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ARTICLE IN PRESS Biomaterials xxx (2010) 1e15 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation Chandana Mohanty, Sanjeeb K. Sahoo* Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023, India a r t i c l e i n f o Article history: Received 19 March 2010 Accepted 29 April 2010 Available online xxx Keywords: Apoptosis Bioavailability Cancer therapy Curcumin Nanoparticle a b s t r a c t Curcumin, the natural anticancer drug and its optimum potential is limited due to lack of solubility in aqueous solvent, degradation at alkaline pH and poor tissue absorption. In order to enhance its potency and improve bioavailability, we have synthesized curcumin loaded nanoparticulate delivery system. Unlike free curcumin, it is readily dispersed in aqueous medium, showing narrow size distribution e192 nm ranges (as observed by microscope) with biocompatibility (confocal studies and TNF-a assay). Furthermore, it displayed enhanced stability in phosphate buffer saline by protecting encapsulated curcumin against hydrolysis and biotransformation. Most importantly, nanoparticulate curcumin was comparatively more effective than native curcumin against different cancer cell lines under in vitro condition with time due to enhanced cellular uptake resulting in reduction of cell viability by inducing apoptosis. Molecular basis of apoptosis studied by western blotting revealed blockade of nuclear factor kappa B (NFkB) and its regulated gene expression through inhibition of IkB kinase and Akt activation. In mice, nanoparticulate curcumin was more bioavailable and had a longer half-life than native curcumin as revealed from pharmacokinetics study. Thus, the results demonstrated nanoparticulate curcumin may be useful as a potential anticancer drug for treatment of various malignant tumors. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Cancer is the most distressing and life threatening disease that enforces severe death worldwide. 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, undesirable side effect to normal tissue and inherent lacking of beneficial response of cytotoxic anticancer drug [1e3]. To this end, the nontoxicity and efficacy of the traditional medicine now-a-day’s open up new avenue for future cancer therapies [4]. 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 [5,6]. Curcumin is a hydrophobic polyphenol, a major yellow phytochemical compound of Turmeric (Curcuma longa, Zingiberaceae). The chemical structure of curcumin is [1,7-bis(4-hydroxy-3* Corresponding author. Laboratory for Nanomedicine, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa 751023, India. Tel.: þ91 674 2302094; fax: þ91 674 2300728. E-mail address: [email protected] (S.K. Sahoo). methoxyphenyl)-1,6-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 [5,7,8]. This is due to its interference in diverse range of cell signaling pathway including cell cycle (cyclin-D1 and cyclin E), apoptosis (activation of caspases and down-regulation of antiapoptotic gene products), proliferation (HER-2, EGFR, and AP-1), survival (PI3K/Akt pathway), invasion (MMP-9 and adhesion molecules), angiogenesis (VEGF), metastasis (CXCR-4) and inflammation (NFkB, TNF, IL-6, IL-1, COX-2, and 5-LOX) [4,7]. So in current research, curcumin has been taken as an imminent herbal drug to instigate multitargeted therapy, which is needed for treatment of various fatal diseases including cancer [7,8]. Clinically, it is considered extremely safe while administered at very high doses. Conversely, systemic toxicity at high dose rendered other anticancer drug unsuitable for cancer therapy [9,10]. Though, curcumin has demonstrated safety and efficacy (as a chemotherapeutic agent) but it 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 [4,11,12]. Current trends in curcumin research have concentrated on the development of potential delivery systems to increase its aqueous 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.062 Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 2 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 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 like piperine that interferes with glucuronidation and the use of other delivery vehicle like liposomal curcumin, curcumin nanoparticles, curcumin phospholipid complex, and structural analogues of curcumin have certainly testified as the comprehensible methods to increase the potentiality of delivered