CANCER THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application is related to and claims the priority benefit of U.S. Provisional Patent Application Serial No. 62/101,275, filed January 8, 2015, the contents of which is hereby incorporated by reference in its entirety into this disclosure.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under CBET-1264336 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to a radio-sensitization method to enhance the effectiveness of radiation therapy for treatment of cancer. Specifically, the invention relates to the use of radio-luminescent particles for sensitizing tumor cells to radiation therapy.

BACKGROUND OF THE INVENTION

[0004] Cancer is one of the leading causes of death worldwide. Radiation therapy is one of the three pillars of cancer therapy alongside chemo therapy and surgery. About two-thirds of all cancer patients receive radiation therapy during their illness. The history of radiation therapy dates back to the late 1890s when physicists discovered X-rays and radioactive compounds. Throughout the 20 ^th century, many advances were made toward enhancing the therapeutic efficacy of radiation therapy. Recent decades have also seen significant progress in understanding the molecular mechanisms of radiation therapy. A current active area of research attempts to develop chemical agents (called "radio- sensitizers") that make cancer cells more sensitive to radiation therapy. Several anticancer drugs have been under study as radio- sensitizers; some drugs sensitize cancer cells to radiation by intercalation with DNA, and others produce sensitization effects by arresting the cell cycle, usually in the G2/M phase. Recently, significant attention has also been paid to metal/metal oxide nanoparticles; these "high-Z" materials produce strong secondary radiation due to the photoelectric Compton and Auger effects of X-rays/γ radiation, and cause localized augmentation of radiation damage. However, the sensitizing effects of these currently available compounds are not satisfactory. Also, current nanoparticle sensitization methods work best with X-rays of the order of 100 kVp in energy, but not as well with more clinically relevant radiations with MV-level photon energies.

[0005] The effects of UV light in killing cells have been known for more than 50 years. However, UV has never before been thought useful for radio-sensitization in clinical radiation therapy because UV light has a very limited penetration distance in tissue (< 1 mm in human tissue).

[0006] Accordingly, there exists a need for improved radio-sensitization methods to enhance the effectiveness of a radiation therapy to treat a cancer.

SUMMARY OF THE INVENTION

[0007] In one aspect, a method of treating cancer in a subject is presented. The method includes providing radio-luminescent particles to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles produce ultraviolet (UV) light in response to said ionizing radiation, thereby treating said cancer in said subject. In another aspect, the treatment improves tumor cell killing without increasing toxicity to normal tissue. In yet another aspect, the particle is a calcium tungstate (CaWC ) particle. In yet another aspect, the calcium tungstate particle provided to said tumor in said subject at a concentration less than about 1.0 mg/ml. In yet another aspect, the mean diameter of said particle material is in the range between about 0.1 and 1000 nm. In yet another aspect, the mean diameter of said particle is ranged between about 1 and 10 μιη. In yet another aspect, the mean diameter of said particle is in the range between about 1 and 200 nm. In yet another aspect, the radiation is high-energy photon radiation. In yet another aspect, the radiation is gamma (γ) ray radiation. In yet another aspect, the radiation is X-ray radiation. In yet another aspect, the particle is functionalized or conjugated with a target agent specific to said cancer. In yet another aspect, the target agent is a biological molecule having specific affinity to said cancer cell so as to enhance the delivery of said particles to said tumor cells. In yet another aspect, the particle is functionalized with folic acid. In yet another aspect, the particle is functionalized with polyethylene glycol (PEG), poly(D,L-lactic acid-ran-glycolic acid) (PLGA), or a combination thereof. In yet another aspect, the cancer is lung cancer. In yet another aspect, the cancer is small-cell lung carcinoma (SCLC). In yet another aspect, the cancer is non-small-cell lung carcinoma (NSCLC).

[0008] In yet another aspect, a method of improving the effectiveness of radiation therapy for treatment of cancer in a subject is presented. The method includes providing radio-luminescent particles to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby improving the effectiveness of said radiation therapy for treatment of said cancer in said subject.

[0009] In yet another aspect, a method of sensitizing tumor cells to radiation therapy for treatment of cancer in a subject is presented, which includes providing radio-luminescent particles to said tumor; and exposing said tumor to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby sensitizing said tumor cells to said radiation therapy for treatment of said cancer in said subject.

[0010] In yet another aspect, a method of improving tumor cell killing for treatment of cancer in a subject is presented, which includes providing radio-luminescent particles to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby improving tumor cell killing for treatment of said cancer in said subject.

[0011] In yet another aspect, a method of generating secondary UV radiation in deep tissue tumors is presented, which includes delivering radio-luminescent particles to said tumor and illuminating said particles with deep-penetrating radiation, thereby generating said secondary UV radiation in said tumor.

[0012] In yet another aspect, a method of treating cancer in a subject is presented, which includes providing radio-luminescent particles or particle aggregates to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles or particle aggregates produce ultraviolet (UV) light in response to said ionizing radiation, thereby enhancing treatment of said cancer in said subject. In yet another aspect, the treatment improves tumor cell killing without increasing toxicity to normal tissue. In yet another aspect, the particle comprises a radio-luminescent metal tungstate crystallite material in the form of Mx(W0 ₄ )y where the metal component (M) can be any compound selected from the "Alkaline Earth Metal", "Transition Metal" or "Poor Metal" group of elements in the periodic table, or an atomic mixture thereof. In yet another aspect, the particle is a composite material comprising a radio-luminescent metal tungstate crystallite in claim 24 and other biocompatible organic or inorganic compound. In yet another aspect, the particle comprises a radio-luminescent metal molybdate crystallite material in the form of M _x (Mo0 ₄ ) _y where the metal component (M) can be any compound selected from the "Alkaline Earth Metal", "Transition Metal" or "Poor Metal" group of elements in the periodic table, or an atomic mixture thereof. In yet another aspect, the particle is a composite material comprising a radio- luminescent metal tungstate crystallite in claim 26 and other biocompatible organic or inorganic compound. In yet another aspect, the particle comprises a radio-luminescent calcium tungstate (CaW0 ₄ ) crystallite. In yet another aspect, the particle is a composite material comprising a radio-luminescent calcium tungstate crystallite and other biocompatible organic or inorganic compound.

[0013] In yet another aspect, the radio-luminescent metal tungstate or molybdate crystallite particle or particle aggregate provided to said tumor in said subject at a concentration between about 0.01 and 100 mg metal tungstate or molybdate active ingredient per cc of tumor. In yet another aspect, the radio-luminescent metal tungstate or molybdate crystallite particle or particle aggregate provided to said tumor in said subject at a concentration less than about 20 mg metal tungstate or molybdate active ingredient per cc of tumor.

[0014] In yet another aspect, the mean largest dimension of said particle material is in the range between about 1 and 50,000 nm in its unaggregated state. In yet another aspect, the mean largest dimension of said particle aggregate is ranged between about 10 and 500,000 nm. In yet another aspect, the mean diameter of said particle or particle aggregate is in the range between about 1 and 500 nm.

[0015] In yet another aspect, the ionizing radiation is high-energy photon radiation. In yet another aspect, the ionizing radiation is gamma (γ) ray radiation. In yet another aspect, the ionizing radiation is X-ray radiation. In yet another aspect, the ionizing radiation is electron beam radiation. In yet another aspect, the ionizing radiation is alpha particles emitted in radioactive decay. In yet another aspect, the ionizing radiation is beta particles emitted in radioactive decay. In yet another aspect, the ionizing radiation has a peak energy between about 1 and 50,000 keV. [0016] In yet another aspect, the particle or particle aggregate is functionalized or conjugated through an organic or inorganic linker with a target agent specific to said cancer. In yet another aspect, the target agent is a biological molecule having specific affinity to said cancer cell so as to enhance the delivery of said particles or particle aggregates to said tumor cells and also the internalization of said particles or particle aggregates by said tumor cells. In yet another aspect, the particle or particle aggregate is functionalized or coupled through an organic or inorganic linker with folic acid.

[0017] In yet another aspect, the particle or particle aggregate is encapsulated within a biocompatible coating material. In yet another aspect, the particle or particle aggregate is encapsulated with a biocompatible amphiphilic block copolymer. In yet another aspect, the particle is coated with polyethylene glycol (PEG), poly(D,L-lactic acid) (PLA), or a copolymeric combination thereof. In yet another aspect, the particle is coated with polyethylene glycol (PEG), poly(D,L-lactic acid-ran-glycolic acid) (PLGA), or a copolymeric combination thereof. In yet another aspect, the particle is coated with polyethylene glycol (PEG), poly(s-caprolactone) (PCL), or a copolymeric combination thereof. In yet another aspect, the particle is coated with polyethylene glycol (PEG), poly(styrene) (PS), or a copolymeric combination thereof. In yet another aspect, the cancer is a solid tumor. In yet another aspect, the cancer is a hematological tumor. In yet another aspect, the cancer is head and neck, breast, prostate, lung, gynecological, cervical or brain cancer.

