divided into: topical (local effect, substance is applied directly where its action is desired); enteral (desired effect is systemic (non-local), substance is given via the digestive tract); parenteral (desired effect is systemic, substance is given by routes other than the digestive tract). The U.S. Food and Drug Administration recognizes 111 distinct routes of administration. The following is a brief list of some routes of administration.

[00115] Topical form of administration includes epicutaneous (application onto the skin), e.g. allergy testing, typical local anesthesia; inhalational, e.g. asthma medications; enema, e.g. contrast media for imaging of the bowel; eye drops (onto the conjunctiva), e.g. antibiotics for conjunctivitis; ear drops - such as antibiotics and corticosteroids for otitis externa; intranasal route (into the nose), e.g. decongestant nasal sprays; vaginal, e.g. topical estrogens, antibacterials.

[00116] Enteral form of administration involves any part of the gastrointestinal tract: by mouth (orally), many drugs as tablets, capsules, or drops; by gastric feeding tube, duodenal feeding tube, or gastrostomy, many drugs and enteral nutrition; rectally, various drugs in suppository or enema form;

[00117] Parenteral form of administration by injection or infusion involves intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous infusion (into the bone marrow) - in effect, an indirect intravenous access because the bone marrow drains directly into the venous system; intradermal (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion (infusion into the urinary bladder). Other parenteral administration include transdermal (diffusion through the intact skin), sublingual, i.e. under the tongue, nitroglycerine, buccal (absorbed through cheek near gumline), vaginal suppositories, inhalational, epidural (i.e., peridural) (injection or infusion into the epidural space), intravitreal.

[00118] Injection encompasses intravenous (IV), intramuscular (IM), and subcutaneous (sub-Q). In acute situations, in emergency medicine and intensive care medicine, drugs are most often given intravenously. This is the most reliable route, as in acutely ill patients the absorption of substances from the tissues and from the digestive tract can often be unpredictable due to altered blood flow or bowel motility.

VI. Hyperproliferative Diseases

[00119] In certain embodiments of the invention, a therapeutically effective amount of the pharmaceutical composition comprising a nanoparticle formulated in a pharmaceutically acceptable nanoparticle formulation may be used to treat a diseases and/or condition in a subject.

A. Definitions

[00120] "Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a parenteral pharmaceutical composition comprising a nanoparticle formulated in a pharmaceutically acceptable nanoparticle formulation. [00121] A "subject" refers to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[00122] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[00123] A "disease" or "health-related condition" can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress.

[00124] A "hyperproliferative disease" includes diseases and conditions that are associated with any sort of abnormal cell growth or abnormal growth regulation, specifically a cancer.

[00125] In some embodiments of the invention, the methods include identifying a patient in need of treatment. A patient may be identified, for example, based on taking a patient history, based on findings on clinical examination, based on health screenings, or by self-referral.

B. Diseases

[00126] The present invention can find application in the treatment of any disease for which delivery of a therapeutic nanoparticle to a cell or tissue of a subject is believed to be of therapeutic benefit. Examples of such diseases include hyperproliferative diseases and quiescent malignant diseases.. In particular embodiments, the disease is a hyperproliferative disease, such as cancer of solid tissues or blood cells. Quiescent malignant diseases that can be treated by the nanoparticles include, for example, chronic lymphocytic leukemia.

[00127] For example, a nanoparticle formulated in a pharmaceutically acceptable nanoparticle formulation may be administered to treat a hyperproliferative disease. The hyperproliferative disease may be cancer, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, pre-neoplastic lesions (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[00128] The cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In certain embodiments, the cancer is ovarian cancer. In particular aspects, the cancer may be a chemo-resistant cancer. [00129] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

VII. Combination Treatments

[00130] In certain embodiments, the compositions and methods of the present invention involve a nanoparticle formulation-based composition as set forth herein with a second or additional therapy. Such therapy can be applied in the treatment of any disease for which treatment with the nanoparticle formulation is contemplated. For example, the disease may be a hyperproliferative disease, such as cancer. [00131] The methods and compositions including combination therapies enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with a therapeutic nucleic acid, such as an inhibitor of gene expression, and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) including one or more of the agents (i.e., inhibitor of gene expression or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an inhibitor of gene expression; 2) an anti-cancer agent, or 3) both an inhibitor of gene expression and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with a chemotherapy, radiotherapy, surgical therapy, or immunotherapy. [00132] A therapeutic nanoparticle formulation-containing composition set forth herein may be administered before, during, after or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the nanoparticle formulation-containing composition is provided to a patient separately from an additional anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two agents would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the inhibitor of gene expression therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more preferably, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between respective administrations.

[00133] Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.

[00134] Various combinations may be employed. For the example below a therapeutic nanoparticle formulation-containing composition is "A" and an anti-cancer therapy is "B":

[00135] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B

B/A/B/B

[00136] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A

B/B/A/A

[00137] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A

A/A/B/A

[00138] Administration of any compound or therapy of the present invention to a patient may follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as radiation and surgical intervention, may be applied in combination with the described therapy.

[00139] In specific aspects, it is contemplated that a standard therapy may include chemotherapy, radiotherapy, immunotherapy, surgical therapy or gene therapy and may be employed in combination with the inhibitor of gene expression therapy, anticancer therapy, or both the therapeutic nucleic acid and the anti-cancer therapy, as described herein.

A. Chemotherapy

[00140] A wide variety of chemotherapeutic agents may be used in accordance with certain aspects of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas. Examples of these agents have been previously set forth. Specifically, a therapeutic agent for combination with nanoparticles in cancer treatment may also be a compound that specifically targets a specific molecule in a cancer cell or on the cancer cell surface. Such target-specific therapeutic agents include, for example, Imatinib (Gleevec), rituximab, cetuximab (erbitux), and herceptin (trastuzumab).

B. Radiotherapy

[00141] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. [00142] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. C. Immunotherapy

[00143] In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[00144] Another immunotherapy could also be used as part of a combined therapy with certain aspects of the present invention. In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al, 2000). Moreover, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.

[00145] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapy, e.g., interferons α, β and γ; IL-1, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, e.g., TNF, IL-1, IL-2, p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2, anti-pl85 (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the nanoparticle therapies described herein. [00146] In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine" is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993). [00147] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988; 1989).

D. Surgery

[00148] Approximately 60% of persons with cancer may undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[00149] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[00150] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

E. Other Agents

[00151] It is contemplated that other agents may be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents.

[00152] Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP- 1, MIP-lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increasing intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.

[00153] In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present invention to improve the anti- hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present invention to improve the treatment efficacy. [00154] Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 106°F). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.

[00155] A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that may be internally heated. Whole -body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.

[00156] Hormonal therapy may also be used in conjunction with certain aspects of the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases. VIII. Examples

[00157] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Synthesis, characterization, and stability [00158] Citrate-coated CuS NPs (Cit-CuS NPs) were readily synthesized in aqueous solution by reacting CuCl₂ and Na₂S in the presence of sodium citrate at 90°C for 15 min. PEG coating was introduced by incubating Cit-CuS NPs with SH-PEG (molecular mass, 5,000 Da) at room temperature overnight.

