Curcumin-loaded nanoemulsions, method of manufacture, and method of preventive treatment using the same

Abstract

A oil-in-water curcumin nanoemulsion that includes curcumin dissolved in at least one miscible solvent and encapsulated in an oil core, wherein the oil core also comprises a stabilizer, wherein the oil core forms an organic phase which is dispersed in an aqueous solvent, and wherein the oil core is selected from a pharmaceutically acceptable oil. A method of manufacturing an oil-in-water curcumin nanoemulsion includes dissolving the curcumin in at least one miscible solvent; encapsulating the curcumin in the oil core to produce an organic phase solution; adding the stabilizer to the organic phase solution; dispersing the organic phase solution in the aqueous solvent; and evaporating the mixture. A method of preventing metastatic cancer using an oil-in-water curcumin nanoemulsion by administering an amount of the nanoemulsion topically to an area of an excised primary tumor, and monitoring any reincidence of metastatic cancer in the excised primary tumor area.

Claims

1. A topical nanoemulsion composition for topical administration, the composition comprising: curcumin encapsulated in an oil core, a stabilizer, and a dispersing aqueous solvent, wherein the oil core is selected from a pharmaceutically acceptable oil, including soybean oil, ethyl oleate, ethyl butyrate, ethyl caprylate, caprylic/capric triglyceride, mineral oil, liquid paraffin, medium-chain triglyceride oil, grapeseed oil, or combinations thereof; the stabilizer is selected from an ionic surfactant, soy lecithin, lecithin, agar, carrageenan, gellan, gelatin, guar gum, sodium alginate, xanthan gum, or combinations thereof; the aqueous solvent is selected from water; wherein said nanoemulsion has a particles of particle size between 203 to 231 nm, and a zeta potential between −30 to −36 mV; and, wherein the nanoemulsion composition is obtained by: dissolving the curcumin in a miscible solvent selected from acetone, ethanol or combinations thereof; encapsulating the curcumin in the oil core to produce an organic phase solution; adding the stabilizer to the organic phase solution; dispersing the organic phase solution in the aqueous solvent; and evaporating the mixture until a volume of about 1 ml to about 10 ml remains, whereby topical administration of the composition treats tumor reincidence, cancer relapse or metastasis after surgery in a subject in need thereof.

2. The nanoemulsion composition of claim 1, wherein the amount of the pharmaceutically acceptable oil added is about 400 μl to about 800 μl per 100 ml of the aqueous solvent.

3. The nanoemulsion composition of claim 2, wherein the amount of pharmaceutically acceptable oil added is about 500 μl to about 700 μl per 100 ml of the aqueous solvent.

4. The nanoemulsion composition of claim 1, wherein at least one dose of said nanoemulsion is applied to the subject in need thereof.

5. The nanoemulsion composition of claim 4, wherein said topical administration is applied to a body area after intervention by surgery for tumor excision.

6. The nanoemulsion composition of claim 5, wherein said subject is a mammal.

7. The nanoemulsion composition of claim 6, wherein said mammal is a human.

Description

BRIEF DESCRIPTION OF THE FIGURES (NON-LIMITING EMBODIMENTS OF THE DISCLOSURE)

(1) The invention will be better understood and aspects other than those set forth above will become apparent when consideration is given to the following description thereof. Such description makes reference to the annexed figures, wherein:

(2) FIG. 1 is a schematic diagram of a general experimental design according to Example 1 according to at least one embodiment of the invention.

(3) FIG. 2 is a scanning transmission electron microscopy (STEM) image of an oil-in-water curcumin nanoemulsion of according to at least one embodiment of the invention.

(4) FIG. 3A is a graph of the viability assay data of metastatic murine melanoma cells (B16F10) after treatment with and according to at least one embodiment of the invention.

(5) FIG. 3B is a graph of the viability assay data of human embryonic kidney cells (HEK293T) after treatment with and according to at least one embodiment of the invention.

(6) FIG. 3C is a graph of the viability assay data of human HeLa uterine cervical cancer cells after treatment with and according to at least one embodiment of the invention.

(7) FIG. 3D is a series of graphs of the viability assay data of different cancerous and non-cancerous cell lines after treatment with and according to at least one embodiment of the invention at different concentrations.

(8) FIG. 3E is a graph summarizing the viability assay data presented in FIG. 3D.

(9) FIG. 4 is a chart of the ROS levels produced in cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(10) FIG. 5A is a graph of the flow cytometry accumulation data of cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(11) FIG. 5B are confocal images of accumulation data of cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(12) FIG. 50 is a chart of co-localization accumulation data of cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(13) FIG. 6A is a chart of the cell migration of cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(14) FIG. 6B is a chart of the cell invasion of cultured metastatic B16F10 murine melanoma cells after treatment with and according to at least one embodiment of the invention.

(15) FIG. 7A is a graph of tumor reincidence data in a pre-clinical mouse model after treatment with and according to at least one embodiment of the invention.

(16) FIG. 7B is a graph of lung metastasis data in a pre-clinical mouse model after treatment with and according to at least one embodiment of the invention.

(17) FIG. 7C is a graph of the persistent fluorescence of the curcumin signal after excision and treatment with and according to at least one embodiment of the invention.

(18) FIG. 7D is a series of is a scanned transmission electron microsope (STEM) images of the persistent fluorescence of the curcumin signal after excision and treatment with and according to at least one embodiment of the invention.

(19) FIG. 8A is a chart of the particle size and zeta potential related to the stability before and after lyophilization of at least one embodiment of the invention.