curcumin [13e17]. However, limited studies have been reported regarding its successful in vivo bioavailability by encapsulating it in different polymeric delivery system. Recently, Anand et al. have achieved twofold increased in bioavailability of curcumin with in PLGA nanoparticle (NP), where as ninefold enhancement was reported by Shaikh et al. in same polymeric NP and furthermore, Maiti et al. observed 2.5 times more bioavailable of curcumin while delivering in phospholipid complex compared to the native curcumin [12,14,18]. Recently much attention was given for bioadhesive delivery systems to enhance the drugs bioavailability by increasing the residence time which subsequently facilitate the absorption of drug through adhesion with the cellular surface [19,20]. In this view, the best considered strategy to achieve enhanced bioavailability of curcumin is to encapsulate it within glycerol monooleate based nanoparticle (GMO NP). The GMO was approved by food and drug administration (FDA) and it is an emulsifier, flavoring agent used in the food industry and well studied excipient agent for antibiotics [19]. These bioadhesive delivery systems are currently gaining interest to augment the systemic bioavailability by encapsulating different hydrophobic drugs [19e22]. However, for preventing aggregation in biological solution and for providing better stabilization to NPs coating of large molecules such as polymers or macromolecules (containing long-chain hydrocarbons) are necessitate [23]. In this scenario, the choice of an ideal polymer or macromolecule is vital as it regulates the essential properties such as solubility, stability, drug loading capacity and drug release profile of NPs [24,25]. Some representative of such material is nonionic block copolymer Pluronic F-127 and polyvinyl alcohol (PVA) which have gained much attentions for providing specific surface charge and chemical functionalization to NP delivery system [26]. Apart from providing stability to NPs, the key attribute of Pluronic F-127 is their ability to enhance drug transport by effective passive targeting towards cancerous tissues and making sensitize the multidrug resistance tumors to various anticancer agents [27]. Due to their amphiphilic character these copolymers display surfactant properties and further offers stability and biocompatibility to NPs. 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. The objective of this study was to develop a nanoparticulate delivery system by the use of GMO and pluronic F-127 that can solubilise curcumin in aqueous media at clinically relevant concentration, protect it from hydrolytic degradation and in vivo biotransformation, and delivered curcumin in a controlled manner. In this regard, it will improve the bioavailability of delivered curcumin for tumor therapeutic treatment. To this end, the potentiality of the formulated nanoparticulate curcumin was determined by observing its in vitro release kinetics, stability, cellular uptake, cytotoxicity and apoptosis inducing properties on tumor cell lines. Further, we studied the in vivo bioavailability of the nanoparticulate curcumin compared to native curcumin in mice. 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 purchased from Sigma Aldrich (St. Louis, MO) without further purification. 2.2. Preparation of nanoparticulate curcumin Preparation of nanoparticulate curcumin was done as per the protocol of Trickler et al., with little modification [20]. 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 10 ml of PVA (0.5% w/v) by sonication (VC 505, Vibracell Sonics, Newton, USA) set at 55 W of energy output for 2 min. The resultant solution was further emulsified with 10 ml of pluronic solution (10% w/v) by sonication (VC 505, Vibracell Sonics, Newton, USA) set at 55 W 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 mm mercury pressure, LYPHLOCK, Labconco, Kansas City, MO) to get lyophilized powder for further use. 2.3. Physical characterization of nanoparticulate curcumin 2.3.1. Particle size and zeta (z) potential measurements Particle size and polydispersity index were determined using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) based on quasi-elastic light scattering. Briefly, e1 mg/ml of nanoparticulate curcumin solution was prepared in double distilled water and sonicated for 30 s in an ice bath (VC 505, Vibracell Sonics, Newton, USA). Size measurements were performed in triplicates following the dilution (100 ml 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. All measurements were performed in triplicates. 