[00159] FIG. 1A is a representative transmission electron microscopy image of the CuS NPs. The optical spectra of Cit-CuS NPs and PEG-CuS NPs are shown in FIG. IB. Both types of NPs displayed a blue-shifted band gap absorption compared to bulk CuS materials (peak >1100 nm) (Zhao et al., 2009), confirming the effect of quantum size confinement. On the basis of the measured absorbance A, the extinction coefficient was calculated to be about 8.66 x 10 7 M -^"1 cm -^"1 at the peak absorption of 930 nm using the following equation: ε = (A^6d³pN_A)/(LC_wt) where d is the average diameter of the CuS NPs assuming the NPs are spherical, p is the density of the NPs assuming it is the same as the bulk (-4.6 g/cm ), NA is Avogadro's constant, L is the path length (1 cm), and Cwt is the weight concentration of the NPs. [00160] The coating of citrate and PEG to the surface of CuS NPs were confirmed using nuclear magnetic resonance analysis (FIG. 6). All X-ray diffraction peaks of the CuS NPs (FIG. 7) could be indexed as covellite-phase CuS with lattice parameters similar to those of the Joint Committee on Powder Diffraction Standards card 79-2321. The diffraction peaks were relatively broad, reflecting the small size of CuS crystals. No obvious impurity peaks were detected, indicating the acquirement of covellite CuS with high quality. The CuS NPs were well dispersed and relatively uniform in size, with an average diameter of 11 nm. The hydrodynamic sizes of the CuS NPs were determined from dynamic light scatting study (FIG 8). CuS NPs which had a TEM diameter of 11 nm showed DLS size of 11.7 nm and 31.6 nm before and after PEG coating, respectively. The increased diameter of PEG- coated NPs in aqueous solution is probably due to the PEG layer that is invisible in TEM measurement. These CuS NPs were significantly larger than previously reported thioglycolic acid-stabilized CuS NPs, which had an average diameter of 3 nm and displayed peak absorbance at 900 nm. The high-resolution TEM (FIG 9) reveals the fringes of hexagonal CuS (102) planes with a lattice spacing of about 0.3 nm. These results are in agreement with the lattice spacing of the {102} plane (0.305 nm) of hexagonal CuS nanostructures described in previous reports (Wu et αί, 2006; Xu et ah, 2006).

[00161] These are typical characteristics of covellite CuS and can be interpreted in terms of valence-band-free carriers (positive holes), which are essentially metallic in character and give rise to NIR plasmon absorption because of high charge density (Zhao et ah, 2009). On the basis of X-ray diffraction, UV-vis spectroscopy, and transmission electron microscopy results, it was concluded that pure and high-quality CuS NPs were produced. It is worth noting that the maximum absorption of the 11 -nm Cit-CuS NPs and PEG-CuS NPs, 930 nm, is 30 nm red-shifted compared to the absorption of the 3-nm CuS NPs previously reported but is 50 nm blue-shifted compared to the absorption of the 15-nm CuS NPs previously reported by Zhao et al. (2009), who studied the composition dependence of plasmonic resonance spectra of Cu₂-_xS. Future studies are needed to identify and clarify the effect of size on the NIR plasmonic resonance spectra for CuS NPs. [00162] The stability of Cit-CuS NPs and PEG-CuS NPs was investigated by incubating these NPs in various media, including water, 0.4 mM citrate solution, 100 mM acetate buffer solution, 100 mM NaCl solution, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid buffer (HEPES), phosphate -buffered saline (PBS), 50 mM bovine serum albumin solution, PBS containing 10% fetal bovine serum (FBS), and 100% FBS at 37°C for up to 7 days. No precipitates were observed and no obvious change of hydrodynamic particle sizes were found for either type of NP in these solutions (FIG. 10 and Table 1), indicating that both Cit-CuS NPs and PEG-CuS NPs possess excellent colloidal stability under a wide range of environmental conditions. Table 1. Stability of Cit-CuS NPs and PEG-CuS NPs in different buffer solutions after 7 days incubation at 37°C*

Water Citrate Acetate NaCl HEPES PBS BSA PBS FBS

Cit- 11.7 17.3 18.6 21.2 18.3 18.4 18.7 20.2 20.2

CuS nm nm nm nm nm nm nm nm nm

NPs

PEG- 31.6 32.6 32.7 41.4 32.9 32.7 34.7 34.7 32.7

CuS nm nm nm nm nm nm nm nm nm

NPs

* Data were obtained by dynamic light scattering study. Solutions were as follows: 1, water; 2, citrate solution (0.4 mM); 3, acetate buffer solution(100 mM); 4, NaCl solution(100 mM); 5, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer (HEPES); 6, Phosphate buffered saline (PBS); 7, bovine serum albumin solution (BSA) (50 mM); 8, PBS containing 10% fetal bovine serum (FBS); and 9, 100% FBS.

[00163] [⁶⁴Cu]-CuS NPs were prepared using the same procedures used for the preparation of plain CuS NPs except that for the synthesis of PEG-[⁶⁴Cu]-CuS NPs, the PEG coating was introduced directly during the course of CuS NP synthesis instead of after CuS NP formation through PEG/citrate substitution. This is necessary to shorten the time of synthesis for [⁶⁴Cu]-CuS NPs. The preparation of the 3-nm CuS described in a previously reported procedure requires more than 24 h for the reaction to complete at room temperature, which is not suitable for Cu labeling because the decay half- life of Cu is only 12.7 h. High reaction temperature was used for rapid radio-synthesis of PEG-[⁶⁴Cu]-CuS NPs, reducing the time of PEG-[⁶⁴Cu]-CuS NP production to ~20 min. The specific activity was readily controlled by varying the radioactivity of ⁶⁴CuCl₂ in the mixture of ⁶⁴CuCl₂ and cold CuCl₂ at the time of [⁶⁴Cu]-CuS synthesis. As shown in Table 2, 100% of radioactivity was associated with Cit-[⁶⁴Cu]-CuS NPs and PEG-[⁶⁴Cu]-CuS NPs at the end of synthesis, indicating that the radiolabeling efficiency approached 100%. After incubation in PBS and FBS at 37°C for 24 h, Cit-[⁶⁴Cu]-CuS NPs lost 12.7% and 15.3% of radioactivity, respectively. However, negligible amount of radioactivity was lost from PEG-[⁶⁴Cu]-CuS NPs. These data indicated that ⁶⁴Cu was more stably integrated in PEG-[⁶⁴Cu]-CuS NPs than in Cit-[⁶⁴Cu]-CuS NPs (Table 2 and FIG. 11).

Table 2. Radiolabeling efficiency and stability of [^b4Cu]-CuS NPs*

Cit-[⁶⁴Cu]-CuS NPs PEG-[⁶⁴Cu]-CuS NPs

CuS NPs 100% 100%

CuS NPs in PBS 87.3% 99.8%

CuS NPs in FBS 84.7% 99.2%

* Representative radio-instant thin-layer chromatograms after incubation in phosphate buffered saline (PBS) or 100% fetal bovine serum (FBS) at 37°C for 24 h. [00164] The cytotoxic effect of CuS nanoparticles on three different cell lines

(MDA MB231 cells, human brain U87 tumor cells, human embryonic kidney 293 cells (HEK293 cells)) are shown in FIG. 13. CuS has no cytotoxic effect on the cells at concentration up to 100 μΜ after 48 hours of incubation.

[00165] Materials. Copper(II) chloride (CuCl₂), sodium sulfide (Na₂S-9H₂0), sodium citrate, and methoxy-PEG-thiol (SH-PEG; molecular weight, 5,000) were purchased from Sigma-Aldrich (St. Louis, MO). Isoflurane was obtained from Baxter (Deerfield, IL). ⁶⁴CuCl₂ was obtained from Wisconsin University at Madison (Madison, WI). PD-10 columns were purchased from Amersham-Pharmacia Biotech (Piscataway, NJ). All the chemicals and solvents were at least ACS grade and were used without further purification. Deionized water (18 ΜΩ) was obtained from a Milli-Q synthesis system (Millipore, Billerica, MA). Human U87 glioblastoma cells were obtained from American Type Culture Collection (Manassas, VA). RPMI-1640 culture medium and calcein AM were obtained from Sigma- Aldrich.

[00166] Synthesis of CuS NPs. The general procedure for the synthesis of CuS NPs in water was as follows. Into 1000 mL of aqueous solution of CuCl₂ (0.1345 g, 1 mmol) and sodium citrate (0.2 g, 0.68 mmol) was added 1 mL of sodium sulfide solution (Na₂S, 1 M) under stirring at room temperature. The pale blue CuCl₂ solution turned dark brown immediately upon the addition of sodium sulfide. Five minutes later, the reaction mixture was heated to 90°C and stirred for 15 min until a dark green solution was obtained. The mixture was transferred to ice-cold water. The Cit-CuS NPs were obtained and stored at 4°C. To introduce PEG coating, about 1 mg of SH-PEG was added into the Cit-CuS NP solution (1.42xl0¹⁵ NPs in 1.0 mL of water). The reaction was allowed to proceed overnight at room temperature.