[0018] In yet another aspect, the subject is a human patient. In yet another aspect, the subject is an animal patient. In yet another aspect, the radio-luminescent particles or particle aggregates are administered to the tumor site associated with said cancer in said subject through local, intratumoral, intravenous, intraarterial or intraperitoneal routes.

[0019] In yet another aspect, a method of improving the effectiveness of radiation therapy for treatment of cancer in a subject is presented. The method includes providing radio-luminescent particles or particle aggregates to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby improving the effectiveness of said radiation therapy for treatment of said cancer in said subject.

[0020] In yet another aspect, a method of sensitizing tumor cells to radiation therapy for treatment of cancer in a subject is presented, which includes providing radio-luminescent particles or particle aggregates to said tumor; and exposing said tumor to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby sensitizing said tumor cells to said radiation therapy for treatment of said cancer in said subject.

[0021] In yet another aspect, a method of improving tumor cell killing for treatment of cancer in a subject is presented, which includes providing radio-luminescent particles or particle aggregates to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles emit ultraviolet (UV) light in response to said ionizing radiation, thereby improving tumor cell killing for treatment of said cancer in said subject.

[0022] In yet another aspect, a method of generating secondary UV radiation in deep tissue tumors is presented, which includes delivering radio-luminescent particles or particle aggregates to said tumor and illuminating said particles with deep-penetrating radiation, thereby generating said secondary UV radiation in said tumor.

[0023] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. 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

[0025] Figure 1. Schematic illustration of the concept of combining γ radiation therapy with UV treatment through the use of radio-luminescent agents. UV light can be generated in deep tissue tumors by delivering radio-luminescent particles (RLP) to the tumor and illuminating them with deep-penetrating γ rays. UV-induced G2/M arrest makes cancer cells more susceptible to γ radiation damage.

[0026] Figure 2. SCC7 cells (grown to about 70% confluency in RPMI 1640 medium (Invitrogen) containing 10% FBS and 100 mg/ml penicilin/streptomycin) were exposed to various radiation environments, γ: 6-MV γ radiation (Clinac 600C, Varian) for 150 seconds (5 Gy total dose). RNP: in the presence of added 0.5 mg/ml CaW0 ₄ radio-luminescent powder (RLP) (> 10 μιη diameter) with no γ radiation, γ + RLP: 6-MV γ radiation at 5 Gy total dose in the presence of 0.5 mg/ml CaW0 ₄ RLP. UV: 365 nm wavelength UV irradiation (20 mW/cm ² power density) for 20 minutes. UV→ γ: 20-minute UV irradiation followed by 5-Gy γ radiation, γ→ UV: 5-Gy γ radiation followed by 20-minute UV irradiation. Control: no γ ray, UV light or RLP treatment. At different times (24, 48 and 72 hours) after these treatments, the apoptotic, early apoptotic and necrotic populations were measured as the percentages of total cell populations by FACS with Annexin V/PI double staining. The original two- dimensional dot plots demonstrating the fluorescence gating criteria used are presented in Figure 4.

[0027] Figure 3. Photographs of stirred suspensions of CaW0 ₄ RLP at various concentrations in PBS.

[0028] Figure 4. FACS analysis with Annexin V/PI staining. Ql: necrosis. Q2: apoptosis. Q3: live. Q4: early apoptosis. See the figure caption of Figure 2 for further details.

[0029] Figures 5A - 5G. (Figure 5A) Schematic illustration of a PEGylated (i.e., polyethylene glycol-block-D,L-lactic acid) or PEG-PLA-encapsulated) CaW0 ₄ (CWO) microparticle/nanoparticle. (Figure 5B) A representative TEM micrograph, (Figure 5C) the X-ray diffraction pattern, and (Figure 5D) luminescence emission spectra (under 200 nm excitation) of PLA-PEG-encapsulated CWO microparticles (MPs) and nanoparticles (NPs). All measurements were performed at an identical CWO concentration of 0.1 mg/ml. (Figure 5E) A representative dynamic light scattering (DLS) correlation function for PEG-PLA-coated CWO NPs. From this data the mean hydrodynamic diameter of the PEG-PLA-coated CWO NPs was estimated to be about 180 nm. The average diameter of the pristine CWO NPs was about 10 nm (as shown in (Figure 5B)). The number- average degrees of polymerization of the PEG and PLA blocks of the PEG-PLA block copolymer used were determined by ¾ NMR spectroscopy to be 113 and 44, respectively. (Figure 5F) A photograph of PEG-PLA-coated CWO NPs (1.0 mg/ml in Milli-Q water) under 6 MV X-ray irradiation demonstrating the radio- luminescence of CWO. (Figure 5G) Luminscence emission spectra of uncoated CWO MP suspensions recorded with 200 nm excitation. At high particle concentrations (> 1 mg/ml), optical turbidity does not seem to compromise the luminescence signal.

[0030] Figures 6A - 6B. (Figure 6A) In vitro cytotoxicity of uncoated CaW0 ₄ (CWO) microparticles (MPs) in HN31 cells assessed by Cell Counting Kit-8 (CCK-8) (n = 5). The diameters of the CWO MPs were in the range of about 2 - 3 μιη. (Figure 6B) In vitro cytotoxicity of PEGylated (i.e., poly(ethylene glycol-block-D,L-lactic acid) or PEG-PLA- encapsulated) CaW0 ₄ (CWO) nanoparticles (NPs) in HN31 cells assessed by CCK-8 (n = 5). The average diameter of the pristine CWO NPs was 10 nm (Figure 6B). The mean hydrodynamic diameter of the PEG-PLA-coated CWO NPs was about 180 nm (Figure 6F). The number- average degrees of polymerization of the PEG and PLA blocks of the PEG-PLA block copolymer used were determined by ¾ NMR spectroscopy to be 113 and 44, respectively. In these cytotoxicity measurements, HN31 cells were seeded on 96- well culture plates at a density of 0.5 x 10 ⁴ per well and incubated for 24 h prior to exposure to CWO. The cells were treated with uncoated CWO MPs or PEGylated CWO NPs for 24 h at the various CWO concentrations indicated above. CCK-8 assays were performed at 24 h post-CWO treatment.

[0031] Figure 7. Effect of γ ray dose on the viability of p53-mutant human head and neck cancer HN31 cells (seeded on 60-mm ² culture plates at densities of 4 x 10 ⁵ , 2 x 10 ⁵ and 1 x 10 ⁵ cells per plate (for 24, 48 and 72-hour experiments, respectively) with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin). After 24 h incubation the cells were exposed to various doses of ¹³⁷ Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every 11 s. At different times (24, 48 and 72 hours) after these radiation treatments, the apoptotic, early apoptotic and necrotic populations were measured as the percentages of total cell populations by FACS (fluorescence-activated cell sorting) with Annexin V/PI double staining.

[0032] Figure 8. Effect of γ irradiation on clonogenic survival of HN31 cells at various radiation doses, γ: γ radiation. MP + γ: γ radiation in the presence of added 0.5 mg/ml uncoated CaWC (CWO) radio-luminescent microparticles (MPs) (2 - 3 μιη diameter). NP + γ: γ radiation in the presence of added 0.125 mg/ml PEG-PLA-encapsulated CWO nanoparticles (NPs) (180 nm hydrodynamic diameter). UV→ γ: 365 nm wavelength UV irradiation (20 mW/cm ² power density) for 20 minutes followed by γ radiation, γ→ UV: γ radiation followed by 20-minute UV irradiation. HN31 cells were seeded on 60-mm ² culture plates at densities of 0.2 x 10 ³ (0 Gy), 1.0 x 10 ³ (3 Gy), 2.0 x 10 ³ (6 Gy) and 5.0 x 10 ³ (9 Gy) cells per plate. After 24 h incubation the cells were exposed to various doses of 6-MV ¹³⁷ Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every l i s. Irradiated cells were cultured for 14 days. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted (n = 4). The survival fraction was calculated based on the number of such colonies relative to (Upper Left) that of the untreated control or (Upper Right) that of the respective ηοη-γ-irradiated subgroup for each group. (Lower) Fits to the exponential-quadratic survival function, S = exp(-aD - βΌ ² ) where S denotes the survival fraction, D denotes the radiation dose, and a and β are fitting parameters. The values of the Sensitization Enhancement Ratio or SER (defined as the ratio of the radiation dose at 10% clonogenic survival in the absence of CWO relative to the radiation dose at 10% survival in the presence CWO) were estimated to be 1.15 for MP + γ (0.5 mg/ml CWO) and 1.13 for NP + γ (0.125 mg/ml CWO). For 0.5 mg/ml CWO MP + γ, the p-values relative to control ("γ") were estimated to be 0.13 at 3 Gy, 0.04 at 6 Gy, and 0.04 at 9 Gy. For 0.125 mg/ml CWO NP + γ, the p-values relative to γ were 0.03 at 3 Gy, 0.11 at 6 Gy, and 0.06 at 9 Gy.