2.3.2. Atomic force microscope (AFM) The shape of nanoparticulate curcumin was further characterized by AFM (Nanoscope III A, Vecco, Santa Barbara, USA). A drop of nanoparticulate curcumin solution (e1 mg/ml) was placed on freshly cleaved mica. After 5 min of incubation the surface was gently rinsed with deionized water to remove unbound NPs. The sample was air dried at room temperature and mounted on the microscope scanner. The shape was observed and imaged in noncontact mode with frequency 312 kHz and scan speed 2 Hz. 2.3.3. Transmission electron microscopy (TEM) The internal structure of NPs was determined by TEM measurements, for which a drop of diluted solution of the nanoparticulate curcumin was placed in carboncoated copper TEM grid (150 mesh, Ted Pella Inc., Rodding, CA), negatively stained with 1% uranyl acetate (w/v) for 10 min and allowed to air-dry. The samples were imaged using a Philips 201 transmission electron microscope (Philips/FEI Inc., Barcliff, Manor, NY) and visualized at 120 kV under microscope. The TEM photograph was taken by using the NIH imaged software. To calculate the mean particle diameter, 50 particles were taken for measurement. 2.3.4. Fourier transform infrared (FTIR) spectral study FTIR spectra were taken in to observation (Perkin Elmer, Model Spectrum RX 1, USA) to investigate the possible chemical interactions between the curcumin and the polymer matrix. Void NPs, native curcumin, nanoparticulate curcumin were crushed with KBr to get the pellets by applying a pressure of 300 kg/cm2. FTIR spectra of the above sample were obtained by averaging 32 interferograms with resolution of 2 cm1 in the range of 1000e4000 cm1. 2.3.5. Differential scanning calorimetric (DSC) studies The physical state of the nanoparticulate curcumin was characterized using a DSC thermogram analysis (STA 6000, Simultaneous Thermal Analyser, Perkin Elmer, MA, USA). Each sample (e5 mg of native curcumin, void NPs and nanoparticulate curcumin) was sealed separately in a standard aluminum pan, the samples were purged in DSC with pure dry nitrogen gas set at a flow rate of 10 ml/ min, the temperature speed was set at 10 C/min, and the heat flow was recorded from 0 to 200 C. 2.3.6. X- ray diffraction (XRD) study XRD analysis was done to know the crystallographic structure of the nanoparticulate curcumin formulation. The patterns of native curcumin, void NPs and nanoparticulate curcumin were obtained using X-ray diffractometer (Bruker 9XS, G8ADVANCE). The measurements were performed at a voltage of 40 kV and 25 mA. The scanned angle was set from 3 2q 40 and the scan rate was 2 min1. 2. Materials and methods 2.1. Materials CUR-500, containing Curcumin (>95%) was purchased from UNICO Pharmaceuticals, Ludhiana, India. Polyvinyl alcohol (PVA, average MW ¼ 31,000e50,000) was purchased from SigmaeAldrich Co. (St Louis, MO), 3-(4, 5-dimethylthiazol- 2.3.7. Quantifying entrapment efficiency of curcumin by high performance liquid chromatography (HPLC) method The entrapment efficiency of curcumin was measured by HPLC method following our previous protocol [19]. Accordingly, the nanoparticulate curcumin was dissolved in methanol (e1 mg/ml) to disrupt its structure. The sample was then subjected to sonication for 3 min at 55 W (Model: VCX750, Sonics and Materials Inc., Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 USA) followed by centrifugation at 13,800 rpm for 10 min at 25 C (SIGMA 1-15K, Germany) to get a clear supernatant. The supernatant obtained was analyzed using reverse phase isocratic mode (RP-HPLC) system of WatersTM 600, Waters Co. (Milford, MA, USA). For this, 20 ml 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 (M600E WATERSTM) at 25 C with a C 18 column (Nova- Pak, 150 4.6 mm, internal diameter). The curcumin levels were quantified by UV detection at 420 nm with dual wavelength absorbance detector (M 2489). The amount of curcumin in the sample was determined from the peak area correlated with the standard curve. Triplicate samples (supernatants) were analyzed and the Curcumin encapsulation efficiency was calculated by dividing the amount of carboplatin entrapped by the total amount of Curcumin added, multiplied by 100. 