[00167] Characterizations of CuS NPs. For transmission electron microscopy, aqueous solution of CuS NPs was deposited on carbon-enhanced copper grids without negative staining. The NPs were allowed to adhere on the grid for 1 h, after which they were briefly rinsed with deionized water and air dried. The samples were then examined using a transmission electron microscope (JEM 2010, JEOL Japan) at an accelerating voltage of 200 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA). The average diameter of CuS NPs was determined by measuring up to 200 individual particles. The UV-Vis spectroscopy of CuS NPs was recorded on a Beckman Coulter DU-800 UV-VIS spectrometer (Brea, CA) with a 1.0-cm optical path length quartz cuvette. The identity and crystallinity, crystalline structure, size, and shape of the NPs were observed by X-ray diffraction and a high-resolution transmission electron microscope (200 kV, JEOL, Japan). X-ray diffraction was performed using a Siemens Kristalloflex 810 D-500 X-ray diffractometer (Siemens, Germany) under an operating mode of 40 kV and 30 niA with λ = 1.5406 Angstrom radiation.

[00168] Stability of CuS NPs. The stability of Cit-CuS NPs and PEG-CuS NPs in various media was investigated by incubating CuS NPs in water, 0.4 mM citrate solution, 100 mM acetate buffer solution, 100 mM NaCl solution, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid buffer, PBS, 50 mM bovine serum albumin solution, PBS containing 10% fetal bovine serum (FBS), or 100% FBS at 37°C for up to 7 days. The appearance of precipitation was observed by visual inspection.

[00169] Synthesis of radioactive [⁶⁴Cu]-CuS NPs. ⁶⁴CuCl₂ (20 pL, 1000 μ( ϊ) was added to 1 mL of CuCl₂ solution (1 mM) containing sodium citrate (0.2 g/L). Then, 10 μΐ, of sodium sulfide solution (100 mM) was added to the CuCl₂ solution under stirring. The mixture was then heated to 90°C for 15 min until a dark green solution was obtained. The reaction mixture was transferred to ice-cold water to give Cit-[⁶⁴Cu]-CuS NPs. The same procedure used for Cit-[⁶⁴Cu]-CuS NPs was used for the preparation of PEG-[⁶⁴Cu]-CuS NPs. Thus, 10 μΐ, of sodium sulfide solution was added into 1 mL of aqueous solution of ⁶⁴CuCl₂/CuCl₂ solution containing 1 mg of SH-PEG.

[00170] The radiolabeling efficiency and the stability of labeled NPs were analyzed using instant thin layer chromatography (ITLC). The ITLC strips were developed with PBS (pH 7.4) containing 4 mM ethylenediaminetetraacetic acid and quantified using a Bioscan IAR-2000 TLC Imaging Scanner (Washington, DC). For the study of labeling stability, Cit-[⁶⁴Cu]-CuS NPs and PEG-[⁶⁴Cu]-CuS NPs were suspended in PBS or mouse serum and incubated at 37°C for 24 h. Free ⁶⁴Cu²⁺ ions moved to the solvent front, and the NPs remained at the original spot. The radioactivity at the original spot was recorded as a percentage of the total radioactivity of the ITLC strip.

[00171] Characterization of CuS nanoparticles. 1H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 300 spectrometer (Billerica, MA). For high-resolution transmission electron microscopy, aqueous solution of CuS NPs was deposited on carbon-enhanced copper grids without negative staining. The NPs were allowed to adhere on the grid for 1 h, after which they were briefly rinsed with deionized water and air dried. The samples were then examined using a high-resolution transmission electron microscope (JEM 21 OOF, JEOL Japan) at an accelerating voltage of 200 kV. Particle size was measured using dynamic light scattering at a 90° scatter angle on a ZetaPLUS particle electrophoresis system (Brookhaven Instruments Corp., Holtsville, NY).

Example 2- Pharmacokinetics, biodistribution, and PET Imaging

[00172] Blood activity-time profiles of Cit-[⁶⁴Cu]-CuS NPs and PEG-[⁶⁴Cu]- CuS NPs are shown in FIG 2 A and their pharmacokinetic parameters are presented in Table 3. Both formulations appeared to have very different in vivo disposition characteristics. The mean systemic clearance was significantly slower with PEG (0.48 mL/h) than with citrate (0.75 mL/h, p = 0.003), suggesting that citrate-coated CuS NPs were cleared faster than PEG- coated NPs following intravenous administration. This may be attributed to higher uptake by reticuloendothelial systems (RES), i.e., liver and spleen, and consequently faster elimination of Cit-[⁶⁴Cu]-CuS NPs than that of PEG-[⁶⁴Cu]-CuS NPs. As a result, PEG-[⁶⁴Cu]-CuS NPs had a significantly higher systemic exposure, i.e., area under the curve (AUC = 213.7 %ID h/mL), than that of Cit-[⁶⁴Cu]-CuS NPs (134.9 %ID h/mL, p = 0.008). The mean volume of distribution at steady- state was higher with citrate coated NPs (5.8 mL) than with PEG coated NPs (3.79 mL, p = 0.018), suggesting that citrate coated NPs has higher uptake in such tissues as liver and spleen. Interestingly, there was no difference between the two CuS NP formulations in the half-lives (ti/_2a = 0.76 h and ίι/₂β = 5.98 for Cit-[⁶⁴Cu]-CuS NPs versus ti 2a = 0.71 h and t_i/2p = 6.06 h for PEG-[⁶⁴Cu]-CuS NPs). This was likely due to the compensatory effect of systemic clearance and the volume of distribution. For each formulation, volume of distribution at steady-state was close to the volume of distribution in the central compartment, indicating that the drug mainly distributed to the central compartment (systemic blood circulation).

Table 3. Pharmacokinetics parameters of Cit-[⁶⁴Cu]-CuS NPs and PEG-[⁶⁴Cu]-CuS NPs after intravenous injection in mice*

Τι_/2α Τι_/2β AUC Vd(ss) Vc CL MRT

(h) (h) (%ID (mL) (mL) (ml/h) (h) h/mL)

Cit-[⁶⁴Cu]- 0.76±0.0 5.98±0.3 134.9±15.5 5.80±0.8 3.57+0.2 0.75±0.0 7.74±0.3

CuS NPs 3 5 6 6 2 9 2

PEG- 0.71+0.3 6.06±1.5 213.7±32.3 3.79±0.8 2.39+0.5 0.48+0.0 8.05+1.9

[⁶⁴Cu]-CuS 6 8 6 5 6 7 0

NPs

Values are means ± standard deviations. Abbreviations: ti/₂a, blood distribution half- life; ίι_/2β, blood terminal elimination half-life; AUC, area under the blood activity-time curve; Vd(ss), volume of distribution at steady-state; Vc, volume of distribution in the central compartment, CL, total body clearance; MRT, mean residence time. [00173] Biodistribution data obtained 24 h after i.v. injection of Cit-[ Cu]-CuS

NPs and PEG-[⁶⁴Cu]-CuS NPs are shown in FIG 2B. Cit-[⁶⁴Cu]-CuS NPs displayed significantly higher uptake than did PEG-[⁶⁴Cu]-CuS NPs in the liver and the spleen, both are RES enriched tissues. Conversely, PEG-[⁶⁴Cu]-CuS NPs were less likely being captured by RES cells, and therefore, displayed higher levels in the heart, kidney, lung, stomach, intestine, and bone. These findings are consistent with the pharmacokinetic findings that PEG-[⁶⁴Cu]- CuS NPs has a slower systemic clearance than that of Cit-[⁶⁴Cu]-CuS NPs, which made it more available to distribute to target tissues. Importantly, uptake in human U87 glioblastoma xenografts in mice was almost 3 times as high with PEG-[⁶⁴Cu]-CuS NPs as with Cit-[⁶⁴Cu]- CuS NPs at 24 hr after i.v. injection (7.6 ±1.4 %ID/g vs. 2.6 ± 0.4 %ID/g, p = 0.011). This may be attributed to the enhanced permeability and retention effect of NPs, and greater systemic exposure of PEG-[⁶⁴Cu]-CuS NPs, which made it more available to the tumor uptake. This effect can be utilized for passive targeting of NPs to areas with increased angiogenesis, where NPs with longer blood circulation time exhibit higher tumor uptake (Zhang et al, 009; Yang et al, 2009).