[0033] Figure 9. Assessment of the radio-sensitization efficacy of intratumorally injected CWO MPs and NPs in mouse HN31 xenografts. HN31 xenografts were prepared by implanting 7 x 10 ⁷ cells (per mouse) to the left flank of female athymic nude mice (Balb/c, 5 weeks old, 20 to 25 g body weight, n = 6). When tumors were grown over a 21-day period to approximately 250 mm ³ , total 0.30 mg of uncoated CWO MPs or PEG-PLA-encapsulated CWO NPs were infused directly into the tumor (total 1.2 mg of CWO per cc of tumor); the procedure involved two injections of 120 μΐ CWO solution in PBS (CWO concentration: 1.23 mg/ml) over a two- day period. After unfractionated ⁶⁰ Co γ radiation with Gamma Knife (total dose: 5 Gy, dose rate: 2 Gy per minute, γ ray energy: 1.17 and 1.33 MeV), tumor volume was measured for 54 days (using the formula (TI/6)XLXWXH to give volume in ml). The plot on the right is the same plot, but without error bars for clarity.

[0034] Figure 10. Apoptotic and necrotic populations measured by FACS with Annexin V/PI double staining in HN31 cells following exposure to γ radiation (5 Gy) in the absence and presence of concomitant UV light generated by CaW0 ₄ (CWO). Control: no γ ray, UV light or CWO treatment. MP: in the presence of added 0.125 mg/ml uncoated CWO radio- luminescent microparticles (MPs) (2 - 3 μιη diameter) with no γ radiation. NP: in the presence of added 0.125 mg/ml PEG-PLA-encapsulated CWO radio-luminescent nanoparticles (NPs) (180 nm hydrodynamic diameter) with no γ radiation, γ: γ radiation. MP + γ: γ radiation in the presence of added 0.125 mg/ml uncoated CWO MPs. NP + γ: γ radiation in the presence of added 0.125 mg/ml PEG-PLA-encapsulated CWO NPs. UV: 365 nm wavelength UV irradiation (20 mW/cm ² power density) for 20 minutes with no γ radiation. UV→ γ: 365 nm wavelength UV irradiation for 20 minutes followed by γ radiation, γ→ UV: γ radiation followed by 20-minute UV irradiation. HN31 cells were seeded on 60-mm ² culture plates at densities of 4 x 10 ⁵ , 2 x 10 ⁵ and 1 x 10 ⁵ cells per plate (for 24, 48 and 72-hour experiments, respectively) with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin). After 24 h incubation the cells were exposed to a total 5-Gy dose of ¹³⁷ Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every 11 s. At different times (24, 48 and 72 hours) after these radiation treatments, the apoptotic, early apoptotic and necrotic populations were measured as the percentages of total cell populations by FACS (fluorescence-activated cell sorting) with Annexin V/PI double staining. The original two- dimensional dot plots demonstrating the fluorescence gating criteria used are presented in Figure 11.

[0035] Figure 11. FACS analysis of HN31 cells with Annexin V/PI staining. Ql: necrosis. Q2: apoptosis. Q3: live. Q4: early apoptosis. See the figure description of Figure 8 for further details.

[0036] Figure 12. Visualization of senescent HN31 cells by β-galactosidase assay following exposure to γ radiation (5 Gy) in the absence and presence of concomitant UV light generated by CaW0 ₄ (CWO). Control: no γ ray, UV light or CWO treatment, γ: γ radiation. MP: in the presence of added 0.125 mg/ml uncoated CWO radio-luminescent microparticles (MPs) (2 - 3 μιη diameter) with no γ radiation. MP + γ: γ radiation in the presence of added 0.125 mg/ml uncoated CWO MPs. UV: 365 nm wavelength UV irradiation (20 mW/cm ² power density) for 20 minutes with no γ radiation. UV→ γ: 365 nm wavelength UV irradiation for 20 minutes followed by γ radiation, γ→ UV: γ radiation followed by 20-minute UV irradiation. HN31 cells (total 4 x 10 ⁵ cells) were seeded on a 4- well plate. After 24 h incubation the cells were exposed to a total 5-Gy dose of ¹³⁷ Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every l i s. Irradiated cells were cultured for 3 or 6 days. Afterward the cells were stained with X-Gal Staining Solution (Sigma- Aldrich). The cells were imaged using an microscope in order to count blue-stained and unstained cells.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention relates to a radio-sensitization method to enhance the effectiveness of radiation therapy for treatment of cancer. Specifically, the invention relates to the use of radio- luminescent particles for sensitizing tumor cells to radiation therapy.

[0038] The invention demonstrates that secondary UV radiation can be generated in deep tissue tumors by delivering radio-luminescent particles to the tumors and illuminating them with deep-penetrating γ rays or X-rays.

[0039] As demonstrated in the Example section below, one can combine radiation therapy with UV treatment by utilizing radio-luminescent particles which emit UV light as a result of exposure to ionizing radiation. Accordingly, as discussed above, secondary UV radiation can be generated even in deep tissue tumors.

[0040] Any suitable radio-luminescent particle, known to one of skilled in the art, capable of emitting UV light in response to high-energy ionizing radiation can be used. In the literature, "radio-luminescent" materials are also referred to as "scintillation crystals". In an exemplary embodiment, the radio-luminescent material is calcium tungstate (CaW0 ₄ ) powder (i.e., particles having diameters greater than a micrometer). In another embodiment, the radio- luminescent material is submicrometer- sized CaW0 ₄ particles (i.e., nanoparticles).

[0041] Other examples of radio-luminescent materials that can be used include other types of metal tungstates, i.e., M _x (W0 ₄ ) _y where the metal component (M) can be any compound selected from the "Alkaline Earth Metal", "Transition Metal" or "Poor Metal" group of elements in the periodic table, or an atomic mixture thereof. For purposes of this disclosure, Alkaline Earth Metals refers to any one of or a combination of the elements found in Group 2 of the periodic table as defined in accordance with the characterization set forth by the Royal Society of Chemistry (e.g., Be, Mg, Ca, Sr, Ba). Similarly, "Transition Metal" refers to a metal as defined by the IUPAC Gold Book as "an element whose atom has an incomplete d sub-shell, or which can give rise to cations with an incomplete d sub-shell" (e.g., Cr, Mn, Fe, Cu, Zr, Hf). A "Poor Metal" refers to metals that are considered in the art to include some metallic elements of the p-block in the periodic table which are more electronegative than transition metals, and, as defined by the Los Alamos National Laboratory, include "post-transition metals" which include "Al, Ga, In, Tl, Sn, Pb and Bi. As their name implies, they have some of the characteristics of the transition elements. They tend to be softer and conduct more poorly than the transition metals." Poor Metals can also include metalloids, which include "B, Si, Ge, As, Sb, Te, and Po. They sometimes behave as semiconductors (B, Si, Ge) rather than as conductors." These various metal tungstates are generally synthesizable in the micro or nano- sized particulate form.

[0042] Other feasible radio-luminescent materials also include metal molybdates, i.e., M _x (Mo0 ₄ ) _y where the metal component (M) can be any compound selected from the "Alkaline Earth Metal", "Transition Metal" or "Poor Metal" group of elements in the periodic table, or an atomic mixture thereof. Metal tungstates and metal molybdates share common tetrahedral atomic coordination geometries and thus similar electronic band structures; as a result, they exhibit similar radio-luminescent characteristics. Metal molybdates are also synthesizable in micro/nanoparticulate form.

[0043] Any non-toxic concentration of radio-luminescent particles can be used. In a particular embodiment, calcium tungstate particles or particle aggregates at a calcium tungstate concentration less than about 1.0 mg/cc of tumor can be used. As discussed in Example 5, in a clinical embodiment of the present technology, it is expected that at least about 5-10 mg of calcium tungstate particles per cc of tumor needs be infused into the tumor prior to delivery of the first fraction of radiation therapy. Therefore, in one possible embodiment, calcium tungstate particles or particle aggregates at a calcium tungstate concentration less than about 20 mg/cc of tumor can be used. In another embodiment, calcium tungstate particles or particle aggregates at any calcium tungstate concentration in the range between about 0.1 and 100 mg/cc of tumor can be used because the particles are non-toxic.

[0044] Any suitable particle size can be used. In one example, the mean diameter of the particles may be about 0.01, 0.1, 1, 10 or 100 μιη. In a particular embodiment, the mean diameter of the particles is ranged between about 1 and 100 μιη. In another embodiment, the mean diameter of the particles is ranged between 0.01 and 1 μιη.

[0045] The radio-luminescent material can be a nanoparticle. The mean diameter of the nanoparticles can range between about 0.1 and about 500 nm. In one example, the mean diameter of the nanoparticles may be about 0.1, 0.5, 1, 5, 10, 50, 100, 150, 200 or 500 nm.