2.3.8. Analysis of photophysical properties of curcumin encapsulated in nanoparticulate curcumin and native curcumin Encapsulation and binding of curcumin in hydrophobic core of GMO in nanoparticulate curcumin formulation was further examined by spectroscopic analysis [28]. We measured the absorbance and fluorescence spectra of both native curcumin (dissolved in methanol) and nanoparticulate curcumin (in aqueous solution) of concentration 3 mg/ml for the spectroscopic experiments. Aqueous nanoparticulate curcumin solution was vortexed followed by sonication for 1 min (Model: VCX750, Sonics and Materials INC., USA) to get well dispersed solution for spectroscopic studies. Curcumin was quantified spectrophotometrically at 425 nm (Synergy HT, BioTekÒ Instruments Inc., Winooski, VT, USA) and fluorescence emission spectra was recorded from 450 to 700 nm with an excitation wavelength 420 nm (Perkin Elmer, Model No. LS 55, Massachusetts, USA). 3 100 ml of solutions (either native curcumin or nanoparticulate curcumin) were taken and added to 900 ml of methanol to quantify the stability of curcumin with time in PBS (0.01 M, pH 7.4) by HPLC. 2.6. Cellular uptake studies 2.6.1. Quantitative cellular uptake study The cellular uptake of curcumin (native and nanoparticulate curcumin) was evaluated in pancreatic cell (PANC-1) following the protocol of Kunwar et al. [32]. Briefly, PANC-1 cells were seeded in a 24 well plate (Corning, NY, USA) at a seeding density of 5 104 cells per well in 1 ml of growth medium. After 24 h of incubation at 37 C the attached cells were treated with equivalent dose (5, 10, 20 and 30 mM) of native curcumin and nanoparticulate curcumin 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 (0.01 M, pH 7.4) and lysed by adding methanol. The cell lysates were centrifuged at 10,000 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 (lex ¼ 420 and lem ¼ 540 nm) (Synergy HT, BioTekÒ Instruments Inc., Winooski, VT, USA). Each measurement was performed in triplicates and the data obtained are mean values from three different experiments. 2.3.9. In vitro release kinetics The release of drug from nanoparticulate curcumin was carried out by dissolving 100 mg of NPs in 15 ml PBS (0.01 M, pH 7.4) and the solution was divided in 30 eppendorf (500 ml each) tubes, as experiment was performed in triplicates [13]. The tubes were kept in a shaker at 37 C at 150 rpm (Wadegati Lab equip, 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 nanoparticulate curcumin. The released curcumin was redissolved in 1 ml of methanol and 20 ml of this solution was injected in the HPLC to determine the amount of curcumin released with respect to different time intervals. 2.6.2. Qualitative cellular uptake study For qualitative cellular uptake study, PANC-1 cells were seeded at a seeding density of 15 104 cells on 35 mm culture plate (Corning, NY, USA) and 1 105 cells in BioptechÒ 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 mM and 1 mM for fluorescence microscopic studies and confocal studies respectively) of native curcumin and nanoparticulate curcumin 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 native curcumin. 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 fluorescence 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 nanoparticulate curcumin, the BioptechÒ tissue culture plates were removed from the incubator at predetermined time intervals and the cells were processed using the above confocal studies protocol. 2.4. Toxicity studies of void polymeric nanoparticle 2.7. Colony soft agar assay An ideal drug delivery vehicle must be biodegradable, biocompatible and not to be associated with incidental adverse effects. In order to justify the nontoxicity of our void polymeric particle two sets of in vitro toxicity studies were conducted. The noncytotoxicity of the void particle was assessed by observing the cell morphology in confocal microscope after staining with lysotracker dye and 40 -6-Diamidino-2phenylindole (DAPI) [19,29]. Briefly, PANC-1 cell suspensions were prepared at a concentration of 1 105 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 cells were washed with PBS (0.1 M, pH 7.4) and treated with lysotracker dye (2 ml of 1 mM in DMSO solution dissolved in 40 ml of media) for 30 min. The cells were washed twice with PBS (0.01 M, pH 7.4), 10% buffered formaldehyde for 15 min and finally stained with DAPI for 30 min. The cells were further washed with PBS (0.01 M, pH 7.4) and imaging was done with confocal laser scanning microscopy (Leica TCS SP5, Leica Microsystems GmbH, Germany) using the 60 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, PANC-1 cells (at density 1 105 cells/ml) were incubated with different concentrations (0.1e0.5 mg/ml) for 24 h [30,31]. 