(20) FIG. 8B is a UV-vis spectrum graph before and after lyophilization of at least one embodiment of the invention.

(21) The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION

(22) Hereinafter, an oil-in-water curcumin nanoemulsion, a method of manufacture of the oil-in-water curcumin nanoemulsion, and a method of preventive treatment are described. The development and the in vitrolin vivo evaluation of a novel anti-melanoma oil-in-water curcumin nanoemulsion are also described. The nanoemulsion is composed of a lipid core containing curcumin and stabilized by an ionic surfactant. The advantages of this invention include its simplicity (minimum amount of components and easy to produce), bio acceptability, and efficacy (demonstrated anti-tumor activity and metastasis inhibition).

(23) The oil-in-water curcumin nanoemulsions contain round shaped, homogeneous in size curcumin particles around 200 nm (PDI≥0.2) with negative zeta potential and a curcumin encapsulation efficiency of 80%. In vitro assays show that this nanoemulsion is safe in non-cancerous human cells (HEK-293T) and preferentially active in murine melanoma cell (B16F10). The oil-in-water curcumin nanoemulsion increases intracellular curcumin accumulation and is demonstrated in vitro to increase reactive oxygen species (ROS) levels while preventing migration and invasion of melanoma cells. In vivo studies demonstrate that a single dose of the oil-in-water curcumin, applied topically in the area of an extirpated primary tumor, is sufficient to prevent reincident melanoma growth and lung metastasis.

(24) Additionally, these nanoemulsions are nanocarriers readily dispersible in biological media thereby permitting direct administration of curcumin. The ability of nanocarriers loaded with curcumin to inhibit the proliferation, migration and invasion of melanoma cells was evaluated in vitro. The safety and biological selectivity of the nanoemulsion was also evaluated in vitro in human embryonic kidney (HEK293T) cells. The ability of these nanoemulsions to improve curcumin accumulation as well as ROS formation in melanoma cells was determined. Finally, these oil-in-water curcumin nanoemulsions were tested in a preclinical animal model of melanoma, whereby tumor reincidence and metastasis to the lung were evaluated following surgical removal of the initial tumor and applying topically the nanoemulsion. The in vitro results showed that such nanoemulsions were safe and effective at inhibiting proliferation, migration and invasion of melanoma cells while increasing ROS levels and promoting intracellular accumulation of curcumin. Importantly, a single dose was determined to be remarkably effective at preventing completely tumor reincidence and lung metastasis in vivo.

(25) The nanoemulsion can be converted to a dry powder by lyophilization and is shown, upon reconstitution, to maintain the size and zeta potential of the colloid as well as the UV-vis spectrum of curcumin. Considering the remarkable biocompatibility of the oil-in-water curcumin nanoemulsion and that the clinically indicated first line of defense against melanomas, and indeed a variety of malignant tumors, is extirpation, the nanoemulsion is an ideal agent to be applied in patients following cancer surgery, to prevent tumor reincidence and metastasis.

(26) One embodiment of the invention is an oil-in-water curcumin nanoemulsion comprising curcumin dissolved in at least one miscible solvent and encapsulated in an oil core, wherein the oil core also comprises a stabilizer, wherein the oil core forms an organic phase which is dispersed in an aqueous solvent, and wherein the oil core is selected from a pharmaceutically acceptable oil.

(27) A. Curcumin

(28) Curcumin (theracurmin, turmeric extract, curry extract) is fat soluble and useful in the oil-in-water nanoemulsion for the anti-tumor properties. Curcumin is known to inhibit cancer cell growth (including melanoma cells and to some extent metastasis) with minimal side effects in normal tissues. The weight of the curcumin should be determined in relation to the total mass/weight percentage of the nanoemulsion, as it is not effectively absorbed to observe the anti-tumor properties in low concentrations, whereas high concentrations may cause DNA damage and suppress the immune system at high concentrations. In one embodiment of the oil-in-water curcumin nanoemulsion, the weight of the curcumin is from about 10 mg to about 20 mg per 100 ml of the at least one miscible solvent. In a more preferred embodiment of the oil-in-water curcumin nanoemulsion, the weight of the curcumin is from about 12 mg to about 15 mg per 100 ml of the at least one miscible solvent.

(29) B. Oil Core

(30) The oil core is added to an aqueous solvent to create an oil-in-water curcumin nanoemulsion. Any pharmaceutically acceptable oil may be used to create the oil core of the oil-in-water curcumin nanoemulsion. This includes but is not limited to soybean oil, ethyl oleate, ethyl butyrate, ethyl caprylate, Miglyol 812, mineral oil, liquid paraffin, medium-chain triglyceride oil, grapeseed oil, and combinations thereof. A neutral oil is preferred as it has a low or medium viscosity and prevents further reactivity with the curcumin between any additional reagents in an organic phase. In one preferred embodiment of the invention, the neutral oil is Miglyol 812. The amount of the pharmaceutically acceptable oil added to create the oil-in-water curcumin nanoemulsion should be enough to overcome the interfacial tension between the two phases as the nanoemulsion is mixed. In one embodiment of the oil-in-water curcumin nanoemulsion, the amount of pharmaceutically acceptable oil added is about 400 μl to about 800 μl per 100 ml of the aqueous solvent. In a more preferred embodiment of the oil-in-water curcumin nanoemulsion, the amount of pharmaceutically acceptable oil added is about 500 μl to about 700 μl per 100 ml of the aqueous solvent.