[0046] In general, any radio-luminescent metal tungstate or molybdate particle with a mean largest dimension between 0.001 and 50 μιη (as determined in the unaggregated state) can be used. Any radio-luminescent metal tungstate or molybdate particle aggregate with a mean largest dimension between 0.001 and 500 μιη can be used. In a preferred embodiment of the present technology, the radio-luminescent metal tungstate/molybdate particle or particle aggregate has a mean diameter in the range between 0.001 and 0.5 μιη.

[0047] The radio-luminescent particles of the invention emit UV light in response to high- energy photon radiation. Examples of high-energy photon radiation include, but not limited to, gamma (γ) ray and X-ray radiations. The radio-luminescent particles can also be excited by other types of ionizing radiation, including electron beam radiation and alpha/beta particles produced during radioactive decay of some radioactive isotopes.

[0048] The radio-sensitization method based on radio-luminescent particles works well with clinically relevant radiations with MV-level X-ray/y-ray photon energies. In a preferred embodiment of the invention, the radio-luminescent particles are used with ionizing radiation with an energy in the range of between about 1 keV and about 50 MeV.

[0049] In one aspect, the radio-luminescent particles of the invention can be conjugated or operably linked to a targeting agent ("ligand") specific to cancer cells. For example, lung cancer specific targeting ligand can be conjugated to the surfaces of the radio-luminescent particles in order to enhance the delivery of the particles to lung cancer cells. The targeting agent can be a biological molecule (e.g., an antibody) having specific affinity to cancer cells. Examples of cancer targeting agents that can be used include, but not limited to, folic acid, transferrin, and monoclonal antibodies against CD123, CD33, CD47, CLL-1 , etc. Any suitable conjugation or linking method, known to one of skilled in the art, can be used.

[0050] In another aspect, the radio-luminescent particles or particle aggregates of the invention can be encapsulated within a biocompatible coating material. The coating material can be a polymer, for example, a biodegradable or biocompatible amphiphilic block copolymer. Any suitable polymer can be used for this purpose. Examples of polymers include, but are not limited to, polyethylene glycol (PEG), poly(D,L-lactic acid) (PLA), poly(D,L-lactic acid-ran- glycolic acid) (PLGA), poly(s-caprolactone) (PCL), poly(styrene) (PS), or a copolymeric combination of two or more of these polymer components. The coating material itself can be functionalized with a cancer-targeting moiety such as folic acid.

[0051] In a particular embodiment, provided herein is a method of treating cancer in a subject, the method comprising: providing radio-luminescent particles to a tumor site associated with said cancer in said subject; and exposing said tumor cells to high-energy ionizing radiation, wherein said particles emit UV light in response to said ionizing radiation, thereby treating said cancer in said subject. The treatment may improve tumor cell killing without increasing toxicity to normal tissue. The tumor to be treated can be a solid or hematological tumor. Said subject can be a human or an animal (e.g., an animal patient presented to an animal hospital). Possible routes of administration of the radio-luminescent particle include, but not limited to, intratumoral, intravenous, intraarterial, and intraperitoneal.

[0052] As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e. , the state in which the disease or condition does not worsen), delay or slowing down of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if a patient were not receiving treatment. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented. In one example, the terms "treat" and "treatment" refer to inhibiting tumor growth.

[0053] The method of the invention can be used to treat any cancer/tumor. Examples of cancer/tumor types which may be treated include, but not limited to, head and neck cancer, breast cancer, prostate cancer, lung cancer, lung cancer, gynecological cancer, cervical cancer, brain cancer, melanoma, and colorectal cancer (including HER2+ and metastatic). Additional examples of cancers/tumors which may be treated include, but not limited to, bladder cancer, ovarian cancer, and gastrointestinal cancer. Examples of lung cancer include, but are not limited to small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

[0054] Cancers/tumors that can be treated include primary, secondary and metastatic tumors (including those metastasized from lungs, breast, prostate, gastrointestinal tract, kidney, and larynx) as well as recurrent or refractory tumors. Recurrent tumors encompass tumors that appear to be inhibited by treatment but recur up to five years, sometimes up to ten years or longer after treatment is discontinued. Refractory tumors are tumors that have failed to respond or are resistant to treatment with one or more conventional therapies for the particular tumor type. Refractory tumors include those that are hormone-refractory (e.g., androgen-independent prostate cancer, or hormone-refractory breast cancer such as breast cancer that is refractory to tamoxifen), those that are refractory to treatment with one or more chemotherapeutic agents, those that are refractory to radiation therapy, and those that are refractory to combinations of chemo and radiation therapies, chemo and hormone therapies, or hormone and radiation therapies.

[0055] Therapy may be "first-line", i.e. , as an initial treatment in a patient who has had no prior anti-cancer treatments, either alone or in combination with other treatments, or "second-line", as a treatment in a patient who has had one prior anti-cancer treatment regimen, either alone or in combination with other treatments, or as "third-line," "fourth-line," etc., treatments, either alone or in combination with other treatments.

[0056] Therapy may also be given to a patient who has had previous treatments which have been partially successful but is intolerant to the particular treatment. Therapy may also be given as an adjuvant treatment, i.e. , to prevent reoccurrence of cancer in a patient with no currently detectable disease or after surgical removal of tumor. [0057] In another aspect provided herein is a method for improving the effectiveness of radiation therapy for treatment of cancer in a subject, the method comprising: providing radio- luminescent particles or particle aggregates to a tumor site associated with said cancer in said subject; and exposing said tumor cells to high-energy ionizing radiation, wherein said particles emit UV light in response to said ionizing radiation, thereby improving the effectiveness of said radiation therapy for treatment of said cancer in said subject.

[0058] In yet another aspect provided herein is a method for sensitizing tumor cells to radiation therapy for treatment of cancer in a subject, the method comprising: providing radio- luminescent particles or particle aggregates to said tumor; and exposing said tumor cells to ionizing radiation, wherein said particles emit UV light in response to said ionizing radiation, thereby sensitizing said tumor cells to said radiation therapy for treatment of said cancer in said subject.

[0059] In yet another aspect provided herein is a method for improving tumor cell killing for treatment of cancer in a subject, the method comprising: providing radio-luminescent particles or particle aggregates to a tumor site associated with said cancer in said subject; and exposing said tumor cells to ionizing radiation, wherein said particles emit UV light in response to said ionizing radiation, thereby improving tumor cell killing for treatment of said cancer in said subject.

[0060] In yet another aspect provided herein is a method for generating secondary UV radiation in deep tissue tumors, the method comprising delivering radio-luminescent particles or particle aggregates to said tumor and illuminating said particles with deep-penetrating radiation (e.g., γ rays or X-rays), thereby generating said secondary UV radiation in said tumor.

[0061] Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety.

[0062] The following examples are presented in order to more fully illustrate preferred embodiments of the present invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES

EXAMPLE 1

Radio-Luminescent Particles for Enhancement of Radiation Cancer Therapy

[0063] Cancer is one of the leading causes of death worldwide. Current data suggest that radiation and concurrent chemotherapy may increase survival of patients with inoperable cancers. However, overall progress in radiation treatment of late stage cancer is still limited. Here, we demonstrate a novel approach for improving the efficiency of radiation therapy in the treatment of cancer without increasing normal tissue toxicity— namely, by simultaneous addition of radio-luminescent particles (which emit UV light under high energy photon radiation), to γ ray/X-ray radiation treatments.

[0064] Synergistic interactions of UV light with ionizing radiation (such as γ rays and X-rays) in killing cells have been known for more than 50 years. However, UV has never before been thought useful for radiation sensitization in clinical radio-therapy because UV light has a very limited penetration distance in tissue (< 1 mm in human tissue). Our recent work demonstrates that one can combine radiation therapy with UV treatment by utilizing radio-luminescent agents which emit UV light as a result of exposure to ionizing radiation; therefore, secondary UV radiation can be generated even in deep tissue tumors by delivering radio-luminescent particles to the tumor and illuminating them with deep-penetrating γ rays or X-rays (Figure 1).

[0065] In order to validate the feasibility of this concept, we first performed in vitro tests in radiation- sensitive cancer models. For this purpose, a murine squamous cell sarcoma SCC7 cell line was used. In one test, SCC7 cells were first exposed to UV light (365 nm) for 20 minutes and then subsequently irradiated with γ rays to a total dose of 5 Gy. As shown in Figure 2 (see data labeled as "UV→ γ"), this UV pre-treatment renders the cells significantly more susceptible to γ radiation damage than non-UV-treated cells (see data labeled as "γ"). At 72 hours after γ radiation, the cells exposed to both UV and γ ("UV→ γ") showed about a 60 percent death rate, whereas the cells exposed only to γ rays ("γ") showed a 30 % death rate; as also shown in the figure (see data labeled as "UV"), the 20-minute UV exposure alone did not influence the viability of SCC7 cells relative to the untreated control cells ("Control"). Even when the sequence of the two types of radiation was reversed (i.e., γ ray first, and then UV light), the combination radiation still produced a synergistic increase in cells undergoing apoptosis (see data labeled as "γ→ UV" in Figure 2); this "γ→ UV" treatment resulted in over 50 percent cell death at 72 hours post-treatment.