[00174] Because they had greater radiolabel stability and a higher tumor uptake value than Cit-[⁶⁴Cu]-CuS NPs, PEG-[⁶⁴Cu]-CuS NPs were further evaluated with regard to their utility for in vivo PET. FIG 3 shows representative whole-body micro-PET/CT images of a mouse acquired at 1, 6, and 24 h after i.v. injection of PEG-[⁶⁴Cu]-CuS NPs. Consistent with the biodistribution analysis, PET/CT images revealed high uptake of PEG-[⁶⁴Cu]-CuS NPs in the liver and the spleen. As expected, PEG-[⁶⁴Cu]-CuS NPs gradually accumulated in the tumor between 1 h and 24 h, permitting remarkably clear visualization of the tumor at 24 h after injection. Quantitative analysis showed that the average tumor-to-muscle ratios at 1 h, 6 h, and 24 h after NP injection were 2.74: 1, 6.14: 1, and 6.55: 1, respectively, indicating that PEG-[⁶⁴Cu]-CuS NPs were deposited in and retained in the tumor over the 24-h period. These values compared favorably with those for ⁶⁴Cu-labeled quantum dots targeted to integrin α_νβ₃ receptors, where RGD-coated quantum dots were found to have a tumor-to-muscle ratio of 4: 1 at 5 h after i.v. injection into nude mice with U87 tumors (Cai et al, 2007). PEG-[⁶⁴Cu]- CuS NPs were excreted by both renal and hepatobilary routes, as indicated by the deposition of radioactivity in the bladder and the gastrointestinal tract (FIG. 3). Instant thin-layer chromatography study revealed that PEG-[⁶⁴Cu]-CuS NPs were cleared from the renal system in the form of nanoparticles (FIG. 12). A previous study by Choi et al. (2007) with quantum dots demonstrated that NPs smaller than 5 nm can be cleared by the renal route. Although the average diameter of the CuS NPs was 11 nm, it is possible that a small fraction of the CuS NPs smaller than 5 nm in diameter were capable of renal clearance.

[00175] Pharmacokinetics. All experiments involving animals were done in accordance with the guidelines of the Institutional Animal Care and Use Committee. For pharmacokinetic analysis, mice were intravenously injected with radioactive CuS NPs (4 x 10¹¹ particles, 50 μθ/π αιβε in 0.2 mL), and blood samples (10 μί) were collected from the tail vein at predetermined time points. The blood pharmacokinetic parameters of the radiotracer were analyzed with a two-compartmental model using WinNonlin 5.0.1 software (Pharsight Corporation, Palo Alto, CA). The animals were euthanized by C0₂ exposure at the end of the study.

[00176] Biodistribution-radioactivity counting. Human U87 glioblastoma tumors were grown subcutaneously in the right thigh of nude mice (20-25 g; Harlan-Sprague- Dawley, Indianapolis, IN) by injecting 1 xlO⁶ viable tumor cells suspended in PBS. When tumors had grown to 5-8 mm in diameter, the mice were randomly allocated into two groups (n = 5). Mice in group 1 were injected with Cit-[⁶⁴Cu]-CuS NPs, and mice in group 2 were injected with PEG-[⁶⁴Cu]-CuS NPs, each at a dose of 8 x 10¹⁰ particles per mouse (20 μθ per mouse in 0.2 mL). Mice were killed by C0₂ overexposure 24 h after injection. Blood, heart, liver, spleen, kidney, lung, stomach, intestine, muscle, bone, brain, and tumor tissues were removed and weighed, and radioactivity was measured with a Packard Cobra gamma counter (Ramsey, MN). Uptakes of ⁶⁴Cu-labeled CuS NPs in various organs were expressed as percentage of injected dose per gram of tissue (%ID/g).

[00177] jm^'croPET/CT imaging. Mice bearing U87 tumors were prepared as before. When tumors reached 8-10 mm in diameter, mice (n = 3) were treated with an i.v. injection of PEG-[⁶⁴Cu]-CuS NPs (8 x 10¹⁰ particles/mouse, 200 μα/mouse; 0.2 mL). The animals were anesthetized with 2% isoflurane and placed in prone position, and micro- PET/CT images were acquired at 1, 6, and 24 h after injection of radiolabeled nanoparticles using an Inveon micro-PET/CT scanner (Siemens Preclinical Solution, Knoxville, TN). The micro-PET and CT images were generated separately and then fused using Inveon Research Workplace (Siemens Preclinical Solution, Knoxville, TN). For data analysis, the region of interest (ROI) was manually drawn covering the whole tumor on CT and copied to the corresponding PET images. Similarly, a circular region of interest was drawn on the muscle of the opposite leg of the mouse on CT images, and copied to the PET images. The mean signal intensities of the tumor and muscle in the ROIs were recorded. The tumor-to-muscle ratio was calculated by dividing signal intensity of the tumor by that of the muscle.

[00178] Statistical analysis. Differences in biodistribution data and extent of necrosis expressed as percentage of necrotic area after treatments were analyzed using two- tailed Student's t test. Differences between groups were considered statistically significant at p < 0.05.

Example 3 - In vitro and in vivo photothermal therapy

[00179] To date, there have been few reports on NPs smaller than 20 nm having NIR absorption. The smallest gold nanostructures reported to date as having plasmon NIR absorption were -40 nm in diameter (Melancon et ah, 2008; Lu et ah, 2009). Because the pharmacokinetics and biodistribution pattern of NPs are strongly affected by their size (Zhang et ah, 2009; Yang et ah, 2009), it is highly desirable that novel nanostructures having diameter less than 20 nm with NIR absorption be identified and evaluated. CuS NPs, which are much smaller than gold nanostructures, may have a better chance of reaching their targets and being cleared from the body through the renal system (Zhang et ah, 2009; Choi et ah, 2007; Burns et al, 2009).

[00180] An important feature of CuS NPs is NIR light-induced thermal effect, which could be used for PTA therapy. To investigate temperature elevation induced by NIR laser irradiation in the presence of CuS NPs, a continuous-wave fiber-coupled diode laser centered at 808 nm was used. FIG 4A shows the temperature change of an aqueous solution containing PEG-CuS NPs as a function of exposure time. Exposure of an aqueous solution of PEG-CuS NPs (500 μΜ CuS molecular units, 7.1 x 10¹⁴/mL [23 nM CuS NP]) to the NIR laser light (16 W/cm ) for 5 min elevated the temperature of the solution from 25°C to 80°C (an increase of 55°C). Under the same conditions, no change in temperature was observed with pure water. The magnitude of increase in temperature of the aqueous solution of PEG- CuS NPs decreased with decreasing PEG-CuS NP concentration (FIG. 4B). These data indicated that PEG-CuS NPs acted as an efficient photothermal coupling agent. Compared with previously reports 3-nm CuS NPs, the new 11 -nm CuS NPs displayed higher photothermal conversion efficiency. For example, the temperature of an aqueous solution of 3-nm CuS NPs increased 12.7°C over a period of 5 min at an output power of 24 W/cm and a concentration of 770 μΜ CuS units (~4.8xl0¹⁶ particles/mL). [00181] To test the cell killing induced by photothermal effect through CuS

NPs, human U87 glioblastoma cells were incubated with PEG-CuS NPs for 2 h. The cells were then irradiated with NIR laser centered at 808 nm. The cell viability after exposure to NIR laser was probed using calcein AM dye, which reports ubiquitous intracellular esterase activity. Twenty-four hours after laser treatment, cells treated with PEG-CuS NPs at a concentration of 500 μΜ CuS plus NIR laser (16 W/cm 2 for 5 min, 40 W/cm 2" for 2 mm, or 40 W/cm for 5 min) had substantially reduced cell density (FIG. 13). No apparent change in cell viability was observed when cells were treated with CuS NPs alone at 100 μΜ or 500

2 2

μΜ or NIR laser alone at 16 W/cm for 5 min or 40 W/cm" for 5 min. These results indicated that PEG-CuS NPs mediated photothermal destruction of U87 cells.