(31) C. Stabilizer

(32) The stabilizer is a molecule that has a hydrophobic (fat soluble) portion and a hydrophilic (water soluble) portion to act as an emulsifier and aid in overcoming the interfacial tension between the oil and the aqueous solvent phases of the nanoemulsion. The stabilizer also aids in decreasing the coalescence of dispersed droplets. The stabilizer can be of any type commonly known, including but not limited to an ionic surfactant, Epikuron 145 V, lecithin, agar, carrageenan, gellan, gelatin, guar gum, sodium alginate, xanthan gum, and combinations thereof. In a preferred embodiment of the oil-in-water curcumin nanoemulsion, the stabilizer is Epikuron 145 V. The weight of the stabilizer added to curcumin dissolved in the at least one miscible solvent is about 100 mg to about 500 mg per 100 ml of the at least one miscible solvent. In a preferred embodiment, the weight of the stabilizer added is about 200 mg to about 400 mg per 100 ml of the at least one miscible solvent.

(33) D. Aqueous Solvent

(34) It is commonly known that there are two basic types of emulsions: oil-in-water (O/W) and water-in-oil (W/O). When oil and water are mixed, the resulting emulsion usually has a higher viscosity than each of the components before the emulsification process. In an embodiment of the nanoemulsion, an aqueous solvent is added to the organic phase solution. In a preferred embodiment, the aqueous solvent is water.

(35) E. Miscible Solvent

(36) The at least one miscible solvent that the curcumin is dissolved in is known for adding bulk to a nanoemulsion, aiding in the facilitation of the drug absorption or solubility of the curcumin by a patient while avoiding interaction with the curcumin, and provides stability to the nanoemulsion to prevent denaturation. The miscible solvent can be of any type commonly known, including but not limited to polar and nonpolar solvents, fillers (plant cellulose, dibasic calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, magnesium stearate), binders (gelatin, cellulose, cellulose derivatives, polyvinylpyrrolidone, starch, sucrose, polyethylene glycol [PEG]), disintegrants (carboxymethyl cellulose), natural and synthetic sorbents, antiadherents, lubricants, glidants, preservatives, antioxidants, buffering agents, chelating agents, viscosity imparting agents, surface active agents, humectants, and combinations thereof. In a preferred embodiment of the oil-in-water curcumin nanoemulsion, the at least one miscible solvent is selected from acetone, ethanol, and combinations thereof. In a more preferred embodiment, the at least one miscible solvent is a combination of acetone and ethanol.

(37) Another embodiment of the invention is a method of manufacturing an oil-in-water curcumin nanoemulsion. The curcumin-loaded nanoemulsions of this application were prepared by a simple method that involve the addition of an organic phase including curcumin to an aqueous phase (water) and then eliminating the organic solvents. Using this strategy, the nanoemulsification process is spontaneously induced, hence avoiding the necessity for energy-consuming steps.

(38) In one embodiment of the method, the method includes dissolving the curcumin in at least one miscible solvent; encapsulating the curcumin in the oil core to produce an organic phase solution; adding the stabilizer to the organic phase solution; dispersing the organic phase solution in the aqueous solvent; and evaporating the mixture until a volume of about 1 ml to about 10 ml remains. In a preferred embodiment, the evaporation of the resulting mixture occurs by rotaevaporation. In a more preferred embodiment, the evaporation of the oil-in-water curcumin nanoemulsion occurs under a vacuum.

(39) In another embodiment of the method, an additional step of freeze-drying (lyophilization) of the oil-in-water curcumin nanoemulsion into a dry powder may optionally occur. Freeze-drying (lyophilization) is one of the most frequent and efficient methods to maintain the properties of nanoparticulate systems during storage, while allowing an easier transport due to the lower weight of the final product (total elimination of water). Additionally, the absence of water significantly diminishes the possibility of contamination by microorganisms, which in turn decreases the quantity of preservatives that need to be used. Nevertheless, this process becomes more complex in the case of oil/water formulations due to the presence of the oil core, which is susceptible of leakage. In this instance (to prevent the collapse of oil nanocarriers), the use of cryoprotectant agents is necessary. Any biocompatible cryoprotectants commonly known can be used, including but not limited to sugars, starches, and polyols. In a preferred embodiment of this method, the cryoprotectant is trehalose because it is less hygroscopic and possesses a higher glass transition temperature.

(40) Another embodiment of the invention is a method of preventing metastatic cancer using the oil-in-water curcumin nanoemulsion comprising administering an amount of the nanoemulsion topically to an area of an excised primary tumor, and monitoring any reincidence of metastatic cancer in the excised primary tumor area.

(41) Referring now to FIG. 1, a general scheme summarizing the procedures in animal experiments, including the time course of tumor formation, the surgery, the treatment post-surgery with a curcumin-loaded nanoemulsion and finally evaluation of reincident tumor growth and metastasis using an oil-in-water curcumin nanoemulsion is shown according to the following non-limiting testing examples. The time course includes tumor inoculation at day 0, primary tumor excision by surgery and local treatment with a curcumin-loaded nanoemulsion in the area of surgery at day 15, and if there is any reincident tumor growth and metastasis post-surgery, there is detection of reincident tumor(s) from day 21-40 in the test animals by examination and evaluation of a subject post-mortem. Specifically, in mice, metastatic foci in the lung was evaluated post-mortem at day 40 after surgery and curcumin treatment.

