WO2011101859A1 - A novel water soluble curcumin loaded nanoparticulate system for cancer therapy - Google Patents

Abstract

The present invention provides a drug delivery system encompassing a water-insoluble drug curcumin for the treatment of cancer. The composition is containing a pharmaceutically acceptable carrier and thus providing a biocompatible drug delivery system. The invention further discloses the nanoparticles composed of glycerol monooleate (GMO), polyvinyl alcohol and pluronic F- 127 and it showed high surface charge (around -32 mV) demonstrating enhanced solubility, stability and bioavailability of entrapped curcumin.

Description

TITLE: A novel water soluble curcumin loaded nanoparticulate system for cancer therapy.

FIELD OF THE INVENTION:

This invention relates to a novel water soluble curcumin loaded nanoparticulate system for cancer therapy.

BACKGROUND OF THE INVENTION:

Cancer is known to be the most distressing and life threatening disease that enforces severe death world wide. American Cancer Society's annual cancer statistics report estimate that there will be about 1, 479, 350 million new cancer cases and about 562, 340 cancer deaths in 2009. To overcome this massive death a successful cancer therapy envisioned which will encompasses its early diagnosis and better therapies for subsequent complete eradication of this fatal disease. The most common option used for treatment of cancer is chemotherapy but it is often associated with number of drawbacks i.e. nonselective distribution of drugs, multidrug resistance, enhanced drug toxicity and undesirable side effect to normal tissue [1]. Further, most of the cancer cells are highly aggressive and show resistance to chemotherapy due to inherent lacking of beneficial response of cytotoxic anticancer drug. To this end, the non toxicity and efficacy of the traditional medicine now a day open up new prospect for future cancer therapies [2]. In this regard, the upcoming anticancer drug modality of natural herbal extracts curcumin gives a solution to the hurdles involved in chemotherapy by showing safety and chemopreventive activities against malignancy. Besides its biocompatibility and no side effect to normal tissue, in recent years it has drawn the attention of research to sensitize cancer cells for chemotherapy by inducing programmed cell death [3, 4],

Curcumin is a hydrophobic polyphenol (molecular formula C₂₁l¾o0₆), a major yellow phytochemical compound of Turmeric (Curcuma longa, Zingiberaceae). The chemical structure of curcumin is [1 , 7-bis (4-hydroxy-3- methoxyphenyl)-l, 6-heptadiene- 3, 5- dione]. Preclinical and clinical studies indicate that curcumin has potential therapeutic value against most chronic disease including neoplastic, neurological, cardiovascular, pulmonary, metabolic and psychological diseases [3, 5, 6] . This is due to its diverse range of molecular targets like transcription factors, growth factors and their receptors, cytokines, enzymes, and genes (regulating cell proliferation and apoptosis) [2, 6]. So, in current research curcumin has been taken as an upcoming herbal drug to instigate multitargeted therapy, which is needed for treatment of various fatal diseases including cancer. Studies have shown that cancer chemopreventive action of curcumin is due to its inhibition of NFKB activation, JNK and AP- 1 transcriptional activity [3, 7]. It has been well studied that curcumin acts as a potent inhibitor of NFKB signaling pathway which is involved in apoptosis as well as its function has been implicated in inflammation, cell proliferation, differentiation and cell survival. Even though curcumin possesses chemopreventive, antineoplastic and anti-inflammatory properties, it is still considered extremely safe when administered at very high doses. Conversely, systemic toxicity at high dose rendered other anticancer drug unsuitable for cancer therapy. It was reported, uptake of curcumin is safe at doses up to 100 mg/day while consumed as a dietary spice [5]. Recently, phase I clinical trials indicate that people shows no discernible toxicities with curcumin doses up to 3600-8000 mg daily for 4 months and can tolerate a dose as high as 8 g/day up to 18 months [8, 9]. In spite of its efficacy and safety, curcumin has restrictive pharmaceutical role because of its extremely low aqueous solubility, rapid systemic elimination, inadequate tissue absorption and degradation at alkaline pH, which severely curtails its bioavailability [2, 10, 11]. With respect to solubility, curcumin shows extremely low solubility in aqueous but fairly soluble in organic solvents such as DMSO, ethanol, methanol and acetones [10]. Its degradation kinetics has also been reported under various pH conditions, showing stable at acidic pH (as normally encountered in stomach) but unstable at neutral and basic pH. It was also reported that most curcumin (> 90 %) is rapidly degraded with in 30 min of placement in phosphate buffer systems of pH 7.2 [12]. Studies to date have suggested that this low aqueous solubility and high degradation of curcumin in physiological pH consequently lead to poor absorption, low tissue distribution and rapid excretion of curcumin that severely restrict its bioavailability. This is due to extensive intestinal and hepatic metabolism and rapid elimination of curcumin which ultimately restraining its bioavailability [9, 10, 12].

Current trends in curcumin research have concentrated on the development of potential delivery systems to increase its aqueous solubility, stability and bioavailability as well as controlled delivery of curcumin at or around cancer tissues. To this end, new avenues like use of adjuvant (piperine) which interferes with glucuronidation or different polymeric delivery system certainly testify a comprehensible method to increase the bioavailability of curcumin [13-15]. Among various strategies the best considered way to achieve this paradigm is to encapsulate curcumin within nanoparticle (NPs). These NPs have been shown to possess significant potential as delivery systems providing several advantages when used as a drug delivery system for hydrophobic drugs like curcumin. The aqueous solubility of a number of anti cancer drugs have been significantly increased by using these NPs as drug delivery vehicle [16-18]. Typically, NPs based on lipid polymer like glycerol monooleate (GMO) are aqueous soluble and more stable in biological fluid leading to prolonged the circulation time and biodistribution of encapsulated drug due to reduced reticuloendothelial system (RES) clearance and renal filtration [19, 20]. Most importantly, it can provide a bioadhesive delivery system to enhance the drugs bioavailability by increasing resistance time and subsequently facilitate the absorption of drug through adhesion with the cellular surface. The GMO was approved by food and drug administration (FDA) and it is an emulisifier, flavouring agent used in the food industry and well studied excipient agent for antibiotics. This GMO based NPs have been used to sustain the delivery of various water soluble and insoluble drugs due to its self- emulsifing property. However, limited researches have been reported to date regarding the encapsulation of hydrophobic anti cancer drugs in GMO based NPs. In spite of several formulation challenges some formulation strategy with this delivery system has already been developed to deliver anticancer drug. As example, GMO/ polyxamer 407 cubic nanoparticles was designed to enhance the bioavailability of water insoluble drug simvastatin but it could not provide the good release profile i.e < 3 % drug released at 10 hours [21]. Similarly, chitosan coating GMO NPs reported much high particle size i.e. 400 nm - 700 nm but demonstrated significantly increase in cellular accumulation and efficacy of entrapped drug paclitaxol; however no in vivo studies for bioavailability of drug have been reported [22]. These bioadhesive delivery systems are currently gaining interest to augment the systemic bioavailability of delivered drugs. However, for preventing aggregation in biological solution and for providing better stabilization to NPs the coating of large molecules, such as polymers or surfactants (containing long-chain hydrocarbons) are necessitate [23]. In this scenario, the choice of an ideal polymer or surfactants is vital as it regulates the essential properties such as solubility, stability, drug loading capacity and drug release profile of NPs. Some representative of such material is nonionic block copolymer Pluro ic F-127 (consists of hydrophilic poly (ethylene oxide) [PEO] and hydrophobic poly (propylene oxide) [PPO]) and PVA which have gained much attentions for providing specific surface charge and chemical functionalization to NP delivery system [24]. Apart from providing stability to NP, the key attribute of Pluronic F-127 is their ability to enhance drug transport by effective passive targeting towards cancerous tissues and can make sensitize the multidrug resistance tumors to various anticancer agents [25]. Due to their amphiphilic character these copolymers display surfactant properties and further offers stability and biocompatibility to NP. Moreover, for intravenous injectable formulation these surface coated hydrophilic polymers are necessary to minimize the opsonization and to prolong the in vivo circulation of NPs.