[00182] In mice bearing subcutaneous U87 tumors, the skin of the mice at the tumor site turned greenish after both i.t. and i.v. injection of PEG-CuS NPs owing to the deposition of the NPs in the tumor (FIG 5A). After NIR irradiation, the skin at the tumor sites in both mice that received i.t. injection and mice that received i.v. injection of PEG-CuS NPs turned brownish or dark red, indicating tissue burn caused by local photothermal effect. In contrast, there was no noticeable change in the skin of mice treated with PEG-CuS NPs alone, saline plus NIR irradiation, or NIR laser alone (FIG 5A).

[00183] Histological examination confirmed that the combination of PEG-CuS

NPs administered by either i.t. or i.v. injection followed by laser treatment caused significantly greater necrotic response than did PEG-CuS NPs without laser, saline plus laser, or saline only (FIG 5B). In the mice treated with PEG-CuS NPs plus laser, common features of thermonecrosis, such as loss of nucleus, cell shrinkage, and coagulation, were found in the tumor tissues. In the mice treated with i.t. injection of PEG-CuS NPs plus laser, almost all of the tumor tissue was necrotized, exhibiting pyknosis, karyolysis, cytoplasmic acidophilia, and degradation and corruption of the extracellular matrix of the tumor. In the mice treated with i.v. injection of PEG-CuS NPs plus laser, about 65% of the tumor tissue was necrotized. In the mice treated with saline plus laser, there was only a baseline fraction of necrosis in tumor tissue (<5%). In the tumors of mice treated with CuS NPs alone (i.t. injection), saline plus NIR laser, or saline alone, there was little pyknosis or karyolysis, confirming the benign nature of these treatments (FIG 5C). Thus, selective in vivo photothermal destruction of the tumors mediated by PEG-CuS NPs was confirmed. [00184] Photothermal effect in aqueous solution. The laser was a continuous- wave GCSLX-05-1600m-l fiber-coupled diode laser with a center wavelength of 808 ± 10 nm. It was powered by a DH 1715A-5 dual-regulated power supply (15PLUS laser, Diomed, Andover, MA). A 5-m, 600-μηι core BioTex LCM-001 optical fiber (BioTex Inc., Houston, TX) was used to transfer laser power from the laser unit to the target. This fiber had a lens mounting at the output that allowed the laser spot size to be changed by changing the distance from the output to the target. The output power was independently calibrated using a handheld model 840-C optical power meter (Newport Corporation, Irvine, CA) and was found to be 1 W for a spot diameter of 3.5 mm (~8 W/cm ) and a 2-amp supply current. The end of the optical fiber was attached to a retort stand using a movable clamp and positioned directly above the sample cell. For measuring temperature change mediated by CuS NPs, NIR laser light (808 nm) was delivered through a quartz cuvette containing the CuS NPs (100 μ ). A thermocouple was inserted into the solution perpendicular to the path of the laser light. The temperature was measured over 10 min. Water was used as a control. [00185] Photothermal ablation of cancer cells with CuS NPs in vitro. U87 cells were seeded onto a 96-well plate with a density of 10,000 per well 1 day before the experiment. Cells were washed three times with Hanks balanced salt solution (HBSS, Sigma- Aldrich) and then incubated with PEG-CuS NPs in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA) at CuS concentrations of 100 μΜ or 500 μΜ at 37°C. Cells without NPs were used as a control. Two hours later, the culture medium was replaced with fresh RPMI-1640 medium without phenol red, and the cells were irradiated with a diode NIR laser centered at

808 nm at an output power of 0, 16 W/cm 2 for 5 min, 40 W/cm 2" for 5 mm, or 40 W/cm 2" for 2 min. The laser was coupled to a 1-m, 2-mm core fiber, which delivered a circular laser beam of 4 mm in diameter, covering the central area of the microplate well. Power calibration was done automatically. After treatment, cells were resupplied with RPMI-1640 containing 10% FBS. Twenty-four h later, the cells were washed with HBSS and stained with calcein AM for visualization of viable cells according to manufacturer's suggested protocol (Invitrogen). Cells were examined under a Zeiss Axio Observer.Zl fluorescence microscope (Carl Zeiss Microimaging GmbH, Gottingen, Germany). [00186] Photothermal ablation of cancer cells with CuS NPs in vivo. Nude mice were inoculated subcutaneously with 5x 10⁶ U87 cells in the right side of the rear leg 21 days before the experiment. When tumor had grown to 7-10 mm in diameter, mice were randomly allocated into 5 groups (n = 5). Mice in group A were injected intratumorally with PEG-CuS NPs (5 uL, 8 mM/mouse, 4xl0¹³ NPs/mouse). Mice in groups B and C were injected intravenously with PEG-CuS NPs (200 μί, 8 mM/mouse). Mice in group D were injected intravenously with saline. Mice in group E did not receive any treatment. After 24 h, the tumors in mice from groups A, B, and D were irradiated with NIR laser at 12 W/cm for 5 min. The mice were killed 24 h after laser treatment, and tumors were removed, snap frozen, and cryosectioned into 1000 μιη. The slides were stained with hematoxylin-eosin. The slices were examined under a Zeiss Axio Observer.Zl fluorescence microscope. The images were taken using a Zeiss AxioCam MRc5 color camera, and the extent of tumor necrosis, expressed as a percentage of the entire tumor area, was analyzed with Zeiss Axio Vision software (version 4.6.3).

Example 4 - Combined radio-photothermaltherapy with [⁶⁴Cu]CuS nanoparticles

[00187] Nude mice bearing s.c. BT474 breast cancer were injected intratumorallyat a dose of 200 μΟ/τιιιηοΓ and 3 x 10¹⁴ particles/tumor in 50 μΐ_^ (FIG. 15). Treatment with [⁶⁴Cu]CuS alone caused significant tumor growth delay (FIG. 15B) with minimal toxicity (FIG. 16A). Treatment with combined [⁶⁴Cu]CuS and near-infrared laser caused tumor cure. RT, radiotherapy. PTT, photothermaltherapy. For PTT, the tumors were exposed to near-infrared laser (808 nm) at 12 W/cm for 2 min. The foregoing studies are described in greater detail in Example 11 below. Example 5 - Deeply Penetrating Photoacoustic Tomography in Biological Tissues Using CuS Nanoparticles and Hollow Gold Nanospheres (HAuNS) as Optical Contrast Agents

[00188] Photoacoustic tomography (PAT) is a developing biomedical imaging modality that is based on the acoustical detection of the optical absorption of laser light by biomolecules. To evaluate new nanoparticles as a potential contrast agent for PAT, the semiconductor copper sulfide nanoparticles (CuS NP, -11 nm in diameter) and hollow gold nanoshells (HAuNS, ~40 nm in diameter) were synthesized and characterized. The nanoparticles were dispersed with 10% polyacryl amide gelatin and used as an imaging object inside fresh chicken breast tissues. Near-infrared laser pulses 800-1064 nm were used to induce photoacoustic signals. PAT images were acquired with a prototype PAT device. Objects containing a concentration of 1 mM of CuS NP and HAuNS could be clearly visualized at ~5 cm depth from the laser illuminating surface. Imaging resolution and sensitivity were estimated to be -800 μιη and ~6 nmol, respectively, at this depth. Significantly, PAT imaging of gels containing CuS NP at 7 cm was attainable under optimal data acquisition conditions.