Examples

(42) Preparation of Curcumin Loaded Nanoemulsions (CUR-NEM):

(43) An oil-in-water curcumin nanoemulsion (NEM) was prepared following a solvent displacement method, which comprised adding 125 μl of Miglyol to an organic phase with 30 mg of Epikuron and 2.76 mg curcumin (CUR) dissolved in 0.5 mL of ethanol and 9.5 mL of acetone. This organic phase was then mixed with 20 mL of water. NEM formation was instantaneous, as evidenced by a milky appearance of the mixture. The above NEM formation was evaporated under vacuum in a rotavap until a volume of 5 mL remained. The final concentration of CUR in the NEM was 1500 μm.

(44) Table 1 summarizes the characteristics of blank NEM and those obtained when CUR was included in the oil-in-water nanoemulsion (CUR-NEM). Observations include nanocarriers in the range of 195-217 nm and showing low polydispersity values (≥0.2), which is indicative of homogeneous populations of nanosystems. The CUR-NEM showed negative zeta potential (−30 to −36 mV) which is attributed to the presence of Epikuron that comprise a mixture of zwitterionic phospholipids, anionic fatty acids, and phosphatidic acid, among others. Inclusion of CUR into the nanocarriers was efficient, as to be expected given the high lipophilicity of the molecule and a high preference for the oil nuclei of the nanocarriers. Association efficiency for CUR was similar to known values by others using other lipophilic compounds.

(45) TABLE-US-00001 TABLE 1 Physicochemical properties of the blank nanoemulsions (NEM) and curcumin-loaded nanoemulsions (CUR- NEM)(mean ± S.D., n□4). Zeta Association Size Polydispersity potential efficiency Formulation (nm) index (mV) (%) NEM 195 ± 10 0.1 ± 0.1 −36 ± 5 — CUR-NEM 217 ± 14 0.2 ± 0.1 −30 ± 3 81.1 ± 0.01

(46) The morphology of such nanoemulsions was evaluated by transmission scanning electron microscopy (STEM), as shown in FIG. 2. Curcumin-loaded nanoemulsions exhibited a round particle shape. Additionally, the nanoemulsions contained curcumin particles that were homogeneous in size, coincident with results obtained by dynamic light scattering (DLS).

(47) Physicochemical Characterization of the Oil-in-Water Curcumin Nanoemulsions:

(48) The size and zeta potential of the colloidal systems were determined by photon correlation spectroscopy and laser Doppler anemometry, with a Zetasizer Nano-ZS (Malvern Instruments, UK). Each batch was analyzed in quadruplicate. Scanning transmission electron microscopy (STEM) images were obtained to analyze the morphology of the carriers. The samples to be analyzed were obtained by depositing one droplet (10 μl) of the oil-in-water nanoemulsion, one droplet of MilliQ-water and one droplet of phosphotungstic acid (1%) on a Parafilm® surface. A copper grid (200 mesh, covered with Formvar®, a polyvinyl formal resin produced by Monsanto Chemical Company) was then incubated with each droplet for 2 minutes. The excess liquid was eliminated with absorbent paper. The grid was allowed to dry for at least 12 h before analysis.

(49) Encapsulation Efficiency of the Oil-in-Water Curcumin Nanoemulsions:

(50) The encapsulation efficiency of curcumin in the oil-in-water nanoemulsions was determined by analyzing the difference between the total amount of curcumin in the nanoemulsion and the free curcumin recovered after an aliquot of the sample was isolated using Vivaspin® tubes (8500G, 20 min, MWCO 100 kD). The total amount of drug was estimated by dissolving an aliquot of non-isolated curcumin loaded nanoemulsions with acetone and measuring their absorbance at 424 nm (Lambda 25, Perkin Elmer-Germany). The amount of isolated curcumin was estimated similarly but using an aliquot of the isolated curcumin and dissolved in acetone. The standard curve of curcumin in acetone was linear (r.sup.2>0.999) in the range of concentrations between 1 and 6 mg/L (molar extinction coefficient was 63291 M.sup.−1cm.sup.−1).

(51) Cell Culture Conditions:

(52) Metastatic B16F10 murine melanoma cells were cultured in RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% FBS, 2 mM glutamine, and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37° C. in 5% CO.sub.2. Human embryonic kidney cells (HEK293T) were cultured in Dulbecco's modified Eagle medium (DMEM) high glucose and supplemented with 10% FBS, 2 mM glutamine, and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37° C. in 5% CO.sub.2.

(53) Viability Assays:

(54) The cultured metastatic B16F10 murine melanoma cells and cultured human embryonic kidney cells (HEK293T) were seeded in 96-well plates at a density of 1×10.sup.4 cells per well and incubated for 24 h in culture medium. The cells were then treated for another 24 h with 10 μL of CUR-DMSO, NEM or CUR-NEM for 24 h. 2% Sodium dodecyl sulfate (SDS) and a standard cell medium were used as positive and negative cell controls, respectively. Cell proliferation was evaluated by the MTS® assay, according to the manufacturer's (Promega, Madison, WI) instructions. Absorbance was evaluated at 490 nm removing any background absorbance (620 nm) in plate reader (Tecan Infinite 200 pro NanoQuant).

(55) Cell viability was assessed in the B16F10 melanoma cells following treatment with CUR-NEM. These cells were selected because they are highly resistant to chemotherapeutic drugs, highly proliferative, and because of their elevated metastatic potential. Murine melanoma cells represent a suitable cellular model to study in C57BL/6 mice tumor appearance after subcutaneous injection, as well as reincident tumor growth and metastasis after the primary tumor has been surgically removed. As shown in FIG. 3A (the data shown is the values averaged from three independent experiments (mean±S.E.M) (**P<0.01; ***P<0.001; ****P<0.0001), the NEM vehicles (blank nanoemulsions) did not affect cell-viability under the conditions employed, evidencing the safety of the vehicles. Importantly, the results also indicate that CUR-NEM were as efficient in decreasing proliferation as curcumin dissolved in the organic solvent DMSO (IC.sub.50=46±5.8 μg/mL and 34.8±7.5 μg/mL, respectively). However, as it is well documented that DMSO is toxic to cells, any strategy to promote DMSO replacement is highly desirable.