In this regard, the current approach was to prepare and characterize curcumin loaded nanoparticulate system (Nano CUR), in a view to get small sized particles with high entrapment of anticancer drug curcumin. To this end, we have successfully synthesized Nano CUR and furthermore, the cellular uptake, cell cytotoxicity, apoptosis studies andw vivo bioavailability of the formulation was done and compared with native curcumin. Results confirmed that Nano CUR was capable of exhibiting enhanced cellular uptake which consequently resulted in reduction of cell viability by inducing apoptosis in tumor cells (as studied in PANC-1 cell) when compared to native curcumin. Thus, we hypothesized that our model drug delivery system, Nano CUR enabled better administration of curcumin in an aqueous phase medium and significantly augmented the potential of this promising anticancer drug in clinical arena. OBJECTS OF THE INVENTION:

An object of this invention is to propose a novel water soluble curcumin loaded nanoparticulate system;

Another object of this invention is to propose a potential curcumin loaded nanoparticulate system for the treatment of cancer clinical arena;

Still another object of this invention is to propose a stable water soluble curcumin loaded nanoparticulate system;

Further, object of this invention is to propose an improved and bioavailable water soluble curcumin loaded nanoparticulate system;

Still further object of this invention is to propose a process for the preparation of a novel water soluble curcumin loaded nanoparticulate system.

BRIEF DESCRIPTION OF THE INVENTION:

According to this invention there is provided a novel water soluble curcumin loaded nanoparticulate system for cancer therapy having narrow monodispersed unimodal size distribution (<200 nm) with high zeta potential around -32 mV.

In accordance with this invention there is provided a process for preparing a water soluble curcumin loaded nanoparticulate system comprising: incorporating curcumin into the fluid phase of GMO;

subjecting the GMO mixture to the step of emulsification with PVA;

emulsifying the resultant solution with pluronic solution ;

lyophilizing the final emulsion by freeze diying to produce lyophilized powder.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Figure 1. a. Mean particle size of Nano CUR measured by light scattering method.

b. Transmission electron micrograph of Nano CUR (bar = 0.19μηι)

c. Size distribution of Nano CUR as measured by AFM.

Figure 2. XRD pattern of native curcumin (Black), void nanoparticles (green) and Nano

CUR (red).

Figure 3. In vitro release kinetics of curcumin from Nano CUR formulation in PBS (0.01

M, pH 7.4) at 37 °C. Data as mean ± s.e.m, n = 3

Figure 4. a. Stability of curcumin (native and Nano CUR) in PBS (0.01 M, pH 7.4) at 37

°C. Data as mean ± s.e.m , -*~ Native curcumin—A— Nano CUR b. Solubility of Nano CUR in PBS.

I. Native curcumin (10 mg) dissolved in PBS (0.01 M, pH 7.4) was insoluble in aqueous media.

II. Equivalent quantity of nanocurcumin was fully soluble in aqueous media. Figure 5. a. In vitro toxicity studies of void polymeric nanoparticles, showing no morphological changes of void nanoparticle treated cell (PANC-1) compared to control cell as observed under confocal microscopy.

Figure 6. Toxicity studies of void polymeric nanoparticle by measuring inflammatory response in TNF-alpha Assay.

i. Anti inflammatory response of different concentration of void nanoparticles(0.1 to 0.5 mg/ml) in PANC-1 cells after 24 h of incubation. ii. Anti inflammatory response of 0.5 mg/ml of void nanoparticles in PANC-1 cells incubated for different time periods.

Figure 7. Nano CUR inliibited the clonogenic potential of pancreatic cancer cell lines.

Colony assays in soft agar were performed comparing the effects of native and Nano CUR in inhibiting the clonogenicity of the pancreatic cancer cell line ( PANC-1). Representative plates are illustrated for (a) control cells (b) void nanoparticle-treated cells (c) native curcumin-treated cells and (d) Nano CUR -treated cells.

Figure 8. Cellular uptake study of native curcumin ( g ) and Nano CUR ( ¾ ) on in vitro cultured of PANC-1 cell. Data as mean ± s.e.m., n =3. (**) p < 0.005,

Figure 9. a) Microscopic observation of PANC-1 cell treated with 10 μΜ curcumin (both native and Nano CUR) and after 1 h of incubation showing maximum fluorescence intensity in Nano CUR treated cell.

(b)Time dependent increase of intracellular fluorescence intensity in Nano CUR treated PANC-1 cell showing sustained release of encapsulated curcumin with incubation time. While decrease in fluorescence intensity was observed in native curcumin treated cell may be due to loss of stability of native curcumin with time

Figure 10. -Dose dependent cytotoxicity of void nanoparticle(e ), native curcumin ( · ), and

Nano CUR (A ) in PANC-1 (A), MIA PaCa-2 (B), K-562 (c), MCF-7(d), A549 (e) and HCT-116 (f) cell lines. The extent of growth inhibition was measured at 5 days by the MTT assay. The inhibition was calculated with respect to respective controls. Data as mean ± s.e.m., n = 6. (**) p < 0.005, native curcumin in solution versus Nano CUR.

Figure 11. Induction of apoptosis in PANC-1 cell line treated with a concentration of όμΜ/ml of native curcumin and Nano CUR. Treated cells are taken for apoptosis analysis as described in Material and Methods. The number shown in the lower right quadrant is the percent of cell staining for apoptosis after 2 days of incubation.

Figure 12. In vitro anti -proliferative effects of curcumin on pancreatic cancer cells.