[00189] Both CuS NP nanoparticles were capable of transmitting and detecting photoacoustic signals (FIG. 17). It is feasible to obtain deeply penetrating PAT images with high ultrasonic spatial imaging resolution in the presence of appropriate contrast agents.

[00190] Various amounts of PEG-CuS NPs were also tested for their ability provide a photoacoustic image using a 1064 nm, 36.5 mJ laser at a depth of 1 cm. Results shown in FIG. 33 demonstrate that the particles provide an excellent signal for photoacoustic imaging. Thus, particles that additional incorporate an integral radionuclide could be used for combined photoacoustic and PET/CT imaging e.g., for sentinel lymph node mapping.

Example 6 - [64Cu]CuS NP in microPET-CT Imaging of Lymph Node

[00191] [⁶⁴Cu]CuS NP was used for lymph node imaging with PET/CT. This technique can be potentially used in noninvasive detection of lymph node metastasis. The ability to detect lymph nodes metastasis is extremely important in cancer staging, determining prognosis, and monitoring treatment outcome. The lymphoscintigraphy could guide metastasis lymph node(LN) detection during the surgery; guide the accuracy resection of sentinel nodes for biopsy; and provide accuracy information to oncologist on guiding postoperation chemoradiotherapy of individual patient. As shown in FIG. 18, MicroPET/CT images of a rat 24 h after subcutanenous injection of the [⁶⁴Cu]CuS NP in the right front paw were shown for lymph nodes as indicated by arrow.

Example 7 - Hyaluronic Acid conjugated CuS NPs Targeting Tumor Cells

[00192] The cell surface receptor CD44 is found on many cancer cells, and has been implicated in various processes such as inflammation and tumor metastasis. CD44 is a major receptor for the hyaluronic acid (HA). [00193] Hyaluronic acid conjugated CuS particles were synthesized as follows:

HA is mixed with CuCl₂ (or a mixture of CuCl₂ and ⁶⁴CuCl₂) in aqueous solution. Then Na₂S solution was added into the above solution at room temperature. The reaction solution was stirred at 90°C for a few minutes. CuS NP coated with HA was isolated by ultracentrifugation. The resulting HA-coated CuS NP (HA-CuS NP) were evaluated with regard to their photothermal conducting effect (FIG. 19), binding to CD44(+) tumor cells (FIGS. 20-21), and selective photothermal ablation of tumor cells (FIG. 22).

Example 8 - Incorporation of additional radio metal ions into CuS NPs

[00194] CuS nanoparticles were synthesized essentially as described or the synthesis of [⁶⁴Cu]CuS NP, however, the positron emitter ⁶⁸Ga was incorporated into the particles in place of ⁶⁴Cu. Briefly, 100 μΐ (2 mCi) of ⁶⁸GaCl₂ was mixed with 1000 μΐ of CuCl₂ containing sodium citrate (0.2 g/L). Then, 10 μΐ, of sodium sulfide solution (100 mM) was added to the CuCl₂ solution under stirring. The mixture was then heated to 90°C for 15 min until a dark green solution was obtained. The reaction mixture was transferred to ice-cold water to give Cit-[⁶⁸Ga]-CuS NPs. The same procedure used for Cit-[⁶⁸Ga]-CuS NPs was used for the preparation of PEG-[⁶⁸Ga]-CuS NPs. As shown in FIG. 23 ⁶⁸Ga was incorporated into CuS nanoparticles with high efficiency without affecting the optical properties of [⁶⁸Ga]CuS NP (see, FIG. 23B).

Example 9 - Further studies to evaluate the properties of CuS NPs

[00195] CuS nanoparticles were synthesized essentially as described. It was

2+ 2- determined that by varying the molar ratio of [Cu ] to [S ^"] the absorption wavelength of the

2+ 2- particles could be tuned. As shown in FIG. 24 altering the ratio of [Cu ] and [S ^"] during nanoparticle synthesis changed the absorbance peak of the resulting nanoparticles.

[00196] The ability of CuS nanoparticles to convert laser energy into heat was also assessed and compared to other agents (see FIG. 25A-B). The indicated particles were dispersed at a concentration of 100 mg/L and exposed to a 3W laser beam (1 cm diameter) having a wave length of 980 nm (FIG. 25A) or 808 nm (FIG. 25B). Results show that CuS nanoparticles were far more efficient at heating the sample as compared to other agents at a wavelength of 980 nm. At 808 nm only hollow gold nanospheres had photothermal conduction properties as great as CuS nanoparticles. It was also found that CuS nanoparticles could be used to repeatedly convert laser light into thermal energy without any appreciable loss of efficiency. As shown in FIG. 26, repeated application of laser light to particle suspensions resulted in virtually identical heating curves. For these studies particles were dispersed at a concentration of 50 mg/L and exposed to a 3W/cm laser beam (1 cm diameter) having a wave length of 808 nm. [00197] Moreover because CuS NPs can be tuned to have an absorbance of between 980-1064 nm, low cost, high powered 1064-nm lasers can be used for photothermal therapies. As shown in FIG. 27, a pulsed 1064-nm laser was able to efficiently kill HeLa cells in the presence of CuS NPs with a high degree of special accuracy. Example 10 - Folic acid coating of CuS NPs

[00198] The general procedures for the synthesis of CuS NP were adapted for the synthesis of folic acid-coated CuS NP (FA-CuS NP). Briefly, into 1000 mL of an aqueous solution containing CuCl₂ (0.1345 g) and folic acid (1.0 mg) was added 1.0 mL of sodium sulfide solution (Na₂S, 1 M) with stirring at room temperature. The pale-blue CuCl₂ solution turned dark-brown immediately upon the addition of sodium sulfide. After 5 min, the reaction mixture was heated to 90 °C and stirred for 15 min until a dark-green solution was obtained. The mixture was transferred to ice-cold water. FA-CuS NP were obtained and stored at 4 °C.

[00199] CuS NPs were produced as detailed above, but in this case coated with folic acid (FA). The physical properties of the particles were then assessed using previously described techniques. The FA coated NPs showed a peak absorbance in a range that would be useful for photothermal therapy (FIG. 28A) and indeed exhibited excellent photothermal conduction properties (FIG. 28B). The CuS NPS with FA coating also maintained a small average particle size of approximately 12 nm as assessed by TEM (FIG. 28C) and showed similar stability in a variety of buffers (FIG. 28D). [00200] Importantly, FA coated NPs were found to specifically bind to KB cells (see FIG. 29) indicating that such particle could be useful in imaging and/or treating related cancers, such as head and neck cancers. Specifically, it was shown in FIG. 29 that FA-CuS NPs, but not PEG-CuS NPs, could specifically bind to KB cells and that such binding was dependent on FA, since excess FA blocked the interaction. Moreover, when FA- CuS NPs were applied to KB cells in conjunction with NIR laser exposure the cells could be selectively killed in the field of the laser pulse (FIG. 30). On the other hand, in the presence of blocking FA, or when PEG-CuS NPs were used, significant cell killing was not observed. FA-CuS NPs are also able to selectively target KB tumors in vivo and tumor accumulation of FA-CuS NPs (Tu) was partially inhibited by excess FA (FIG. 31). The targeted FA-CuS NPs were far more effective (relative to untargeted PEG-CuS NPs) at killing tumor cells by photothermal ablation (FIG. 32). Example 11 - Radio-photothermal therapy and imaging with PEG-[ Cu]CuS NP

[00201] PEG-[⁶⁴Cu]CuS NPs were synthesized essentially as described above.