(56) Cell viability was also evaluated following treatment of the non-cancerous cell line (human embryonic kidney cell line) HEK-293T. At the highest doses tested, CUR-NEM diminished viability of these cells by only about 25% as shown in FIG. 3B, while NEM did not affect this parameter—confirming the safety of the nanocarrier. The results demonstrate that CUR-NEM preferentially reduced melanoma cell viability compared with non-cancerous cells.

(57) To further determine the effect of the formulation on cell viability, the calorimetric method of tetrazolium salt reduction (CellTiter 96®) was used to establish a linear relationship between the number of metabolically active cells and the absorption intensity. To demonstrate the effective anti-carcinogenic activity of the CUR-NEM, the uterine cervical cancer cell line HeLa was used. In addition, the same concentrations of curcumin dissolved in DMSO were evaluated, in order to compare CUR-NEM and curcumin in native state, using an innocuous amount of the solvent for the culture. 2500 HeLa cells were seeded per well, in a 96-well microplate, and the cells were incubated with 100 μL of Dulbecco's Modified Eagle Medium (DMEM) culture medium enriched with 10% fetal bovine serum (FBS), at 37° C. and 5% CO.sub.2. The day after seeding, the 100 μL of medium was removed, and it was replaced by 80 μL of clean culture medium. Then 20 μL of a treatment was applied, and the cells were incubated under the same temperature and CO.sub.2 conditions. 24 hours after the treatment was applied, the treatment was removed, the cells were subsequently washed three (3) times with phosphate-buffered saline (PBS), and 80 μL of medium without indicator was added to the cells and 20 μL of MTS. The cells were incubated for 1 hour, and the absorbance was measured at a wavelength of 490 nm in a microplate reader. The treatments applied were CUR-NEM and CUR dissolved in dimethyl sulfoxide (DMSO). The cells were treated with concentrations of 6.25, 12.5, 25, 50, 75, 100 and 200 μM of CUR in each well of the microplate. The concentration of DMSO did not exceed 0.5% v/v. As controls, nanoemulsions without curcumin (NemBco), 0.5% v/v DMSO, sodium dodecyl sulfate (SDS) and Milli-Q water were used. Each assay was performed in triplicate. In addition, within each assay the same treatment was applied to a minimum of 5 wells. FIG. 3C shows that cytotoxicity is dependent on the concentration of curcumin, with a maximum effectiveness of 200 μM for CUR-NEM and 50 μM for the non-associated CUR, after 24-hour exposure. The CI.sub.50 was calculated for CUR-NEM and curcumin in DMSO, being 30.5±2.3 and 61.8±18.1 μM respectively (even with NemBco not having intrinsic anticancer activity). These results also indicate that from the point 12.5 μM, the differences of CUR-NEM and CUR in DMSO are statistically significant, and when comparing the curves to life control (Milli-Q water) and NemBco, from 50 μM and 150 μM of Cur in DMSO and CUR-NEM respectively, the data is not statistically significant when compared to the SDS control.

(58) Cell viability was also assessed in different cancer cell lines, following treatment with CUR-NEM for 24 hours. These cell lines include human gastric adenocarcinoma (AGS); colorectal adenocarcinoma (HT29); aggressive cells with elevated metastatic potential (HT29US) as compared to HT29 ATCO cells; and cells derived from a human mammary gland adenocarcinoma (MDA-MB-231). Controls corresponds to: culture medium (negative control) SDS 2% (positive control), NEM (empty nanoemulsion). (n=3, **P<0.01, ***P<0.001). In order to further analyze the safety of the formulations, cell viability was also evaluated following treatment of the noncancerous human embryonic kidney cell line (HEK-293T). At the highest doses tested, CUR-NEM diminished viability of these non-cancerous cells by only about 25%, while NEM (empty nanoemulsions) did not affect this parameter (in any cell line), thus confirming the safety of our nano-carrier system. These results indicate that CUR-NEM reduces viability of the different carcinoma cell lines more effectively than the HEK293T control cell line.

(59) ROS Determination by Flow Cytometry (FACS):

(60) The cultured metastatic B16F10 murine melanoma cells were seeded in 24-well plates at a density of 4×10.sup.4 cells per well and incubated for 24 h in culture medium. Then, cells were treated with 12.5 μg/mL of CUR-NEM containing vehicles or empty vehicles (50 μL blank) for another 24 h. Once the cells and medium were recovered, ROS production was evaluated employing 2′,7′-dichlorofluorescein diacetate (H.sub.2DCFDA—0.1 μM) added to cells suspensions 30 min prior to completing the experiment. As a positive control, cells were incubated with freshly prepared H.sub.2O.sub.2 (0.1 mM) for 20 min at 37° C. prior to adding H.sub.2DCFDA. The fluorescence of cells was analyzed by flow cytometry (Becton Dickinson, USA) at an excitation wavelength of 485 nm and at emission wavelength of 530 nm. The non-specific fluorescence of curcumin was employed as the base-line value.