PANC -1 were exposed to 6 μΜ/ml curcumin (both in native and in formulation) for 24 h and the targets were detected by immunoblotting with specific antibodies mentioned in details in materials and methods. Figure 13. In vivo bioavailability of native curcumin and Nano CUR. The mice were divided in to two groups (n=3). Equivalent concentration of native curcumin and Nano CUR (30 mg/kg) was given to group 1 and group 2 mice respectively. Native curcumin and Nano CUR were administered intravenously and blood was collected at different time intervals. Serum was separated and the concentration of curcumin was determined by HPLC analysis.

DETAILED DESCRIPTION OF THE INVENTION:

CUR- 500, containing Curcumin (> 95%) was purchased from UNICO Pharmaceuticals, India. Polyvinyl alcohol (PVA, average MW = 31,000-50,000 was purchased from Sigma-Aldrich Co. (St Louis, MO), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), Pluronic F-127 were purchased from Sigma Aldrich Chemicals, Germany. GMO was procured from Eastman (Memphis, TN). All other chemicals used were of reagent grade and used as purchased without further purification.

Preparation of Nano CUR:

Nano CUR formulation was prepared by the following protocol. Briefly, 100 mg of curcumin was incorporated in to the fluid phase of GMO (1.75 ml at 40 °C). The GMO mixture was emulsified with PVA (0.5 % w/v) by sonication (55 watts for 2 min). The resultant solution was further emulsified with pluronic solution (10 % w/v) by sonication (VC 505, Vibracell Sonics, Newton, USA) set at 55 watt of energy output for 2 min over an ice bath. The final emulsion of the formulation was lyophilized by freeze drying methods (-80 °C and <10 μηι mercury pressure, LYPHLOCK, Labconco, Kansas City, MO) to get lyophilized powder for further use.

Characterization of Nano CUR:

Particle size analysis and zeta potential measurement and topography

Particle size and polydispersity index were determined using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) based on quasi-elastic light scattering. Briefly, 1 mg/ml of NPs solution was prepared in double distilled water and sonicated for 30 s in an ice bath.

Size measurements were performed in triplicates following the dilution (100 μΐ diluted to 1 ml) of the NPs suspension in MilliQ water at 25 °C. Zeta potential was measured in the same instrument at 25 °C using the above protocol. The shape of NP was further characterized by AFM (Nanoscope III A, Vecco, USA). A drop of solution (lmg /ml) was placed on freshly cleaved mica. After five min of incubation the surface was gently rinsed with deionized water to remove unbound NP. The sample was air dried at room temperature and mounted on the microscope scanner. The shape was observed and imaged in non-contact mode with frequency 312 KHz and scan speed 2 Hz. Similarly, the internal structure of NPs were determined by TEM measurements, for which a drop of diluted solution of the Nano CUR (in water) was placed in carbon-coated copper TEM grid (150 mesh, Ted Pella Inc, rodding, CA) and allowed to air-diy. The samples were imaged using a Philips 201 transmission electron microscope (Philips/FEI Inc, Barcliff,

Manor, NY). The TEM photograph was taken by using the NIH imaged software. To calculate the mean particle diameter, 50 particles were taken for measurement.

Drug loading and determination of encapsulation efficiency by HPLC method

The concentration of curcumin from the sample was measured by high performance liquid chromatography (HPLC) method according to Ma et al. with slight modification[26]. In order to estimate the entrapped curcumin content the Nano-CUR was dissolved in methanol (lmg /ml w/v) to disrupt its structure. The sample was then subjected to sonication for 3 min at 55 watt (Model: VCX750, Sonics and materials INC, USA) followed by centrifugation at 13, 800 rpm for 10 min at 25 °C (SIGMA 1-15 , Germany) to get a clear supernatant. The supernatant obtained was analysed using reverse phase isocratic mode (RP-HPLC) system of Waters™. For this, 20 μΐ of the of the sample was injected manually in the injection port and analyzed in the mobile phase consisting of a mixture of 60 % acetonitrile and 40 % citric buffer [1 % (w/v) citric acid solution adjusted to pH 3.0 using 50 % (w/v) sodium hydroxide solution] which was delivered at flow rate of 1 ml/min with a quaternary pump (WATERS™ M600E) at 25 °C with a C 18 column(Nova- Pak, 150 x 4.6 mm, i.d). The curcumin levels were quantified by UV detection at 420 nm with dual wave length absorbance detector (M2489). The amount of curcumin in the sample was determined from the peak area con-elated with the standard curve. Samples (in triplicate) were analyzed and the curcumin encapsulation efficiency was calculated from the following equation:

Mass of curcumin in nanoparticle

Entrapment efficiency (%) = χ 100

Mass of curcumin used in formulation In vitro Release Kinetics

A known amount of lyophilized Nano CUR (100 mg) encapsulating curcumin was dispersed in 15 ml PBS (0.01 M, pH 7.4) and the solution was divided in 30 microfuge (500 μΐ each) tubes, as experiment was performed in triplicates. The tubes were kept in a shaker at 37 °C at 150 rpm (Wadegati Labequip, India). Free curcumin is completely insoluble in water; therefore, at predetermined intervals of time, the solution was centrifuged at 3000 rpm for 10 min (SIGMA 3K30, Germany) to separate the released (pelleted) curcumin from the loaded NPs. The released curcumin was redissolved in 1 ml of methanol and 20 μΐ of this solution was injected in the HPLC to determine the amount of curcumin released with respect to different time intervals.

X- ray diffraction (XRD) study

The patterns of native curcumin, lyophilized void NPs and Nano CUR were obtained using X- ray diffractometer (Bruker 9XS, G8ADVANCE) with source of curcumin radiation. Measurements were performed at a voltage of 40 kv and 25 mA. The scanned angle was set from 3 ° < 2Θ > 40 ⁰ and the scan rate was 2 ° min .

Stability study of curcumin

The stability of native curcumin and Nano CUR in PBS (0.01 M, pH 7.4) was estimated by HPLC method [26]. Nano CUR at a total of 10 ml solution at a fixed concentration of ~ 40 μg /ml was prepared in PBS (0.01 M, pH 7.4) and incubated in a shaker rotating at 150 rpm, 37 °C (Wadegati Lab equip, India) for 6 h. For control, same concentration of native curcumin was dissolved in PBS with the help of methanol (final methanol concentration < 5 % v/v) and incubated under similar conditions in the shaker. At predetermined time points, 100 μΐ of solutions (either native curcumin or Nano CUR) were taken and added to 900 μΐ of methanol for quantitative analysis of curcumin content by HPLC.