Briefly, CuCl₂ (10 jxL, 4000 μCi) was added to 190 μΐ. of CuCl₂ solution (4 mM) containing PEG-SH (0.2 g/L), after which 8 μΐ, of sodium sulfide solution (100 mM) was added to the CuCl₂ solution with stirring. The mixture was then heated to 90 °C for 15 min until a dark- green solution was obtained. The reaction mixture was transferred to ice-cold water to give PEG-[⁶⁴Cu]CuS NP. The non-radioactive analogue NPs, PEG-CuS NP, were synthesized using the similar protocols for synthesizing radioactive nanop articles. A general procedure for the synthesis of CuS NP in water is described as follows. Into 10.0 mL aqueous solution of CuCl₂ (4 mM) and PEG-SH (1.0 mg) was added 40 μΕ sodium sulfide solution (Na₂S, 1M) under stirring at room temperature. Five min later, the reaction mixture was heated to 90 °C and stirred at for 15 min until a dark green solution was obtained. The mixture was transferred to ice-cold water. The PEG-coated CuS NP (PEG-CuS NP) were obtained and stored at 4°C. [00202] As detailed above, PEG-[64Cu]CuS NP were readily synthesized in aqueous solution by introducing ⁶⁴CuCl₂ to the CuCl₂ solution, then reacting mixture and Na₂S in the presence of PEG-SH. The synthesis was completed with 20 min. Transmission electron microscopy image of the PEG-CuS NP confirmed approximately uniform size distribution of the NPs with an average diameter of 11 nm. The optical spectra of PEG-CuS NP showed a strong absorption band in near-infrared region (peak at approximately 930 nm) as previously demonstrated (see, FIG. IB). This important feature of CuS NP is their NIR light-induced thermal effect, which could be used for photothermal therapy. The temperature change of an aqueous solution containing PEG-CuS NP as a function of exposure time was also determined. Exposure of an aqueous solution of PEG-CuS NP [2 mM CuS molecular units, (23 nM CuS NP)] to the NIR laser light (1.5 W/cm²) for 10 min elevated the temperature of the solution from 25 to 48 °C (an increase of 23 °C). Under the same conditions, no change in temperature was observed with pure water.

[00203] The radiolabeling efficiency and stability of labeled NP were also analyzed using instant thin-layer chromatography (ITLC). The ITLC strips were developed with PBS (pH 7.4) containing 4 mM ethylenediammetetraacetic acid and quantified using an IAR-2000 TLC imaging scanner (Bioscan, Washington, DC). To study the labeling stability, the PEG-[⁶⁴Cu]CuS NP were suspended in PBS or mouse serum and incubated at 37 °C for 24 h. Free ⁶⁴Cu²⁺ ions moved to the solvent front, and the NP remained at the original spot. The radioactivity at the original spot was recorded as a percentage of the total radioactivity of the ITLC strip. Results showed that >99% of the radioactivity was associated with PEG-[⁶⁴Cu]CuS NP at the end of synthesis, indicating that the radio-labeling efficiency approached >99%. These results are consistent those detailed above.

[00204] For the in vivo studies twenty four nu/nu mice ( - 6 weeks old) were bought from Charles River Laboratories (Wilmington, MA). All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Texas, MD Anderson Cancer Center. The BT-474 tumor models were generated by s.c. injection of 2 x 10⁶ cells in 50

PBS into the right front arm of female nude mice. T he mice were used for treatment when the tumor volume reached 50 to 150 mm (about 10 days after tumor inoculation). The tumor-bearing mice were randomly allocated into 4 groups (n = 6). Mice in group A were injected intratumorally (i.t.) with PEG- [⁶⁴Cu]CuS NP ([CuS] = 2 mM, 10 μΐ, 200 μα/mouse), Group B with PEG-CuS NP (i.t., 4 mM, 10 μΕ), Group C with PEG-[⁶⁴Cu]-CuS NP (i.t., [CuS] = 2 mM, 10 μΕ, 200 μα/mouse). Mice in Group D were not treated and served as controls. After 24 h postinjection, the tumors of mice in Groups A & B were irradiated with NIR laser (808nm) at 1.5 W/cm for 2 min.

[00205] The tumor sizes were measure by a caliper 2 or 3 time every week and calculated as the volume = (tumor length) x (tumor width) 12. Relative tumor volumes were calculated as V/Vo (V₀ was the tumor volume when the treatment was initiated). Mice were sacrificed on day 30. The tumors were placed into Optimal Cutting Temperature (OCT) medium immediately after being taken out from the mice, frozen by dry ice and stored at -80 °C. Frozen tissues were sectioned into 5-μιη slices. The slices were stored at -80 °C until use. Other major tissues including liver, spleen, kidney, lung, and heart were dissected for frozen sectioning. The adjacent slice received H&E staining. In all cases quantitative data were expressed as mean ± SD. Means were compared using Student's t test. P values of <0.05 were considered statistically significant.

[00206] Initially, micro-positron emission tomography/computed tomography (μΡΕΤ/CT) study was carried out to investigate the distribution of PEG-[⁶⁴Cu]CuS NP in the body. Mice with BT-474 tumor were injected i.t. with 200 μθ 64CuC12 or 200 μθ PEG- [⁶⁴Cu]CuS NP. As shown in FIG. 34, almost all the radio-isotope (>99%) was retained in the tumor 24 h after injection of PEG-[ Cu]CuS NP. In contrast, only -20% of radioactivity remained at the tumor site 24 h after i.t. injection of ⁶⁴CuCl₂ solution. These data indicate that PEG-[⁶⁴Cu]CuS NP was retained in the tumor and is suitable for radiotherapeutic applications. [00207] FIG. 15A shows experimental design for in vivo anticancer effects of various treatments with CuS NP in s.c. BT-474 breast cancer model. FIG. 15B shows tumor growth curves. Compared to untreated control group, significant growth delay was achieved with PEG-[⁶⁴Cu]CuS NP alone (RT), PEG-CuS NP-plus NIR laser (PTT), and PEG- [⁶⁴Cu]CuS NP-plus-NIR laser (RPTT) treatment groups. The mean tumor volumes of untreated control mice reached 998 ± 354 mm by day 30. In animals treated with radiotherapy alone, photothermal therapy alone, and combined radio-photothermal therapies,

3 3 3 the mean tumor volumes were 392 ± 96 mm , 264 ± 345 mm , and 75 ± 105 mm , respectively. At 30 days after treatment, the tumor volumes in the combined treatment group were approximately 14-fold, 5-fold, and 3.5-fold smaller than those in the control group (p=0.003), radiotherapy group (p=0.02), and PTA group (p=0.2), respectively.

[00208] Histological examinations of tumor tissues further confirmed the successful inhibition of tumor cells by the therapy of CuS NP (FIG. 15D). For the control group, tumor mass is localized in the subcutaneous tissue, with an overlying epidermis. At higher magnification, the tumor demonstrates a solid growth pattern with a vague nesting formation. No unequivocal intracellular mucin or glands are present. The tumor is a high- grade breast carcinoma with metaplastic features. Tumor cells display marked pleomorphism, with enlarged nuclei and prominent nucleoli. Atypical mitotic figures are seen readily. However, for the radiotherapy group, tumor mass shows marked degenerative and necrotic changes. Approximately 40% of tumor cells are viable. At higher magnification, the tumor displays an array of treatment effects characterized by clear cell/ballooning degeneration and coagulative tumor necrosis. Histological examinations of tumor tissues also confirmed the successful destruction of tumor cells by the photothermal effect of PEG-CuS NP. According to the hematoxylin and eosin (H&E) staining results, common features of thermonecrosis such as loss of nucleus, cell shrinkages, and coagulation were found from the tumor tissues treated with PEG-CuS NP + NIR. Tumor mass is shrunken, with a small focus of tumor nests that occupy <10% of the nodule. At higher magnification, the nodule mainly consists of fibroblasts, histocytes, and lymphoplasmocytes, which surround a small focus of tumor nests. Tumor cells reveal degenerative changes and single-cell necrosis. All features are consistent with therapeutic effect.