[0066] For generating secondary UV light by γ ray photons, a common radio-luminescent material, calcium tungstate (CaWCU), was tested in powder form (particle diameter 2 - 3 μιη). This CaWCU material exhibits a strong luminescence emission with a maximum at 420 nm wavelength under high energy photon excitation at room temperature (Figures 5D, 5F and 5G). The concept of using CaW0 ₄ radio-luminescent powder (RLP) for the γ ray sensitization of cancer cells was tested again using SCC7 cells. CaWCU powder is itself non- toxic up to a concentration of about 5 mg/ml (Figure 6). When SCC7 cells were exposed to γ radiation at 5 Gy total dose in the presence of 0.5 mg/ml CaWCU RLP (see data labeled as "γ + RLP" in Figure 2), the death rate was significantly higher than in the absence of the particles ("γ"); the "γ + RLP" treatment caused close to 50 percent cell death at 72 hours post-treatment. These results prove that radio-luminescent particles can indeed be used for UV-induced γ ray sensitization of cancer cells.

[0067] Concurrent UV irradiation produces sensitization effects because DNA damage by UV light may likely initiate repair sequence, and arrest progression of the cell cycle from G2 to mitosis; cancer cells are likely most susceptible to radiation damage when they are in the G2/M phase. Even the un-optimized radio-luminescent material is already able to induce about a factor of two enhancement of the cytotoxic effect of γ rays, a level of increase not easily seen with conventional radio-sensitizers. The sensitization effect may become even greater when CaWCU is used in nanoparticle form; smaller nanoparticles have larger surface areas and can produce higher induced UV emissions; also, nanoparticles can easier be internalized by cancer cells.

EXAMPLE 2

Synthesizing CaW0 ₄ nanoparticles of various sizes (20 - 200 nm), functionalizing these particles with different functional groups (PEG, folic acid), and investigating the radio- luminescent and cell interaction properties of these particles.

[0068] CaWCU radio-luminescent nanoparticles (RLNP) having various sizes in the range of 20 to 200 nm can be first synthesized by the solvothermal reaction method. The detailed atomic structures of these CaWCU nanoparticles can be determined by X-ray diffraction and transmission electron microscopy. CaW0 ₄ nanoparticles can be surface functionalized by encapsulation with poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) or poly(lactic acid- co-glycolic acid)-poly(ethylene glycol)-folate. The size and size distribution characteristics of the uncoated, PEG-encapsulated, and folate-functionalized CaW0 ₄ nanoparticles can be characterized by dynamic light scattering and analytical ultracentrifugation. How size and surface characteristics affect the luminescence properties of CaW0 ₄ RLNP can be evaluated under X-ray excitation by spectrophotometry. The effects of size and surface chemistry on the cellular internalization and intracellular trafficking of the nanoparticles can also be investigated in human lung cancer cell lines by flow cytometry and confocal microscopy.

[0069] Synthesis and characterization of CaW0 ₄ nanoparticles. CaW0 ₄ nanoparticles can be prepared by the solvothermal reaction of sodium tungstate dihydrate (Na2W0 ₄ -2H20) with calcium salt (such as calcium chloride (CaCl2-2H20), calcium nitrate (Ca(N03)2-4H20) or calcium acetate (Ca(CH3COO)2)). Surfactant (such as CTAB, Triton X or PEG) can be used to control the particle nucleation and growth kinetics. A hydrothermal post-treatment method can be used to fine tune the sizes of final CaW0 ₄ materials to be studied. The radio-luminescence of CaW0 ₄ can be determined by the crystallinity of the material. The detailed structural characterizations can be performed using the X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HR-TEM) techniques. The cellular internalization and intracellular trafficking of CaW0 ₄ nanoparticles can be significantly influenced not only by the mean size but also by the size distribution of the nanoparticles. The mean particle size data can be obtained using the dynamic light scattering (DLS) technique. The precise size distribution characteristics of the CaW0 ₄ nanoparticles can be examined using the analytical ultracentrifugation (AUC) technique.

[0070] Surface functionalization of CaW0 ₄ nanoparticles with PEG and folic acid. The as-synthesized CaW0 ₄ particles can be further stabilized by encapsulation with a biodegradable block copolymer (BCP), poly(lactic acid-co-glycolic acid) -poly (ethylene glycol) (PLGA-PEG), via the solvent exchange procedure. DLS can be used to measure the mean size, and AUC to determine the size distribution of the BCP-coated CaW0 ₄ nanoparticles. Folate surface functionalized CaW0 ₄ nanoparticles can also be prepared. The folate receptor is overexpressed in many cancel cell types including lung cancer cells. Therefore, folate ligands can enhance the internalization of the nanoparticles in cancer cells. For this purpose, a folate-functionalized PLGA-PEG BCP can be synthesized using the fluorenylmethyloxycarbonyl chloride (FMOC) protection chemistry described in our previous publication.

[0071] Confirmation of the radio-luminescence and cell internalization of CaW04 nanoparticles. The radio-luminescence/phosphorescence properties (emission wavelength, luminescence timescale, etc.) of CaW0 ₄ nanoparticles of various sizes (20 - 200 nm) and surface functionalities (uncoated, PEG-encapsulated, and folate-functionalized) can be characterized by spectrophotometry under the specific X-ray excitation conditions that can be used in Example 3 studies described below (i.e., 250 kVp and 6 MV X-rays). The effect of PEG/folic acid surface functionalization on cellular uptake of CaWCU nanoparticles can be studied using the model lung cancer cell lines described in Example 3 and the fluorescence- activated cell sorting (FACS) technique. Intracellular trafficking properties can be analyzed in the lung cancer cells by confocal microscopy, known in the art. For these studies, a fluorescently labeled (AlexFluor647) version of the PLGA-PEG block polymer can be prepared using the FMOC protection method.

EXAMPLE 3

Determining whether concurrent CaWC>4 radio-luminescent nanoparticles (RLNP) alter the biological effectiveness and/or mode of cell death in human lung cancer cell lines treated with fractionated doses of X-rays in vitro and in tumor xenografts.

[0072] Whether UV irradiation inhibits cell growth of small cell lung cancer (SCLC) and non- small cell lung cancer (NSCLC) cell lines and/or alter cell cycle distribution can be first investigated. If this inhibition is p53-dependent or involves the induction of apoptosis can be determined. One can then test whether UV light generated by CaW0 ₄ RLNP under X-ray radiation increases the biological effectiveness after small and large radiation fractions and determine whether any observed enhancement is dependent on the size and surface characteristics of the CaW0 ₄ RLNP which can determine the cellular uptake rate and intracellular location of the nanoparticles. Also, one can investigate whether the radio- sensitization effect of CaW0 ₄ RLNP is related to the suppression of constitutive and/or radiation- induced NF-κΒ activity in the various lung cancer cell lines. Finally, one can study whether double strand break DNA repair is altered after small or large fraction irradiation in the presence (and absence) of CaW0 ₄ RLNP. If this is observed, one can investigate whether this inhibition involves the homologous recombination or non-homologous end joining pathways.

[0073] Control experiments to confirm the sensitivity of model cell lines against fractionated X-rays. In this study, the amount of cell killing can be quantitated by measuring in vitro clonogenic survival by our standard techniques in two available human lung cancer cell lines isolated from SCLC (SBC-3 and SBC-5) and two NSCLC cell lines (H1792 and H1838) that have been irradiated with radiation fractions of 5, 10, 15, 20, or 40 Gy of 160 kVp X-rays. The mode of cell death (apoptosis, necrosis, mitotic death) can be investigated by methods, known in the art, to test whether it changes versus fraction size. In addition, the kinetics of DNA double strand break repair and amount of residual unrepaired DNA damage can be determined by pulse field gel electrophoresis and the comet assay by established methods after large radiation fraction sizes (10, 15, 20, and 40 Gy) and one can test whether any of the above endpoints are dependent on the constitutive level of NF-κΒ in the cells. NF-κΒ can be determined by gel shift assays according to methods known in the art. [0074] Determination of the efficacy and mechanism of the CaW04 RLNP-induced radio- sensitization of the human lung cancer cells. One can first investigate whether 48 hour incubation with various concentrations (1, 0.5, 0.25, 0.125, 0.0625 and 0 mg/ml) CaW0 ₄ RLNP inhibits cell growth of SCLC and NSCLC cell lines or alters cell cycle distribution. Separately, one can also investigate whether UV irradiation alone causes growth inhibition of the SCLC and NSCLC cell lines and/or alter cell cycle distribution. Whether this inhibition is p53- dependent or involves the induction of apoptosis can be determined. One can then determine whether CaW0 ₄ LRNP increases the biological effectiveness after small and large radiation fractions by the published assays listed above. Furthermore one can test whether any observed enhancement is dependent on the size and surface characteristics of the nanoparticles which can determine their cellular uptake rates and intracellular distributions. Whether the radio- sensitization effect of CaW0 ₄ RLNP is related to the inhibition of the level of constitutive and/or radiation-induced NF-κΒ activity in the various lung cancer cell lines can be investigated and whether double strand break DNA repair is altered after small or large fraction X-ray irradiation in the presence (and absence) of CaW0 ₄ RLNP can be determined. If one observes an alteration in the kinetics of repair or in the number of residual strand breaks, one can begin investigating whether this inhibition involves the homologous recombination or non- homologous end joining pathways through the use of chemical kinase inhibitors and appropriate siRNA knockdown of proteins involved in these pathways.