Toxicity studies of void polymeric nanoparticle

An ideal drug delivery vehicle must be biodegradable, biocompatible and not to be associated with incidental adverse effects. In order to justify the non toxicity of our void polymeric particle two sets of in vitro studies was conducted. The biocompatibility of the void particle was assessed by observing the cell morphology in confocal microscope after staining with lysotracker dye and DAPI [27]. Briefly, PANC-1 cell suspensions were prepared at a concentration of 1 x 10⁵ per ml of media and seeded in bioptech plate (Bioptechs, Butler, PA) for 24 h prior to experiment. 1 ml of void NP suspension at the concentration of 2.5 mg/ml media were added to each well and incubated for 2 and 24 h. After incubation the cell were washed with phosphate buffered saline (0.1 M, pH 7.4) and treated with lysotracker dye (2μ1 of ImM DMSO solution dissolved in 40 ml of media) for 30 min. The cells were washed twice with PBS (0.01M, pH 7.4), 10 % buffered formaldehyde for 15 min and finally stained with DAPI for 30 min. The cells were further washed with PBS (0.01M, pH 7.4) and imaging was done with confocal laser scanning microscopy (Leica TCS SP5, Leica Microsystems GmbH, Germany) using the 60 x oil immersion lens with argon laser at 488 nm to detect the nuclei and HeNe laser at 543 nm to detect the lysosome. To measure the inflammatory response induced by void NP, MIA PaCa-2 cells at density l x 10 ⁵cells/ml were incubated with different concentrations of void NP (0.1 mg/ml to 0.5 mg/ml) for 24 h [28]. The highest concentration of void NP taken in the experiment i.e. 0.5 mg/ml was further observed for a period of 72 h to observe any discrepancy. After incubation the supernatant was collected and centrifuged at 8,000 rpm (Sigma microcentrifuge- 16PK, Germany) for 30 min to remove cell debris. TNF- a protein concentration in the cell supernatant were measured using the ELISA kit (Human TNF- a ELISA KIT, Bender MedSystem Inc., USA) according to the manufacturer's instruction.

Colony soft agar assay

The antiproliferative effect of native curcumin and Nano CUR was analysed based on the methodology of Bisht et al. with slight modification [14]. Briefly, 2 ml mixture of serum supplemented media and 1 % agar containing 15 μΜ of native curcumin and equivalent concentration of Nano CUR was added in a 35 mm culture dish and allowed to solidify. Next, on top of the base layer was added a mixture of serum supplemented media and 0.7 % agar (total 2 mL) containing 10,000 PANC-1 cells with either native curcumin or Nano CUR. Control plate contained PANC-1 cells without any additives. The plates were allowed to solidify and the dishes were kept in tissue culture incubator maintained at 37 °C and 5 % C0₂ incubator (Hera Cell, Thermo Scientific, Waltham, MA), for 7 days to allow for colony growth. All assays were performed in triplicates. The colony assay was terminated at 7^th day and plates were stained with crystal violate (0.005 % w/v).

Cellular uptake studies

For the cellular uptake studies, pancreatic cell (PANC-1) were seeded in a 24 well plate (Corning, NY, USA) at a seeding density of 5 x 10⁴ cells per well in 1 ml of growth medium [29]. After 24 h of incubation at 37 °C the attached cells were treated with equivalent dose (5, 10, 20 and 30 μΜ) of native curcumin and Nano CUR and kept at 37 °C in a cell culture incubator (Hera Cell, Thermo Scientific, Waltham, MA). After 6 h, the cells were washed twice with PBS (0.01M, pH 7.4) and lysed by adding methanol. The cell lysates were centrifuged at 10000 rpm for 10 min at 4 °C (SIGMA 3K30, Germany). The concentration of curcumin from collected supernatant was measured by the use of fluorescence spectrophotometer (X_ex = 420 and _em = 540 nm) (Synergy HT, BioTek^® Instruments Inc., Winooski, VT, USA). Each measurement was performed in triplicates and the data obtained are mean values from three different experiments. For qualitative cellular uptake study, PANC-1 cells were seeded at a seeding density of 15 ^χ 10 ⁴ cells on 35 mm culture plate (Corning, NY, USA) and 1 10 ⁵ cells in Bioptech® tissue culture plates (Bioptechs, Inc, Butler, PA) for fluorescence microscopic studies and confocal studies respectively. The cells were incubated for 24 h at 37 °C for attachment. The attached cells were then treated with a constant concentration (10 μΜ and 1 μΜ for fluorescence microscopic studies and confocal studies respectively) of native curcumin and Nano CUR for 2 h at 37 °C in a cell culture incubator (Hera Cell, Thermo Scientific, Waltham, MA). After incubation the cell monolayers were rinsed three times with 1 ml PBS (0.01 M, pH 7.4) to remove excess NPs or free dye. Fresh PBS (0.01 M, pH 7.4) was added to the plates and the cells were viewed and imaged under a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Germany) equipped with an argon laser using FITC filter (Ex 488 nm, Em 525 nm). The images were processed using Leica Application Suite software. For fluorespence microscopic studies the photographs were taken by excitation of curcumin with a blue filter. Similarly, for time dependant cellular uptake studies of native curcumin and Nano CUR, the Bioptech® tissue culture plates were removed from the incubator at predetermined time intervals and the cells were processed using the above confocal studies protocol. In vitro Mitogenic Assay

The antiproliferative effects of curcumin both in native form and Nano CUR were analyzed by the MTT assay [17]. The assay was based on the cleavage of a yellow tetrazolium salt (MTT) to insoluble purple formazan crystals by the mitochondrial dehydrogenase enzyme of viable cells. Briefly, different pancreatic cell lines (PANC-1 and MIA PaCa-2), breast cell line (MCF-7), leukemic cell line (K-562), human colon cancer cell line (HCT-116) and human alveolar basal epithelial cell line (A549) were seeded at 4000 cells per well density in 96-well plates (Corning, NY, USA). Next day cells were treated with different concentration of (0, 5, 10, 15, 20, 25, 30 and 40 μΜ) either native curcumin dissolved in DMSO or equivalent concentration of Nano CUR.

Concentration of DMSO in the medium was kept < 0.1 % w/v, so that it has no effect on cell proliferation [16]. Cells were incubated for 5 days for assessing the toxicity of curcumin. Medium treated cells and void NPs were used as respective control and a standard MTT based colorimetric assay was used to determine cell viability. After the specified incubation time, 10 μΐ of MTT reagent (Sigma) was added, and the plates were incubated for 3 h at 37 °C in a cell culture incubator (Hera Cell, Thermo Scientific, Waltham, MA), following which the intracellular formazan crystals were solubilized in DMSO and the color intensity was measured at 540 nm using a microplate reader (Synergy HT, BioTek^® Instruments Inc., Winooski, VT, USA). The antiproliferative effect of different treatments was calculated as a percentage of cell growth with respect to respective control.