(61) CUR is known to specifically increase ROS levels in cancer cells. As shown in FIG. 4, ROS levels increased in murine melanoma cells (B16F10) following administration of CUR-NEM. All formulations containing CUR (CUR-NEM and CUR-DMSO) increased ROS levels 2-fold at a dose of 12.5 μg/mL of curcumin. The DMSO solvent significantly increased the basal ROS levels, while this was not the case for the NEM (blank NEM) control. The data shown in FIG. 4 is the values averaged from three independent experiments (mean±S.E.M) (**P<0.01; ***P<0.001; ****P<0.0001).

(62) Confocal Fluorescence Microscopy:

(63) The cultured metastatic B16F10 murine melanoma cells and cultured human embryonic kidney cells (HEK293T) were seeded in 24-well plates at a density of 4×10.sup.4 cells, on sterile coverslips (12 mm round coverslips glasses-Deckglässer), and grown for 24 h in complete medium. Then, cells were treated with vehicles containing 12.5 μg/mL of curcumin or empty vehicles (50 μL) for another 24 h. Cells were subsequently washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton® X-100 (Sigma-Aldrich) in PBS followed by incubation with a monoclonal anti-EEA1 antibody. For detection, an Alexa Fluor 546-labeled goat anti-mouse IgG secondary antibody was used. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei of the cells. Coverslips were mounted on a glass slide using Fluoromount G® Mounting Medium (Southern Biotechnology Associates, USA) and sealed. Confocal images were acquired on a Zeiss LSM700 microscope (Zeiss, NY, USA) with a 63×, 1.3 water glycerin objective with a pinhole of 70 μm. The images were analyzed using the software ImageJ and Imaris.

(64) Although it is known that CUR exerts its beneficial anti-cancer effects at the intracellular level, this invention investigates whether the CUR-NEM nanoemulsion permits the interaction with and then internalization of CUR into melanoma cells. In identifying if CUR formulations (CUR-NEM and CUR-DMSO) permitted intracellular accumulation of the molecule, cells were analyzed by confocal microscopy. When added as CUR-NEM, higher levels of CUR were detectable inside cells after 24 h compared with CUR-DMSO treated cells (as shown in FIG. 5B).

(65) Flow Cytometry:

(66) The cultured metastatic B16F10 murine melanoma cells and cultured human embryonic kidney cells (HEK293T) were seeded in 24-well plates at a density of 4×10.sup.4 cells per well and incubated for 24 h. Afterwards, cells were exposed to vehicles containing 12.5 μg/mL of curcumin or empty vehicles (50 μL) for another 24 h. Then, cells and cell medium were recovered in borosilicate glass tubes, centrifuged to remove trypsin with PBS and resuspending in FAGS buffer (PBS with 1% serum and 5 mM EDTA). The mean relative fluorescence of cell populations from the different treatment groups for different time periods (after 0.25, 0.5, 1, 2 and 24 hours) was determined and reported as mean±standard deviation of values from three independent experiments (N=3, **P<0.01).

(67) Flow cytometry (FACS) assays indicated that CUR-NEM was rapidly and efficiently taken up by melanoma cells (detectable as early as 15 min after incubation) because CUR levels after 2 and 24 h, increased by only 36% and 22%, respectively (as shown in FIG. 5A). As can be observed, cell-associated CUR levels are similar initially for CUR-NEM and CUR-DMSO formulations (maximum after 2 h). However, while this level remained similar after 24 h following CUR-NEM incubation, the CUR values decreased by 55% in CUR-DMSO treated cells. Interestingly, co-localization (the Pearson coefficient) between CUR and the endosomal marker (EEA1 protein) was significantly higher for cells treated with the nanoemulsion (as shown in FIG. 5C), indicating that NEM may promote internalization via this pathway (N=3, *P<0.05). Importantly, in both experiments (FACS and confocal microscopy), higher levels of CUR were detectable after 24 h when administered as CUR-NEM. The results reasonably suggest that NEM formulations could significantly improving biological CUR effects in comparison with other formulations.

(68) Transwell Migration Assays:

(69) Prior experimental to testing, the bottom side of each insert of a Boyden Chambers system (Transwell costar, 6.5 mm diameter, 8 μm pore size) was coated with 2 μg/mL fibronectin. B16F10 cells (5×10.sup.4) previously treated 24 h with CUR-NEM, CUR and control, CUR-DMSO or NEM, were resuspended in serum-free medium and added to the top chamber of the insert, while serum-free medium was added to the bottom chamber. After 2 h, inserts were removed, washed, and cells that had migrated to the lower side of the inserts were stained with 0.1% crystal violet in 2% ethanol and counted in a microscope.

(70) Matrigel Invasion Assay:

(71) B16F10 cells (5×10.sup.4) were seeded, allowed to grow for 24 h, and treated with CUR-NEM, CUR and controls. Then, serum-starved cells (2×10.sup.5) were seeded (24 h) over 8 μm-porous inserts covered with Matrigel (Matrigel Invasion Chamber 8.0 lm; BD Biosciences Bedford, MA, USA). Inserts were fixed in cold methanol and stained with 0.5% toluidine blue in 2% Na.sub.2CO.sub.3. The membranes were mounted in Mowiol, and observed under a light microscope. At least 10 fields were evaluated (at 40× magnification) to determine the number of cells per field. The values obtained were normalized to those obtained for cells without treatment (control). Averages of values from three independent experiments are shown (mean±SEM). Statistically significant differences compared with the corresponding control group are indicated (**P<0.001; ****<0.0001).