EXAMPLE 4

Clinical Impact

[0075] Lung cancer is the leading cancer death in the United States and worldwide. The current evidence suggests that radiation and concurrent chemotherapy such as cisplatin and etoposide may increase median survival in inoperable Stage III NSCLC with good performance status. In limited stage SCLC, combined chemo-radiation also appears to have some benefit; however, overall progress is still limited. A novel concept that the addition of concurrent, tumor-targeted radio-luminescent nanoparticles (which emit UV light under high energy photon radiation), to fractionated radio-therapy approaches may improve tumor cell killing without increasing normal tissue toxicity. The preclinical studies can determine whether the biological efficacy of fractionated radiation treatments can be enhanced by concurrent treatment with this new type of radio-sensitizer, CaW0 ₄ radio-luminescent nanoparticles (RLNP), and can also determine the detailed biological mechanism of the UV-induced radio- sensitization effect, i.e., whether this enhancement is due to alteration of the mode of cell death or DNA repair, and whether NSCLC or SCLC respond in a similar manner.

EXAMPLE 5

Utilizing Radio-Luminescent Particles (RLPs) as a means to achieve radio-sensitization and study the ability to affect head and neck cancer cells (in vitro) and xenografts (in vivo). This Radio Luminescence Therapy (RLT) technology enables cancer patients to achieve the benefits of radiation treatment with reduced negative effects.

[0076] Radiation therapy is one of the three pillars of cancer therapy alongside chemotherapy and surgery. About two-thirds of all cancer patients receive radiation therapy during their illness, and in the US annually nearly a million patients are treated with radiation therapy. Unfortunately, radiation therapy generally carries significant side effects in patients, both acute and chronic, because of the damage high-energy ionizing radiation (such as X-ray or γ ray radiation) causes to normal cells and tissues. Due to the side effects, patients receive small amounts of radiation over weeks of period which increases cost of treatment not only for the patients but also for hospitals and insurance companies; in the US, hospitals annually spend $5.8 billion in radiation equipment, and insurance companies annually spend $15 billion in

radiation therapy. Therefore, radiotherapy is also an economic burden to the entire health care system.

[0077] There is therefore an unmet need for using radiation therapy to treat cancer more safely, effectively, and more rapidly. A current active area of research attempts to develop chemical agents ("radio-sensitizers") that make cancer cells easier to kill with radiation therapy. Many anticancer drugs have radio-sensitization effects. Some drugs (such as doxorubicin, 5- fluorouracil, cisplatin) sensitize cancer cells to radiation by intercalation with DNA, and others (e.g., paclitaxel, etanidazole) produce sensitization effects by arresting the cell cycle, usually in the G2/M phase. For this reason, "chemo radio therapy" has now become standard care for some cancers. However, chemotherapy agents, even if injected locally, typically diffuse out of the site of delivery after a relatively short time, causing systemic side effects. Nanoparticles (NPs) offer improvements, because, due to their larger sizes, nanoparticles are easier to maintain at the tumor sites for longer periods of time. Recently, significant attention has been paid to metal/metal oxide nanoparticles; these "high-Z" materials (particularly, those derived from gold, silver, iron, gadolinium, hafnium) produce strong secondary electron radiation due to the photoelectric, Compton and Auger effects, and thus cause localized augmentation of radiation damage. However, the sensitizing effects of these currently available compounds are not satisfactory; none of these previous methods is able to increase the radiation's potency by more than a factor of about 1.5. Particularly, in in vivo situations, these photo-electric nanoparticles are limited because of the small mean free paths of electrons. Nanoparticles that produce photons (instead of electrons) with X-ray should be better materials for radio- sensitization, because photons have two to three orders of magnitude larger mean free paths than electrons. Also, current radio-sensitization methods based on photo-electric nanoparticles work best with X-rays of the order of 100 kVp in energy, but not as well with more clinically relevant radiations with MV-level X-ray/y-ray photon energies because of the significantly reduced absorption cross-sections at the higher energies. Also, unfortunately the most studied material in this regard, i.e., nanoparticulate gold, is known to cause genotoxic and mutagenic effects in exposed tissues in vivo.

[0078] In this Example, a completely new method of radio-sensitization (named "Radio Luminescence Therapy (RLT)") that addresses all limitations of the conventional nanoparticle radio-sensitization approaches is demonstrated. This method utilizes a new type of radio- sensitizers, namely, "Radio-Luminescent Particles (RLPs)". These RLPs emit UV light, instead of secondary electrons, as a result of exposure to ionizing radiation (such as X-ray or γ ray). The combination of X-ray and UV light significantly enhances the cancer cell-killing effectiveness of X-rays. This RLT works even better with clinically relevant MV- level X-rays. This RLT technology can be applied to the animal radio- sensitizer market (for companion animals with malignant cancers.

[0079] UV light itself has genotoxic effects on cancer cells; it causes damage to DNA in cancer cells. DNA damage by UV light initiates repair sequence, and arrests progression of the cell cycle from G2 to mitosis. Therefore, it is beneficial to combine radiation therapy with UV treatment, because cancer cells are most susceptible to radiation damage when they are in the process of separating the replicated chromosomes during cell division (i.e., in the G2/M phase). This is the underlying hypothesis which the herein disclosed RLT technology is based upon. Synergistic interactions of UV light with X-rays in killing cells have been known for more than 50 years. However, UV has never before been thought useful for X-ray/γ ray sensitization in clinical radiation therapy because UV light has a very limited penetration distance in tissue (< 1 mm). As disclosed in the Results section below, work by our laboratory demonstrates that it is possible to combine radiation therapy with UV treatment by utilizing radio-luminescent agents which emit UV light under ionizing radiation (Figure 1). Secondary UV radiation can be generated even in deep tissue tumors by delivering radio-luminescent particles to the tumor and illuminating them with deep-penetrating γ rays or X-rays. This is a new paradigm in cancer radiation therapy.

[0080] The herein disclosed technology utilizes a naturally- abundant radio-luminescent mineral, calcium tungstate (CaW0 ₄ ) in its micro or nanoparticulate form for generating secondary UV light by γ ray/X-ray photons. This CaW0 ₄ material exhibits a luminescence emission with a maximum at 420 nm wavelength under high energy ionizing radiation such as γ rays, X-rays or short wavelength UV light at room temperature (Figures 5A - 5G). Our RLP formulations are designed to be non-toxic to the human body. The RLP core is composed of CaW0 ₄ , which is chemically stable and non- toxic, and this CaW0 ₄ core is further coated with an FDA-approved polymer (poly(ethylene glycol-block-D,L-lactic acid) (PEG-PLA)) to make it inert to proteins; encapsulated CaW0 ₄ (CWO) has previously been shown to possess no detectable cytotoxicity against HeLa cells. Unlike gold or silica, CaW0 ₄ does not have any reactive sites, and the PEG surface functionalization can be achieved only by the method developed by our laboratory. Both nanoparticle fabrication and encapsulation processes are scalable for production of large quantities. These RLPs are formulated in solution form, and can be injected into the tumor without loss.

[0081] In vitro tests demonstrate that even an un-optimized RLP materials (uncoated CWO microparticles (MPs) with 2 - 3 μιη diameter and PEG-PLA-coated CWO nanoparticles (NPs) with about 180 nm hydrodynamic diameter) are able to induce a significant enhancement of the tumor suppressive effect of γ rays in radio-resistant cancer models. Further optimization of the size and surface functional characteristics is expected to further significantly increase the radio-sensitization effect. Specifically, a study is in progress in which various RLP samples having different sizes (greater vs. less than 50 nm diameter) and surface functionalities (folate acid-functionalized vs. non-folate-functionalized) are prepared by the techniques developed by our laboratory. Particle size and surface functionality are two important parameters that need to be optimized in order to maximize the intratumoral retention and distribution, the cellular internalization, and thus the overall radio-sensitization efficiency, and also the eventual pharmacokinetic fate of the material can be determined by these parameters.

[0082] RLPs can be used in human cancer patients. For regulatory reasons, we will pursue the animal market first. The product will be RLPs in a solution that can be injected directly to tumor prior to radiation treatment; there is no need to change the existing radiation-treatment platform.