Apoptosis analysis by Flow Cytometry

The induction of apoptosis by native curcumin and Nano CUR were studied by flow cytometry. Briefly, PANC-1 cells at density 3 10 ⁵cells/ml of were grown in 25 cm² culture flasks (Corning, NY, USA) containing 5 ml of growth medium in triplicate and allowed to attach overnight at 37 °C. Next day, 5 ml of media containing 6 μΜ/ml concentration of native curcumin and equivalent concentration of the Nano CUR were added to the flasks and the cells were incubated in C0₂ incubator (Hera Cell, Thermo Scientific, Waltham, MA). Medium treated cells and cells treated with void NPs were used as controls for the experiment. After 2 days, the cells were washed twice with PBS (0.01 M, pH 7.4) and collected by trypsinization. The pelleted cells were resuspended in 100 μΐ of 1 X binding buffer (Clontech Laboratories, Inc., Palo Alto, CA), 5 μΐ Annexin V-FITC (final concentration, 1 μg/ml; BD Biosciences Pharmingen) and 5 μΐ propidium iodide (10 μg μl; MP Biomedicals, Inc, Germany) and incubated at room temperature in dark for 20 min. Before flow cytometric analysis, 400 μΐ of 1 X binding buffer was added to the cells. Stained cells were analysed on flow cytometer (FACSCalibur; Becton- Dickinson, San Jose, CA) using Cell Quest software with a laser excitation wavelength at 488 nm.

Western blot analysis

Western blot analysis was done to study the molecular mechanism by which the native and Nano CUR exert antiproliferative effect on pancreatic cancer cells [18]. The pancreatic cancer cell PANC-1 (5x 10 ⁵cells/ml) were treated with 6 μΜ/ml curcumin (both in native and in formulation) for 24 h and next day cell extracts were collected by scraping the cells, washing in IX PBS followed by detergent lysis [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1 % SDS, containing protease and phosphatase inhibitor (Sigma, St. Louis, MO) cocktails]. The protein concentration was determined by the Pierce BCA protein assay (Pierce, Rockford, IL). Equal amount of total cell lysates (50 μg) of each sample were solubilized in 2X sample buffer and electrophoresed on 8-12 % SDS-PAGE. Protein immunodetection was done by electrophoretic transfer of SDS-PAGE separated proteins onto PDVF membrane (Millipore) followed by incubation with primaiy antibody (antibodies used were against phosphor- Akt, ΙκΒα, NFKB P65, p21, c-Myc, cyclin Dl and β actin in 1 :1000 dilutions) for one hour and secondary antibody (1:5000 dilution) for 40 min. Antigen-antibody complex were visualized by chemiluminescent ECL detection system (Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies (primary and secondary) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.

In Vivo Pharmakokinetics

Animal experiment studies were canied out to analyze the pharmacokinetic study of delivered curcumin (in native and NP form) [30]. For in vivo pharmacokinetic study, Balb/c mice weighing 20-25 gm were used. These mice were divided into two groups (n=3), group 1, received native curcumin dissolved in distilled water with Tween 20 (1 %, v/v) and group 2, received Nano CUR. Native curcumin or Nano CUR was given intravenously (30 mg/kg) to each mice and the peripheral blood from retro orbital plexus was collected at different time intervals. The collected blood was allowed to clot at room temperature for about 1 h, centrifuged at 5000 rpm for 5 min and serum was separated and kept at - 20 °C prior to analysis. For extracting the curcumin from serum sample, 0.1 ml of it was taken and diluted to 5 ml with methanol. The content was shaken vigorously and heated at 70-75 °C for 30 min. Then the volume was made up to 5 ml with methanol and the turbid solution was subjected for centrifugation (500 rpm for 10 min). The separated supernatant was taken for quantitative analysis of curcumin by HPLC. Cell culture

All the cell lines were purchased from American Type Culture Collection (Manassas, VA) and cultured using DMEM with 10 % FBS, 1 % L-glutamine and 1 % penicillin- streptomycin at 37 °C in a humidified, 5 % C0₂ atmosphere maintained in an incubator (Hera Cell, Thermo Scientific, Waltham, MA). All chemicals for cell culture were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India.

Statistical analysis

Data are presented as mean ± standard deviation, and analyzed by one-way ANOVA with the Tukey's test applied post hoc for paired comparisons of means (SPSS 10, SPSS Inc. Chicago, IL, USA). Values of p < 0.05 were indicative of significant differences and p < 0.005 were indicative of a veiy significant difference.

3. Physiochemical characterization of Nano CUR

The therapeutics potential of curcumin could be certainly enhanced by the development of an efficient drug delivery system. Hence, in a quest of developing an ideal formulation for achieving small size, maximum entrapment and for enhanced bioavailability of curcumin, we prepared Nano CUR based on GMO in a view to get maximum solubility and bioavailability of curcumin. In this view curcumin was successfully encapsulated inside the Nano CUR with entrapment efficiency of 78.59 ± 3.8 %. The results of the mean particle size and size distribution of NPs as measured by laser light diffraction technique was 192 nm with a negative zeta potential of - 32 mv. TEM characterization showed a distinct spherical particle of uniform smooth surface with an average diameter of 185 nm. Similarly, AFM analysis showed the formulated nanoparticle had an average diameter of 182 nm (Figure.1 a,b,c). So, TEM and AFM data demonstrated a good agreement with the size observed by DLS. As the physical state of the drug in the polymeric matrix reported to influence drugs release characteristics. In this view, the XRD pattern of native curcumin, Nano CUR and void NP were further studied to understand the nature of curcumin in our NP formulation (Figure.2). As shown from figure, the peak of native curcumin showed the traits of high crystalline structure and simultaneously there were no such characteristics peaks were observed when it was entrapped in NP formulation indicating the state of entrapped curcumin is in amorphous state. Similarly, while observing the release profile, we observed a biphasic release pattern of entrapped curcumin from Nano CUR formulation. The biphasic drug release occurred by diffusion followed by degradation of polymer. Herein, we observed a rapid release of curcumin at about 45.94 % in 24 h followed by a sustained release of about 65.6 % over 10 days of our observation (Figure.3).