[00209] In radio-photothermally treated tumors, most residual tissues removed from the treated area displayed features similar to normal skin tissue (FIG. 15D). A rounded cellular nodule is present in the subcutaneous tissue. Notice the surrounding adipose tissue and hair sheaths. At higher magnification, the nodule consists of reactive mesenchymal cells, small vessels, and dense plasma cells and lymphocytes. No tumor cells are present. The absence of tumor cells in the reactive nodule represents a complete response.

[00210] To evaluate of toxicity total body weight was used as a surrogate of toxicity, in addition to general observation of well being of animals. In this work, no obvious sign of toxic side effects for CuS NP injected mice was noted within 30 days after injection. Neither mortality nor noticeable body weight loss for mice treated with CuS NP were observed in untreated control and treatment groups (FIG. 16A). Furthermore, histopathology analysis of major organs (heart, liver, spleen, kidney, and lung) did not show abnormal changes (FIG. 16B). Thus, preliminary toxicity data indicate that there was minimal toxic effect after intratumoral injection.

[00211] The foregoing studies demonstrate a multifunctional nanoparticle system, PEG-[⁶⁴Cu]CuS NP, that can be used for radiotherapy, photothermal therapy, and combined radio-photothermal therapy. In addition, PET imaging can be used to determine tumor retention of the nanoparticles and quantitative dosimetry analysis. Tumor growth of the BT-474 tumor-bearing mice were inhibited significantly by radio-photothermal therapy mediated by PEG-[⁶⁴Cu]CuS NP as compared to the other treatments.

* * *

[00212] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

I . A copper chalcogenide nanoparticle, wherein the copper chalcogenide comprises a radioisotope as an integral component of the nanoparticle. 2. The nanoparticle of claim 1, wherein the copper chalcogenide comprises copper sulfide or copper oxide.

3. The nanoparticle of claim 2, wherein the copper sulfide comprises CuxS where x = 1- 2.

4. The nanoparticle of claim 3, wherein the copper sulfide comprises CuS, Cui.₅S or Cu₂S.

5. The nanoparticle of claim 1, wherein the copper chalcogenide comprises a copper

-7 -1

radioisotope and a stable copper isotope at a ratio of about 10^" to 10^" .

6. The nanoparticle of claim 1, wherein the radioisotope is a gamma emitter, a positron emitter or a beta-emitter. 7. The nanoparticle of claim 1, wherein the radioisotope is Cu-64, In-I l l, Tc-99m, Ga- 67, Ga-68, Y-90 or Lu-177.

8. The nanoparticle of claim 1, wherein the radioisotope is a copper radioisotope.

9. The nanoparticle of claim 8, wherein the copper radioisotope is Cu-64, Cu-67, Cu-62, Cu-63 or Cu-61. 10. The nanoparticle of claim 1, further comprising a surface stabilizer.

I I . The nanoparticle of claim 10, wherein the surface stabilizer comprises citrate, cysteine, folic acid, polyacetylene glycol, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol, polylysine, polyvinyl alhocol, human serum albumin, bovine serum albumin, hyaluranic acid, polyethyleimine (PEI), or polyvinylprrolidone (PVP). 12. The nanoparticle of claim 11, wherein the polyacetylene glycol is polyethylene glycol (PEG).

13. The nanoparticles of claim 12, wherein the polyethylene glycol has molecular weight ranging from 500-20,000 dalton.

14. The nanoparticles of claim 13, wherein the polyethylene glycol has molecular weight ranging from 1000-5000 dalton. 15. The nanoparticle of claim 1, wherein the nanoparticle has an average diameter of about 6 nm to 200 nm.

16. The nanoparticle of claim 15, wherein the nanoparticle has an average diameter of more than 10 nm.

17. The nanoparticle of claim 15, wherein the nanoparticle has an average diameter of less than 100 nm.

18. The nanoparticle of claim 17, wherein the nanoparticle has an average diameter of about 11 or 50 nm.

19. The nanoparticle of claim 1, wherein the nanoparticle has a near-infrared absorption.

20. The nanoparticle of claim 19, wherein the nanoparticle has a maximum absorption at about 800 to 1100 nm.

21. The nanoparticle of claim 1, wherein the nanoparticle is further coupled with a tumor targeting moiety, a targeting ligand, a therapeutic, a peptide, an antibody, a nucleic acid, a small molecule, or a polymer.

22. The nanoparticle of claim 21, wherein the targeting ligand binds to CD44. 23. The nanoparticle of claim 22, wherein the targeting ligand comprises hyaluronic acid (HA).

24. The nanoparticle of claim 21, wherein the targeting ligand binds to folate receptors.

25. The nanoparticle of claim 24, wherein the targeting ligand comprises folic acid.

26. A method of imaging and treating an angiogenic or a malignant tissue in a subject, comprising administering to a subject an effective amount of a nanoparticle, wherein the nanoparticle has a metal radioisotope as an integral component of the nanoparticle and enters an angiogenic or a malignant tissue in the subject.

27. The method of claim 26, wherein the nanoparticle is metal chalcogenide comprising a metal radioisotope. 28. The method of claim 26, wherein the metal radioisotope comprises copper radioisotope.

29. The method of claim 26, further comprising imaging the nanoparticles in the subject after a period of time that is sufficient for the nanoparticles to enter an angiogenic or a malignant tissue. 30. The method of claim 29, wherein the imaging comprises positron emission tomography (PET), single photon emission computed tomography (SPECT), or photoacoustic imaging.

31. The method of claim 29, wherein the imaging comprises imaging of lymph nodes in the subject. 32. The method of claim 26, further defined as a radiotherapy or brachytherapy.

33. The method of claim 26, further comprising administering a photothermal ablation therapy to the tissue having the nanoparticles.

34. The method of claim 33, wherein administering a photothermal ablation therapy comprises applying a continuous-wave or pulsed near-infrared laser to the tissue. 35. The method of claim 34, wherein the pulsed near-infrared laser is a nanosecond, microsecond or millisecond pulsed laser.

36. The method of claim 26, wherein the subject has cancer.

37. The method of claim 36, wherein the cancer is melanoma, leukemia, ovarian cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, colon cancer, head and neck cancer, esophagus cancer, synovium cancer, brain cancer, or bronchus cancer.

38. The method of claim 26, wherein the nanoparticle is administered to the subject at an amount of about lxlO⁷ to lxlO¹⁷ nanoparticles/kg body weight.

39. The method of claim 26, wherein the nanoparticle is administered to the subject with a radioactivity of about 1 mCi to 30 mCi. 40. A method of treating a subject comprising administering to a subject an effective amount of a nanoparticle, wherein the nanoparticle has a metal radioisotope as an integral component of the nanoparticle and enters an angiogenic or a malignant tissue in the subject.

41. The method of claim 40, wherein the nanoparticle is metal chalcogenide comprising a metal radioisotope. 42. The method of claim 40, wherein the metal radioisotope is a beta-emitter.

43. The m,ethod of claim 40., further comprising administering a photothermal ablation therapy to the tissue having the nanoparticles.

44. A method of preparing a nanoparticle, wherein the method comprises the steps of: a) providing a mixture comprising a copper radioisotope, a stable copper salt and a non-copper chalcogenide at a reaction condition having a temperature of about 0 °C to about

100 °C; and

b) preparing a nanoparticle according to any of claims 1 to 25.

45. The method of claim 44, wherein the mixture is aqueous.

46. The method of claim 44, wherein the ratio of copper radioisotope and a stable copper salt is about 10^"7 to 10^"1.

47. The method of claim 44, wherein the mixture further comprises a source of a surface stabilizer for the nanoparticle.

48. The method of claim 44, wherein the reaction condition has a pH of about 2 to 12.

49. The method of claim 48, wherein the reaction condition has a pH of about 6 to 8. 50. The method of claim 44, wherein the reaction condition lasts about 1 to 1440 min.

51. The method of claim 44, wherein the mixture further comprises a tumor targeting moiety, a targeting ligand, a therapeutic, a peptide, an antibody, a nucleic acid, a small molecule, or a polymer.