(72) Metastasis is a complex process that is known to require migration and invasion of malignant cells. Considering that known CUR treatments have been shown to reduce migration and invasion of cancer cells, it was untested whether these beneficial characteristics were maintained in curcumin loaded nanoemulsions. As shown in FIG. 6A, migration of B16F10 cells was substantially decreased (by approximately 50%) upon cell pre-incubation for 24 hours with either 12.5 μg/ml CUR-NEM, NEM, or CUR-DMSO, while empty nanoemulsions (NEM) did not show any significant effects in this respect. Additionally, as shown in FIG. 6B, a highly significant 10-fold reduction in the invasiveness of either of the curcumin containing formulations CUR-NEM or CUR-DMSO was observed, while again no effects were detectable for NEM alone. Importantly, the nanoemulsions of the present invention are shown to maintain the beneficial effect of CUR to the same magnitude as seen for CUR-DMSO, without the necessity of employing a toxic organic solvent like DMSO. This advantageous effect strongly suggests success in vivo treatment of melanoma and potentially other kinds of metastatic cancer.

(73) Animal in vivo Studies:

(74) C57BL/6 mice were obtained from the Instituto de Salud Publica (Santiago, Chile) and housed in the animal facility of the Centro de Estudios Moleculares de la Celula (Instituto de Ciencias Biomedicas, Universidad de Chile). Mice between 8 and 12 weeks of age and average weight of 25 g were used for experiments. As shown in FIG. 1, a general experimental design for in vivo studies is provided.

(75) Reincident Tumor Growth and Lung Metastasis in Animal Models

(76) Subcutaneous tumor growth and metastasis assays in C57BL/6 mice were conducted. B16F10 cells (3×10.sup.5) in 100 μL physiological saline (0.9% NaCl) were injected subcutaneously into the flanks of mice. The largest perpendicular diameters of the resulting tumors were periodically measured, and tumor volumes and mass were calculated according to the following formula: width.sup.2×length×π/6. Tumors were excised by surgery after 15 days or when they reached the bioethically permitted limit of 1500 mm.sup.3-2500 mm.sup.3. A single dose of CUR-NEM (1500 μg/mL) and controls (NEM and physiological serum) were administered after excising the tumor to the wound zone. Volumes to be employed were calculated by correlating tumor volumes with those used for in vitro cell viability studies. Data shown (mean±S.E.M.) is averaged from results obtained with a total of N=10 mice per group (*P<0.05; **P<0.01). For example, in vitro 10 μl of CUR-NEM was administered to each well (0.3 cm.sup.2 per well considering plates of 96 wells). If the area of a tumor was on average 2.5 cm.sup.2 the volume of CUR-NEM added post-surgery was 83.3 μL (2.5 cm.sup.2/0.3 cm.sup.2×10 μL). Animals were maintained for 21-30 days after surgery and then sacrificed. Reincident tumors were detected at or near the original site of tumor growth, and tumor volumes were calculated according to the previously mentioned formula. Lungs were fixed in Feketes solution and black tissues (corresponding to metastatic tumors) were separated from the rest of the lung, and weighed. Lung metastasis was expressed as black tissue mass/total lung mass in percent (%) post-fixation.

(77) This model provides an excellent opportunity to evaluate the efficacy of pharmacological therapies mimicking the situation in humans where surgical removal of tumors is still considered the first line of defense in cancer treatment. As one of the advantages of the CUR-NEM nanoemulsions of the invention is that they deliberately avoid the use of organic solvents like DMSO to solubilize CUR, and specifically with FDA-approved excipients, the CUR-NEM should be suitable in pre-clinical/clinical therapy against melanoma reincidence and metastasis. Several reasons favor using the CUR-NEM nanoemulsions of this invention, including a) reduced melanoma growth without significantly affecting normal cells; b) increased ROS levels; c) decreased migration as well as invasion of melanoma cells; and d) more persistent accumulation of CUR in malignant cells. Administration of one dose of CUR-NEM post-surgery completely prevented tumor reincidence (as shown in FIG. 7A), while in NEM treated animals, a modest approximately 20% decline was detectable. Additionally, CUR-NEM treatment also completely abolished lung metastasis (as shown in FIG. 7B).

(78) CUR-NEM persistency after tumor excision and treatment is further shown in FIGS. 7C and 7D (curcumin fluorescence as assessed post-surgery). The results obtained were after excision of dorsally localized, sub-cutaneous tumors and treatment with CUR-NEM, and FIG. 7C compares the persistance of the curcumin signal when applied dissolved in a solvent (DMSO) or as a nanoemulsion. The signal of curcumin is maintained for a longer time in nanoemulsion than dissolved in DMSO, as shown in the fluorescence in the region of interest (ROI) values measured in vivo at different time points post-surgery (as shown in FIG. 7D) by a previously established in vitro color code.

(79) Curcumin emits fluorescence at approximately 540 nm after excitation at 430 nm. By observing fluorescence decay over time, one may infer how long CUR-NEM remains active at the site of treatment. More extended retention may relate to a better protection of curcumin from degradation. It was observed that curcumin after CUR-NEM treatment was still detectable as fluorescence even 16 days after treatment (1.6-fold basal levels). The most significant differences compared with CUR-DMSO treatment were detected after four (4) days, when fluorescence due to CUR-NEM preparations was still 2.6-fold over basal levels, while CUR-DMSO was not detectable. Extended presence at the site of application is likely responsible for the observed inhibition of reincident tumor growth and suppression of metastasis.