Stability study of curcumin

One of the major challenges of drug delivery to cancerous tissue is its instability and biodegradation in physiological pH. In this view, the ideal drug delivery system should retain the entrapped drug in a stable condition for prolonged time while in circulation. In an attempt to study the biodegradation and instability properties of curcumin, we incubated curcumin (native and Nano CUR) in PBS (0.01 M, pHH 7.4) and estimated its concentration with time by HPLC. It was observed that native curcumin underwent rapid degradation in PBS (only 6 % of curcumin remained intact after 6 h of incubation). However, Nano CUR were stable under the same condition (~ 90 %) (Figure. 4 a). It is noteworthy that our formulation increased the stability of curcumin in PBS by protecting the encapsulated curcumin against hydrolysis and biotransformation for a longer time. Toxicity studies of void polymeric nanoparticle

In order to exclude the possibility of lethality shown by majority of NPs (from its polymeric constituents), we further intended to evaluate its toxicity profile by treating the cell with void particle. The biocompatibility test confirmed the treated cell did not show any obstruction in cell proliferation and illustrated the similar trend of cell population as observed by control cell (Figure 5). Similarly, there is no sort of morphological as well as internal aberration was observed like blebbing of the nucleus, condensation of the chromatin and jagged cell membrane. Its non toxicity feature was further confirmed by TNF- a assay ( Figure 6). The result demonstrated same trend release of TNF- a from both void treated and control cell. It suggested the formulated void NP was not able to elicit the production of pro-inflammatory cytokines TNF- a, indicating no toxicity and biocompatibility of void particle.

Colony soft agar assay

Colony soft agar assay is an anchorage independent growth assay in soft agar, which was taken into consideration to determine the antiproliferative efficacy of native curcumin and Nano CUR on pancreatic cell line. Herein PANC-1 pancreatic cell line was treated with curcumin (both native curcumin and Nano CUR) at a dose of 15 μΜ for 7 days. The result showed that Nano CUR profoundly inhibited the pancreatic colony formation compared to the colony observed from native curcumin treated cell (Figure 7). This suggests that curcumin entrapped in Nano CUR has comparative better antiproliferative activity as it was effectively blocked the clonogenicity of PANC-1 cell compared to native treated cell. Cellular uptake studies

Taking the advantage of photochemical properties of curcumins, the intracellular uptake of Nano CUR was compared with native curcumin by fluorescence spectroscopy. The result of quantitative cellular uptake demonstrated Nano CUR was internalized more efficiently by PANC-1 cell compared to native curcumin (Figure 8). By measuring the fluorescence intensity of curcumin, a concentration dependent increase in cellular uptake of Nano CUR and native curcumin was observed. However, cellular uptake of Nano CUR at lower concentration i.e. at dose 5 and 10 μΜ was 5.9 and 7.7 times more than native while at higher concentrations (30 μΜ) 4.08 times increase in uptake values was observed in comparison to native curcumin. This shows that at lower concentration the Nano CUR uptake is more effective. Similar results were also observed by Sa u et ai. where they have reported a concentration dependent increase in cellular uptake of native curcumin and curcumin encapsulated bovine casein micelle in HeLa cell line [31]. Further, at a particular concentration (10 μΜ) the intracellular uptake of native curcumin and Nano CUR after 1 h of incubation was investigated qualitatively by microscopic (fluorescence and confocal microscope) observation in the same cell line Figure 9. a). The microscopic studies demonstrated the cell treated with Nano CUR showed profound fluorescence intensity compared to the cell treated with native, indicating Nano CUR were internalized more efficiently by the cells than native curcumin. The time dependant cellular uptake studies of curcumin ( both native and Nano CUR ) observed from confocal image studies, demonstrated the fluorescence intensity was initially restricted to cell membrane as shown by 15 min treated cell, while with time it enhanced and extended to cytoplasm (Figure 9). In native curcumin treated case maximum fluorescence was observed at initial treatment but gradually the fluorescence intensity was decreased with time (as observed after 24 h of incubation). However, an increase in fluorescence intensity with time was observed in NP treated cell indicating entrapped curcumin is slowly released from NP for a longer period of time.

In vitro Mitogenic Assay

MTT based colorimetric assay demonstrated that the cytotoxicity of both native and Nano CUR were dose dependant in all the observed cell lines (Figure 10). The curcumin concentration (in native and in Nano CUR formulation) that killed the 50 % of cell (IC50) studied in various cell line were depicted in Table 1. The extent of growth inhibition by the phenolic curcumin was found to be effective in all the studied cell line. As evidenced from Figure 10, curcumin treated pancreatic cell showed its cytotoxicity trend in between 10 to 20 μΜ concentration of curcumin treatment. However, the toxicity was observed early i.e. around 10 μΜ concentration in Nano CUR treated cells.

Comparable inhibition of cell proliferation was clearly observed on in vitro cytotoxicity studies, which demonstrated that Nano CUR was more effective than native in solution in controlling the growth of observed tumor cell line.

Table 1. IC 50 values of native curcumin and Nano CUR in different tumor cells as assayed by MTT cytotoxicity assay. T/IN2010/000618

Apoptosis analysis by Flow Cytometry

Apoptosis results through activation of pre-programmed pathway of biochemical events, which eventually leads the cell to dead. Available evidence suggests that apoptosis may represent a mechanism to counteract neoplastic development, which is essential for cancer therapy [32,33]. The explosion of studies on apoptosis in recent years has described that native curcumin was responsible for eliciting apoptosis signals in a varied number of tumor tissues including colorectal, lung, breast, pancreatic and prostrate carcinoma [26,33]. To determine the ability of curcumin for promoting apoptosis in PANC-1 cell line, we investigated its apoptosis inducing efficiency by staining the cells with annexin V-FITC. On treated cells we have reported the presence of early apoptotic advanced apoptotic and necrotic cell population (Figure 11). However, the Nano CUR treated cell showed more number of cell i.e. 22.37% in apoptosis compared to 5.81% of cell found in native treated cells. So, the apoptosis result clearly indicating curcumin encapsulated Nano CUR treated PANC-1 cell showed 3.5 times more apoptosis than native curcumin.

Western blot analysis

Our apoptosis result clearly demonstrated that curcumin showed its antiproliferative activity against caner cell by inducing apoptosis. This activity has been well studied before and it is mostly due to inhibition of the Akt-NFkB signaling pathway [34-36]. So, to substantiate our finding we studied the molecular basis of apoptosis by accessing the Akt-NF_kB pathway. Our western blot results as shown in Figure 12 demonstrated a decrease in phosphorylation of Akt (band at 60 KD) in curcumin treated compared to control. The band intensity is further decreased in Nano CUR treated cell compared to native curcumin treated, indicating more inhibition of Akt phosphorylation. Further, high intense NF_kB (at 64 Kda) and I_kB (at 34 Kda) band were observed in curcumin treated cell compared to untreated cells. This suggest that Akt, a target of P13K was phosphorylated and thus activated under basal conditions. The work conducted by Schlieman et. al also reported its activation in variety of pancreatic cell line including PANC-1 cell [37]. However, curcumin dephosphorylated Akt which consequently inhibited NF_kB signaling pathway. Whereas, NF_kB is a transcription factor present in the cytoplasm, as an inactive heterodimer consisting of p50, 065 and ¾Βα subunits. So, curcumin restrained Akt activation and consequently blocked phosphorylation of ¾Βα and p65. Which in turn inhibited the activation and translocation of NFKB in to nucleous and as well as transcription of NFKB regulated gene. So our results confirmed the presence of more cytosolic NFKB (in an inactive state) in curcumin treated case. However, in untreated case NFKB gets translocated in to nucleous and hence less intense band was observed. Similarly, high intense band of ΙκΒα in cytosol from curcumin treated cell confirmed the presence of more ΙκΒα in cytosol and the inhibition of NFKB pathway. Nano CUR treated cell further intensified the NFKB and ΙκΒα bands compared to native curcumin treated cell suggesting the Nano CUR is more efficient in delivering the curcumin to tumor cell. We also investigated whether curcumin can modulate NFKB regulated gene products like cyclin-D and c-Myc involved in proliferation and anti apoptosis respectively in tumor cells. In this view, we observed less intense cyclin-D (at 36 Kda) and c- Myc (at 65 Kda) bands in Nano CUR treated cell compared to control and native curcumin treated cell. These results supported our postulate that Nano CUR are more efficiently blocks the NFKB activation and NFKB regulated gene expression through inhibition of ΙκΒα and Akt activation compared to native curcumin.