[0083] Preparation of block copolymer(BCP)-encapsulated CaW0 ₄ (CWO) nanoparticles (NPs). The CWO NPs were synthesized by a micro-emulsion method. First, 20 ml of cyclohexane was mixed with 2 ml of hexanol. CTAB (2 mmol) (> 99%, Sigma) was added to this solvent mixture, and then the solution was heated to 70 °C or until the solution became transparent (Solution 1). Meanwhile, 0.4 mmol of Na2W0 ₄ (99%, Acros Organics) was dissolved in 0.6 ml of Milli-Q water (Solution 2). Next, 0.4 mmol of CaCk was dissolved in a mixture of 0.564 ml of Milli-Q water and 0.036 ml of 0.1 M HC1 solution (Solution 3). Solutions 2 and 3 were immediately injected to Solution 1, and the resulting mixture was vigorously stirred. After about a minute, the mixture was transferred into a Teflon-lined stainless steel autoclave, and the autoclave was heated to 160 °C and maintained at that temperature for 24 hours. Afterwards, the autoclave was gradually cooled to room temperature. The product was collected by centrifugation and washed twice with ethanol to remove residual cyclohexane and excess CTAB.

[0084] The PEG-PLA diblock copolymer was synthesized by l,8-diazabicyclo[5.4.0] undec- 7-ene(DBU, 98%, Aldrich)-catalyzed ring-opening polymerization of lactide (LA, a racemic mixture). 0.45 g of PEG-ME was dissolved in DCM (22 ml) dried with molecular sieves. After a day LA (0.35 g) was added into the PEG-ME solution. The polymerization was initiated by adding 2 ml of a DBU solution (3.35 mmol of DBU dissolved in 30 ml of DCM) to the LA/PEG-ME mixture at room temperature. The polymerization reaction was run for 1 hour at room temperature. Afterward the reaction was terminated by adding 10 mg of benzoic acid (> 99.5%, Sigma- Aldrich). The polymerization mixture was added drop-wise to 1000 ml petroleum ether for precipitation. After the PEG-PLA product settled to the bottom, the supernatant was decanted. The polymer was dried in a vacuum oven.

[0085] PEG-PLA-encapsulated CWO samples were prepared as follows. 1.0 mg of CWO (purified by centrifugation) was dispersed in 1.0 g of DMF. 100.0 mg of PEG-PLA was added to 2.9 g of the above nanoparticle suspension. This mixture was stirred using a high speed overhead mechanical stirrer (at 15000 rpm) with simultaneous sonication. 2.1 ml of Milli-Q water was added to the DMF solution. The resulting mixture was emulsified with a mechanical stirrer and then ultrasonicated in a sonication bath for 30 minutes. This emulsion was placed in a dialysis bag (molecular weight cutoff 50 kDa) and dialyzed for 3 days against a total of 1.0 liter of Milli-Q water (regularly replaced with fresh Milli-Q water) to remove DMF.

[0086] Cell viability. HN31 cells were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS, 100 Units/ml penicillin and 100 μg/ml streptomycin in humidified atmosphere with 5% CO2 37°C. For cytotoxicity measurements HN31 cells were seeded on 96-well culture plates at a density of 0.5 x 10 ⁴ cells per well and incubated for 24 h prior to exposure to CaWC (CWO). The cells were treated with uncoated CWO microparticles (MPs) or PEGylated CWO nanoparticles (NPs) for 24 h at the various CWO concentrations indicated in Figure 6. To quantitate the viability of the CWO-treated cells, Cell Counting Kit- 8 (CCK-8) (Dojindo Molecular Technologies, Rockville, MD, USA) assays were performed at 24 h post-CWO treatment using the manufacturer's procedures (n = 5).

[0087] Flow cytometry. The mode of cell death (apoptosis, early apoptosis, necrosis) following γ radiation in the presence and absence of CWO particles was investigated by flow cytometry analysis. HN31 cells were seeded on 60-mm ² culture plates at various designated densities with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. After 24 h incubation the cells were exposed to various designated doses (see figure captions) of ¹³⁷ Cs γ radiation (IBL 437C, CSI Bio International, France) at a rate of 1 Gy per every 11 s. At designated times following γ ray treatments in the presence and absence of various loadings of CWO MPs or NPs, the cells were collected by trypsin treatment, double

ts- stained with Annexin V-FITC and PI (BD Biosciences, San Jose, CA, USA), and analyzed using the fluorescence- activated cell sorting (FACS) technique.

[0088] In vitro clonogenic assay. HN31 cells were seeded on 60-mm ² culture plates at various designated densities for different radiation doses and incubated for 24 h prior to exposure to γ rays; see Figure 8 for details of the radiation conditions. The irradiated cells were incubated for 14 days. Colonies resulting from radio-resistant cells were stained with Crystal Violet. Colonies of more than 50 daughter cells in culture were counted and the survival fraction was calculated based on the number of such colonies relative to that of the untreated or unradiated control (n = 4).

[0089] In vivo tumor growth. HN31 xenografts were prepared by implanting 7 x 10 ⁷ cells (per mouse) to the left flank of female athymic nude mice (Balb/c, 5 weeks old, 20 to 26 g body weight, n = 6) (Orient Bio, Korea). When tumors were grown over a 21-day period to approximately 250 mm ³ , total 0.30 mg of uncoated CWO MPs or PEG-PLA-encapsulated CWO NPs were infused directly into the tumor (total 1.2 mg of CWO per cc of tumor); the procedure involved two injections of 120 μΐ CWO solution in PBS (CWO concentration: 1.23 mg/ml) over a two-day period. After unfractionated ⁶⁰ Co γ radiation with Leksell Gamma Knife Perfexion TM (Elekta Instrument, Sweden) (total dose: 5 Gy, dose rate: 2 Gy per minute, γ ray energy: 1.17 and 1.33 MeV), tumor volume was measured for 54 days (using the formula (TI/6)XLXWXH to give volume in ml).

[0090] UV treatment. UV exposure was performed using a UVP's B-100AP lamp (wavelength = 365 nm, power density = 20 mW/cm ² ) mounted on an illumination environmental chamber designed for homogeneous illumination and better heat dissipation via the vent at the bottom.

[0091] Statistical analysis. All data values represent averages from independent series of experiments. Error bars represent standard deviations. Student's i-test was used to analyze statistical significance between control and treatment.

[0092] Synergistic interactions of UV light with ionizing radiation (such as γ rays and X-rays) in killing cells have been known for more than 50 years. However, UV has never before been thought useful for radiation sensitization in clinical radio-therapy because UV light has a very limited penetration distance in tissue (< 1 mm in human tissue). Here, we report our recent work that demonstrates that it is possible to combine radiation therapy with UV treatment by utilizing radio-luminescent agents which emit UV light as a result of exposure to ionizing radiation; therefore, secondary UV radiation can be generated even in deep tissue tumors by delivering radio-luminescent particles to the tumor and illuminating them with deep- penetrating γ rays or X-rays (Figure 1).

[0093] CaWCU (CWO) RLPs of various sizes (2 - 100 nm diameters) have been successfully prepared by the reaction of sodium tungstate dihydrate with calcium salt (Figures 5A - 5G). Surfactant was used to control the particle nucleation and growth kinetics. A post-reaction thermal treatment method was used to fine-tune the final size. The radio-luminescence of CaWCU is due to the unique crystal structure and resulting electronic band gap of the material. Detailed structural characteristics (grain size, shape, and crystallinity) of the synthesized CaWCU nano crystals were characterized using the X-ray diffraction and high-resolution TEM techniques (Figures 5B and 5C). The blue emission of CaWCU at around 420 nm (due to the electron transfer from the tungsten 5d conduction band to the oxygen 2p valence band) under high-energy radiation was confirmed (Figures 5D, 5F, and 5G). It was also confirmed by comparison with CaWCU power (i.e., microparticles (MPs) of 2 - 3 μιη diameter) that for a given mass of material nanoparticles (NPs) produce significantly higher intensities of UV light (Figure 5D); therefore, the RLT is most feasible with NPs. Depending on the synthesis method used, the as-synthesized CaWCU NPs were coated with different surfactant materials (e.g., citric acid, cetyl trimethylammonium bromide, a mixture of oleic acid and oleylamine). Regardless of the type of surfactant, the original surfactant coating could be replaced with a new enclosure formed by block copolymer (BCP) materials using the method developed by our laboratory. Two types of BCPs have been tested: PEG-PLA, and poly(ethylene glycol- block-n-butyl acrylate) (PEG-PnBA). Both polymers were found to be able to produce fully PEGylated CaWCU RLP products that are essentially devoid of surfactant and are stable against aggregation under physiological salt concentrations for indefinite periods of time. The mean hydrodynamic diameters of the resulting PEG-PLA and PEG-PnBA-encapsulated CaWCU RLPs were determined by dynamic light scattering (DLS) to be about 180 and 50 nm, respectively (Figure 5E); the bare CaWCU nano crystal material used has an average diameter of 10 nm and was monodisperse (Figure 5B). In all studies discussed in this manuscript, the PEG-PLA-encapsulated CaWCU sample with a diameter of 180 nm have been used; in the proposed project PnBA-PEG-encapsulated CaW0 ₄ RLNPs having diameters of about (or less than) 40 nm will also be studied to establish how size (and chemistry) affects the radio- sensitization efficiency and also the pharmacokinetic behavior of CaW0 ₄ RLNPs. As demonstrated in Figures 5D and 5G, the polymer encapsulation does not alter the luminescence activity of CaW0 ₄ ; a more detailed discussion can be found in Reference [13].