(80) Freeze-Drying Studies of Curcumin Loaded Nanoemulsions

(81) Concentrations of NEM (0.5 and 1% w/v) loaded with CUR and the cryoprotectant trehalose (5% and 10%) were considered as the variables for a lyophilization study. 1 mL dilutions of CUR-NEM were transferred into vials of 2 mL (Eppendorf tubes) and frozen at −20° C. Samples were lyophilized (FreeZone1, Labconco Corp., USA) overnight at −53° C. and 0.1 mBar. CUR-NEM were recovered by adding 1 mL of ultrapure water to the freeze-dried powders followed by manual resuspension and such resuspended material was characterized in terms of size and zeta potential.

(82) The UV-vis spectra of CUR from fresh nanoemuision formulations (similar to those tested in vitro and in vivo) and the freeze-dried preparations reconstituted in water were evaluated in quartz vessels and scanned at wavelengths ranging from 350 to 550 nm (Lambda 25, Perkin-Eimer-USA). For analysis, aliquots (200 μL-400 μL) of CUR-NEM, either freshly prepared or freeze dried and reconstituted in acetone (final volume 5 mL) were vigorously mixed in a vortex. The curcumin-loaded nanoemulsion formulations were then centrifuged for 30 min at 12000G (Hermle Labortechnik, Germany) and the supernatant was analyzed in the spectrophotometer.

(83) The particle size and zeta potential of CUR-NEM upon reconstitution of the freeze-dried product as compared to a fresh formulation (Mean±S.D.; N=3) are shown (FIG. 8A). Overall, the results indicate that at relatively high concentrations of the CUR-NEM (0.5 and 1% w/v), it is possible to achieve an optimal resuspension of the dried product without altering the size and zeta potential of the original (fresh) CUR-NEM. At higher concentrations of CUR-NEM, more of the cryoprotectant trehalose is required to maintain the original characteristics of the formulation. Indeed, the spectrum of CUR is similar for fresh CUR-NEM and for the lyophilized formulations after reconstitution (as shown in FIG. 8B). These results indicate that the CUR-NEM nanoemulsions of the invention may be converted to a dry product, which will promote colloidal stability as well as the stability of the encapsulated CUR.

(84) Acute Toxicological Assay Following Curcumin Nanoemulsion Administration

(85) Five (5) mice underwent surgery, imitating the resective surgery of a melanoma model. The animals were all between 19.9 and 21.2 grams in weight, and approximately 6 weeks old with negative microbiological and viral patterns. A maximum volume of 300 μL of the curcumin nanoemulsion (concentration of 0.01 M of curcumin was injected into each animal. On the Six days later (post-surgery and administration), euthanasia was performed. Tissues were fixed and a complete blood analysis was made by COPD using an Epoc® system. As illustrated in Tables 2, 3 and 4, all tested parameters were normal, and thus further indicate the safety of the curcumin formulations even at doses 22 times higher than those administered in animal patients.

(86) TABLE-US-00002 TABLE 2 Essential systemic metabolites measurements GLu mg/dL Creatinine mg/dL Samples 98-152 Lact mmol/L 0.4 a 1.5 mg/dL Mouse 1 100 2.88 1.58 Mouse 2 110 3.23 1.20 Mouse 3 118 2.67 0.36 Mouse 4 140 1.88 0.58 Mouse 5 157 1.91 0.56

(87) TABLE-US-00003 TABLE 3 Measurements of metabolic chemistry in plasma Na K Ca Cl cHgb 140-156 4.3-5.2 0.2-0.4 100-106 cTCO2 Hct 3-25 BE(b) Samples mmol/L mmol/L mmol/L mmol/L mmol/L 10-75% g/dL mmol/L Mouse 1 139 4.2 0.33 103 13.6 10 Cnc −11.4 Mouse 2 129 4.4 0.31 102 13.2 9.6 cnc −12.5 Mouse 3 102 4.9 0.20 104 12.5 25 22 −13 Mouse 4 111 4.8 0.28 100 15 11 Cnc −11 Mouse 5 125 4.7 0.27 106 12 12 cnc −12.0

(88) TABLE-US-00004 TABLE 4 Measurements of plasma gases in plasma samples pCO2 pCO2(T) cHCO3— BE(ecf) pH 32-41 pO2 pH 15-85 pO2(T) 14-24 −30 a cSO2 Muestra 7.2-7.5 mmHg mmHg (T) mmHg mmHg mmol/L 30 0-100% Mouse 1 7.2 43.2 34 7.21 31 21 21 9 76 Mouse 2 7.02 32.9 42 7.12 28 20 15 3 75 Mouse 3 7.1 38.1 31 7.23 29 10 17 10 62 Mouse 4 7.1 53.3 51.8 7.299 31.5 22.4 18.2 −10.7 74.7 Mouse 5 7.2 42.5 45.6 7.333 29 21.0 21 −12 70.4

(89) While certain embodiments of the nanoemulsion and methods of manufacture and treatment have been described in detail with reference to the accompanying tables and figures, it should be apparent that modifications and variations thereto are possible, all of which fall within the true spirit and scope of the invention. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

(90) As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based may readily be utilized as a basis for other compositions, formulations, nanoemulsions, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

(91) Further, the purpose of the foregoing Abstract is to enable the U.S. Patent and Trademark Office and the public generally, and especially the scientists, engineers and practitioners in the art who are not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the invention of the application, which is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.

(92) These together with other aspects of the invention, along with the various features of novelty that characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific aspects attained by its uses, reference should be had to the accompanying figures and description matter in which there are illustrated preferred embodiments of the invention.

(93) Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.