Pharmacokinetic study

Nano CUR was designed with a notion to improve the systemic bioavailability of delivered curcumin. In this view, the Nano CUR and native curcumin with a dose of 30 mg/kg were intravenously injected in mice to monitor the systemic bioavailability of delivered curcumin. The mean curcumin concentration in the serum of mice after i.v administration of both native and formulations at single dose of 4 mg/ml are illustrated in Figure 13. Result showed maximum availability of 25 μg/ ml of curcumin was observed after lh of Nano CUR administration. In contrast a maximum of 0.53 μg /ml was detected after 15 min administration of native curcumin. In this way, high availability as well as sustainded serum concentration of curcumin for 24 h of our observation was noticed in Nano CUR case. This result suggested a sustained release of curcumin from our Nano CUR formulation consequently increased the bioavailability of delivered curcumin. Whereas, in native case the level was subsequently decreased with time and not detectable beyond 1 h, indicating rapid metabolism of native curcumin in physiological pH. This observation indicated intravenous injection of curcumin using NP formulation could facilitate its successful delivery by efficiently preserving its stability.

Nanostructure lipid-based drug delivery system based on GMO holds many promises for delivering hydrophobic drug like curcumin. The selfemulsifing properties of GMO can form a hydrophobic core (assuming a micellar structure), which enhance the solubility of hydrophobic drug and can provide a foundation for successful surface modification. To this end, we have developed a novel nanoparticulate delivery system consisting of GMO to overcome major obstacle associated with delivery of curcumin like poor solubility, rapid degradation and poor bioavailability.

The physicochemical characterization of delivery system is very much essential to achieve an ideal drug delivery vehicle for successful therapeutics. In this regard, the particle size is an important parameter which will directly influence the physical stability, cellular uptake, biodistribution and release of drug from NP. The DLS size measurement showed 192 nm size of our formulation having narrow monodispersed unimodal size distribution pattern and TEM images showed discrete spherical outline and monodispersed size distribution (-190 nm). Further, AFM observation confirmed the notion that the process of NPs preparation was highly reproducible and the resultant particles were spherical in shape. As we know small size of particles are advantageous for passive targeting to tumor tissue by enhanced permeability and retention effect [1, 31]. Hence, we can anticipate that the small size of our Nano CUR formulation could enhance circulation half lives as well as reduced reticuloendothelial system (RES) uptake. Besides, achieving small size its XRD analysis demonstrated, the drug incorporated inside the NP is in amorphous state. The major challenges of curcumin delivery in therapeutics grounds involves while defining its stability. During systemic drug delivery the stability of delivered drug is one of the prime parameter to be considered for achieving better therapeutics. In this view, while demonstrating the stability of entrapped curcumin, our results showed unlike native curcumin the curcumin encapsulated within Nano CUR were dramatically explained its stability in PBS (0.01 M, pH= 7.4). Consistent to our results, studies conducted by Ma et al. also reported high degradation and instability property of native curcumin in PBS (0.01 M, pH= 7.4) and approximately 30.41 % of native curcumin remained intact after 20 min of their incubation [26]. Similarly, Wang et al. reported 90 % of native curcumin degraded after 30 min in phosphate buffer (0.01 M, pH 7.4 [12]. We attribute this degradation could be due to rapid presystem hydrolysis and biotransformation of curcumin into its glucuronide and sulphate conjugates within a short period of time [3, 10, 26]. Hence, it can be said our formulated Nano CUR system can efficiently increased the stability of curcumin even in PBS by protecting the encapsulated curcumin against hydrolysis and biotransformation for a longer time. While observing the in vitro release profile, we observed a biphasic release pattern of encapsulated curcumin from Nano CUR. The slow and constant release of curcumin in our study after initial burst release is mainly due to the slow diffusion of drug molecules through the polymeric matrix of the NPs [17, 38]. Consequently the slow and sustained release of the drug at later stages can be attributed to the diffusion/ erosion of polymeric matrix which releases the encapsulated drug. Studies conducted by Dash et al. in cubic phase chitosan/GMO NPs observed the similar trends of release for paclitaxel and dexamethasone, showing an initial burst release of ~10 % and ~ 45 % respectively in 1 h, followed by a slower and constant release thereafter [22].

Toxicity studies of void polymeric NP were performed to evaluate the preliminary safety profile of our delivery vehicle. In general, apoptosis induced by the toxic polymeric particle showed a typical signs like blebbing of the nucleus and condensation of the chromatin etc. [39]. However, these aberrations were not at all observed in our void treated cell confirming its biocompatibility to PANC-lcell line. Its non toxicity profile was further confirmed by getting the same trend of TNF-a released from both treated and control cell. Here, TNF-a was taken as a parameter to quantify the cell toxicity (induced by void NP), as TNF is released from cells when the cells are damaged and it is a marker cytokines for inflammation which promotes antitumor and immune responses [28].

Cellular uptake study is an important parameter needs to be explained for justifying successful drug delivery of our formulation to cancer tissue. In this study, we observed a concentration dependent increase in curcumin uptake in both the Nano CUR and native curcumin treated cells. The native curcumin treated cell showed maximum fluorescence initially for few hours of treatment but gradually the fluorescence intensity decreased with time as observed after 8 and 24 h of incubation by PANC-1 cell. In contrast, the subsequent enhanced uptake (as studied qualitative and quantitative experiments) of our formulation with time proved succeeding internalization and sustained release of encapsulated curcumin for the period of our observation. Our MTT results confirmed that Nano CUR demonstrated a lower IC₅₀ values compared to native curcumin as studied in all the observed cancer cell lines. This could be due to difference in uptake profile (as observed in PANC-1) which was reflected in the current cytotoxicity profile. As, it is well known that the antiproliferative effect of drug is very well correlated with the duration of its intracellular retention and drugs stability [26]. In general, native drug always diffuses across the cell membrane of uptake cell (when used as a solution) [18]. Herein, after attaining saturation inside the cytoplasm further diffusion was restricted. These small fractions of diffused native drug showed its antiproliferative effect for a short time of its existence. However, through endocytosis enough Nano CUR can be available inside the cell and released the encapsulated therapeutic agent in a sustained manner to exert profound cell toxicity.