[0094] It has been confirmed that both uncoated and PEG-coated CaW0 ₄ materials are nontoxic to cultured human cells (p53 -mutant human head and neck cancer HN31 cells) at therapeutically relevant concentrations (Figures 6A and 6B). A study is currently in progress to confirm the maximum tolerated dose (MTD) of the 180-nm diameter PEGylated CaW0 ₄ RLNPs in normal (BALB/c) mice. As of the time of this writing, it has been confirmed that the MTD of this material is, at least, greater than 200 mg per body weight following a single i.v. administration; this prototype material is thus as safe as, for instance, commercially available dextran-coated iron oxide nanoparticles that are currently used clinically as MRI contrast agents (MTD in mice ~ 168 mg/kg per dose i.v.).

[0095] In this study, we quantitated the amount of cell killing by measuring in vitro clonogenic survival in an available human head and neck cancer HN31 cell line (p53 mutant, radioresistant) that has been irradiated with varying radiation doses); note that head and neck cancer accounts for the largest share of the radiation therapy application market among all cancer types. The mode of cell death (apoptosis, necrosis, mitotic death) was investigated to test whether it changes versus total dose. As shown in Figure 7, the extent of apoptotic death increased with radiation dose. The effect of radiation on cell death becomes more pronounced at elevated doses.

[0096] We then investigated whether uncoated CaW0 ₄ RLPs (CWO MPs and PEG-PLA- encapsulated CWO NPs) increase the biological effectiveness after various radiation doses by clonogenic survival analysis. The results are displayed in Figure 8. As shown in the lower figure, the clonogenic survival curves for γ-irradiated HN31 cells (both in the presence and absence of CWO RLPs) were seen to follow the standard exponential-quadratic decay formula. From these data, the values of the Sensitization Enhancement Ratio or SER (defined as the ratio of the radiation dose at 10% clonogenic survival in the absence of CWO relative to the radiation dose at 10% survival in the presence CWO) were estimated to be 1.15 for MP + γ (0.5 mg/ml CWO) and 1.13 for NP + γ (0.125 mg/ml CWO). For 0.5 mg/ml CWO MP + γ, the /^-values relative to control ("γ") were estimated to be 0.13 at 3 Gy, 0.04 at 6 Gy, and 0.04 at 9 Gy. For 0.125 mg/ml CWO NP + γ, the p-values relative to γ were 0.03 at 3 Gy, 0.11 at 6 Gy, and 0.06 at 9 Gy.

[0097] We also assessed the radio- sensitization efficacy of intratumorally injected PEGylated CWO NPs and uncoated CWO MPs with mouse HN31 xenografts. We tested whether UV light generated by CWO RLPs under γ ray radiation increases the biological effectiveness after radiation by repeated measurements of tumor volume on physical examination. The volume of the tumor was measured by physical examination using calipers. The results are presented in Figure 9. As shown in Figure 9, the data clearly demonstrated that both uncoated CWO MPs and PEGylated CWO NPs are effective in producing radio-sensitization effects. Further, the radio-sensitization effect appears to be not related to the cellular uptake and intratumoral location of the CWO RLPs because the CWO MPs should not have been internalized by the tumor cells due to their large size. It should be noted that in both these initial in vitro clonogenic and in vivo tumor growth assays (Figures 8 and 9, respectively) we used intratumoral CWO RLP concentrations that were at least five-fold lower than the target clinical CWO dose level of intratumoral 5 - 10 mg per cc of tumor; it is remarkable that despite the small amounts of CWO used significant radio-sensitization effects of CWO RLPs were clearly visible. The above clinical dose value (i.e., 5 - 10 mg CWO/cc of tumor) was estimated using the following information. In a previous publication, we have shown that the minimum amount of total deposited photon energy (at a wavelength of 365 nm) required to cause immediate (within less than 24 hours) damage in cancer cells (HeLa cells) is about 16 J/cm ² per cancer cell (equivalent to an exposure of a cancer cell to UV radiation from a UVP's B-100AP lamp (power density = 20 mW/cm ² ) at 10-inch distance for 30 minutes); see Figure S 14 of Reference [15]. CaW0 ₄ has a radio-luminescence energy transfer efficiency of about 5% for X-ray/γ ray radiation. Therefore, in order for 10 nm diameter CWO NPs to produce the same level of UV emission (16 J/cm ² within each cancer cell) under 5 Gy X-ray excitation (at a standard clinical X-ray dose rate of 0.4 Gy per minute), each cell should receive 6.3 x 10 ¹² nanoparticles. Considering that the cell density within a solid tumor is known to be about 1.7 x 10 ⁵ cells per cc of tumor, the above information now translates into the likely minimum amount of CWO NPs to be administered by direct intratumoral infusion into the tumor, that is, about 3.3 mg CWO per cc of tumor. Therefore, in the clinical embodiment of the RLT technology, it is desirable that about 5-10 mg of CWO NPs per cc of tumor be infused into the tumor prior to delivery of the first fraction of radiation therapy. Preclinical testing of this protocol is also currently underway.

[0098] We investigated whether the radio-sensitization effect of CWO RLPs is related to any change in the mode of cell death (particularly, apoptosis vs. necrosis) in the HN31 cell line. As shown in Figures 10 and 11 (see, for instance, the "72 h" data), we detected only a marginal difference in the overall apoptosis plus necrosis levels between γ ray irradiation in the presence versus absence of CWO RLPs (i.e., between "γ" vs. "MP + γ" or between "γ" vs. "NP + γ").

[0099] X-Gal assays were also performed in order to determine whether the population of senescent cells generated by γ radiation increased under the influence of CWO RLPs (Figure 12. As shown in the optical micrographs of Figure 12, only negligible numbers of γ-irradiated HN31 cells were found to be X-Gal-stained regardless of concomitant use of CWO RLPs. Therefore, this indicates that the mechanism of the CWO RLP-induced radio-sensitization of the HN31 human head and neck cancer cells involves an increased level of mitotic catastrophe.

[00100] Further modifications, such as decreasing particle size, and surface-functionalization with folate, further enhance the radio-sensitization effects of CaW0 ₄ RLNPs, mainly for three reasons. One, for a given total amount of CaW0 ₄ material, smaller nanoparticles have larger surface areas and thus will produce higher UV emissions (Figure 5D). Secondly, smaller nanoparticles can easier be internalized by cancer cells. Folate ligands will also enhance the cellular internalization of the nanoparticles. Nanoparticles localized in cytoplasm and nuclear compartments will be more effective in causing cell cycle arrest and inducing radio- sensitization of cancer cells. The third reason is that the folate receptor is overexpressed not only in many cancer cell types including head and neck cancer cells but also in immunosuppressive tumor-associated macrophages; folate ligands will enhance the internalization of the nanoparticles in both of these cell types. With intratumoral nanoparticle injections and localized radiation therapy our proposed therapy will have three arms of potential effectiveness, namely, radiotherapy, UV therapy, and immunotherapy. Therefore, the size and surface functionality need to be optimized for maximum radio-sensitization efficiency.

[00101] Although radiation therapy is a key component of cancer treatment, there are significant side effects. Thus there is great interest in the development of ways to achieve the benefits of radiation treatment with reduced negative effects. Our laboratory is developing a new type of radio-sensitizers (namely, "Radio-Luminescent Particles (RLPs)) that make cancer cells easier to kill with less radiation. Data obtained with prototype formulations (uncoated CaW0 ₄ (CWO) microparticles (MPs) and PEGylated CWO nanoparticles (NPs)) already demonstrated that our RLPs are non-toxic and effective in producing radio-sensitization effects. Specifically, in in vitro tests using radiation-sensitive cells (a murine squamous cell sarcoma SCC7 cell line) the un-optimized CaW0 ₄ MPs induced about a factor of two enhancement of the apoptotic effect of 6-MV γ rays (Figure 2). CWO treatment also significantly decreased the clonogenicity of γ ray-treated radiation-resistant cells (p53-mutant human head and neck cancer HN31 cells) (Figure 8). The 180-nm diameter PEGylated CWO NP formulation showed an indication of radio-sensitization effects in mouse HN31 xenografts (Figure 9). It should noted that these promising in vitro and in vivo results were obtained even with intratumoral CWO RLP concentrations that were at least five-fold lower than the target clinical CWO dose level of intratumoral 5 - 10 mg per cc of tumor. Ongoing studies are in progress to test further optimized (i.e., renal-clearable and folate-functionalized) RLP formulations for their efficacy in radio-

sensitizing head and neck cancer cells (in vitro) and xenografts (in vivo) at more clinically relevant intratumoral CWO RLP loading conditions.

[00102] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.

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