Apoptosis is one of the pathway by which chemotherapeutic agents can induce cell death in tumor tissue. A plethora of experimental evidence suggested that curcumin eliciting apoptosis signals in a varied number of tumor tissues including colorectal, lung breast, pancreatic and prostrate carcinoma [4, 32, 40]. Our result supports these finding that truly curcumin has the potency to induce apoptosis on cancer cell as we observed in pancreatic cancer cell line (PANC-1). In our studies, native curcumin treated cells demonstrated 59.38 % of cell in necrotic stage and 5.81 % of cell in apoptosis stage. However, Nano CUR treated cells showed less number of cells i.e. 32.39 % in necrosis and more number of cells i.e. 22.37 % in apoptosis stage compared to native treated cell. Here, we suggest that native curcumin may have diffused and accumulated directly at its site of action thus resulting in more fractions of cells in necrotic stage rather than in apoptotic stage. However, better uptake of Nano CUR resulted in greater accumulation of delivered curcumin inside tumor cell accompany with its sustained release exerted more percentage of cells in apoptotic phase and with time resulted reduction of cell viability, as observed in MTT assay. Our cytoplasmic localization of curcumin supports the fact that caspase might play an important role in induction of apoptosis. Caspase is the cytoplasmic aspirate-specific cysteine proteases responsible for apoptosis. Activation of caspase leads to many molecular and structural changes in apoptosis including degradation of DNA repaired enzyme poly (ADP) ribosepolymerase (PARP). Furthermore, apoptosis induction properties of curcumin have been attributed to its ability to inhibit COX-2 because it is well know that curcumin is a natural COX-2 inhibitor. Our previous study demonstrated 7.16 % of apoptotic cells were observed following treatment with 30 μΜ of native curcumin in PANC-1 cell [41] . Lev-Ari et al. reported curcumin (25 μΜ) treated PANC-1 cells showed approximately 8 % of total population cell arrest in sub Gl phase (apoptotic phase) [32] . Similarly, Ma et al. reported high population apoptotic cells (81.87 %) in B 16- F10 cell line following treatment with 40 μΜ curcumin [26] . Our current studies demonstrated only 5.81 % of apoptotic cells following treatment with native curcumin. This modest level of apoptotic signals could be due to low level of COX-2 expression in PANC-1 cell line as compared to other cell lines [32]. Further, we hypothesize the role of cytoplasmic NFKB for induction of apoptosis in tumor cell, as reported previously that curcumin showed potent anti proliferative activity in tumor cell including pancreatic cancer cell by inhibiting NFKB DNA binding activity [34, 35]. In this regards, consistent to apoptosis result our western blot analysis confirmed our proposition that NFKB pathway was really inhibited by curcumin as observed in PANC- 1 cell. In addition to P-Akt down regulation, curcumin blocks the classical NFKB pathway which regulates inflammation, cell proliferation and apoptosis in normal cell [40]. Our results also explained the involvement of NFKB path way and its control in the expression of gene involved in proliferation and anti apoptotic process in tumor cell. Here we attribute the better uptake, sustained release and stability of curcumin encapsulated in Nano CUR, results greater accumulation of it inside cancer cell and consequently showed more pronounced down regulation of NFKB compared to native curcumin treated cell. One of the major interests lying in formulating Nano CUR is to improve curcumins in vivo bioavailability. Interestingly, our results showed, Nano CUR is 50 folds more bioavailable and displayed a substantially longer half-life compared to native curcumin. Such sustained release in vivo was observed in a series of pharmacokinetic studies conducted in various laboratories [10, 30, 38, 42, 43]. In agreement with Bisht et al. and Anand et al. [2, 14], we also observed that our formulation was not toxic to the animals. Hence, our discussed results suggest Nano CUR possess better chemopreventive, chemotherapeutic properties than native curcumin due to its better bioavailability and it consequently exert induction of apoptosis in tumor cells, advocates their potential use in a strategy for cancer control.

The enhancement of water solubility as well as stability will undoubtedly bring curcumin to the forefront of existing anticancer therapeutic agents. In this regard, the encapsulation of curcumin within Nano CUR brought about a new avenue to improve the bioavailability of curcumin and can make the drug amenable to intravenous dosing for the treatment of cancer. Most importantly, the observed comprehensible results justified the Nano CUR was comparatively more effective than native curcumin under in vitro condition against pancreatic cell line with time due to greater cellular uptake, sustained intercellular drug retention and enhanced antiproliferative effect. Consequently, the enhanced cellular internalization resulted in reduction of cell viability by inducing apoptosis. Thus, the Nano CUR provided an efficient delivery for encapsulated curcumin and proved a promising carrier candidate by increasing its water solubility and improving its stability for tumor therapeutic treatment in near future.

Claims

WE CLAIM:

1. A novel water soluble curcumin loaded nanoparticulate system for cancer therapy having narrow monodispersed unimodal size distribution (<200 nm) with high zeta potential around -32 mV.

2. The water soluble curcumin loaded nano particulate system as claimed in claim 1, wherein the said system has enhanced solubility, stability and bioavailability.

3. The method for preparing curcumin loaded nanoparticles system comprising: incorporating curcumin into the fluid phase of GMO;

subjecting the GMO mixture to the step of emulsifi cation with PVA;

emulsifying the resultant solution with pluronic F-127 solution;

lyophilizing the final emulsion by freeze drying to produce lyophilized powder.

4. The method as claimed in claim 3, wherein the step of emulsification is preferred by sonication for 2 min at 55 watt.

5. The method as claimed in claim 3, wherein the lyophiiization is preferred by freeze drying method at -80°C and <10 μπι mercury pressure.

6. The method as claimed in claim 3, the stabilizer/ surfactant used are polyvinyl alcohol (0.5 %w/v) and pluronic F-127 (10 % w/v), wherein the pluronic solution (comprises of hydrophilic poly (ethylene oxide) [PEO] and hydrophobic poly (propylene oxide) [PPO]).

The method as claimed in claim 3, wherein nanoparticles include a hydrophobic core comprising GMO, where the hydrophobic agent curcumin tightly bound.

The method as claimed in claim 3, wherein the GMO nanoparticle surrounded by a hydrophilic surface layer including polyvinyl alcohol and pluronic F-127.