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 8000 rpm (Sigma microcentrifuge-16PK, Germany) for 30 min to remove cell debris. TNF-a protein concentration in the cell supernatant was measured using the ELISA kit (Human TNF-a ELISA KIT, Bender MedSystem Inc., USA) according to the manufacturer’s instruction. The antiproliferative effect of native curcumin and nanoparticulate curcumin was analyzed based on the methodology of Bisht et al. with slight modification [13]. Briefly, 2 ml mixture of serum supplemented media and 1% agar containing 15 mM of native curcumin and equivalent concentration of nanoparticulate curcumin was added in a 35 mm culture dish (Corning, NY, USA) 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 nanoparticulate curcumin. 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% CO2 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 7th day and photographs were taken after staining the cell with crystal violate (0.005% w/v). 2.5. Solubility and stability study of curcumin Equivalent quantity of native curcumin and nanoparticulate curcumin were dissolved in PBS (0.01 M, pH 7.4) to observe the aqueous solubility of our formulation. Further, the stability of our formulation and native curcumin in PBS (0.01 M, pH 7.4) was estimated by HPLC method [19]. Native curcumin and nanoparticulate curcumin at a fixed concentration ofe40 mg/ml (at a total of 10 ml solution) were 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. Native curcumin was dissolved in PBS with the help of methanol (final methanol concentration 5% v/v). At predetermined time points, 2.8. In vitro mitogenic assay The antiproliferative effects of curcumin both in native form and nanoparticulate curcumin were analyzed by the MTT assay [25]. The assay was based on the cleavage of a yellow tetrazolium salt 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 (0, 5,10,15, 20, 25, 30 and 40 mM) of either native curcumin dissolved in DMSO or equivalent concentration of nanoparticulate curcumin. Concentration of DMSO in the medium was kept <0.1% w/v, so that it has no effect on cell proliferation [33]. Cells were incubated for 5 day 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 ml 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, BioTekÒ Instruments Inc., Winooski, VT, USA). The antiproliferative effect of different treatments was calculated as a percentage of cell growth with respect to respective control. Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 4 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 2.9. Apoptosis analysis by flow cytometry The induction of apoptosis by native curcumin and nanoparticulate curcumin was studied by flow cytometry [24]. Briefly, PANC-1 cells at density 3 105 cells/ml 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 mM/ml concentration of native curcumin and equivalent concentration of the nanoparticulate curcumin were added to the flasks and the cells were incubated in CO2 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 ml of 1 binding buffer (Clontech Laboratories, Inc., Palo Alto, CA), 5 ml Annexin V-FITC (final concentration, 1 mg/ml; BD Biosciences Pharmingen) and 5 ml propidium iodide (10 mg/ml; MP Biomedicals, Inc., Germany) and incubated at room temperature in dark for 20 min. Before flow cytometric analysis, 400 ml of 1 binding buffer was added to the cells. Stained cells were analyzed on flow cytometer (FACSCalibur; BectoneDickinson, San Jose, CA) using Cell Quest software with a laser excitation wavelength at 488 nm. 2.10. Western blot analysis Western blot analysis was done to study the molecular mechanism by which the native and nanoparticulate curcumin exert antiproliferative effect on pancreatic cancer cells [24]. The pancreatic cancer cell PANC-1 (5 105 cells/ml) were treated with 6 mM/ml curcumin (both in native and nanoparticulate curcumin) for 24 h and next day cell extracts were collected by scraping the cells, washing in 1 PBS followed by detergent lysis [50 mmol/l TriseHCl (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 mg) of each sample were solubilized in 2 sample buffer and electrophoresed on 8e12% SDS-PAGE. Protein immunodetection was done by electrophoretic transfer of SDS-PAGE separated proteins onto PVDF membrane (Millipore, corp.) followed by incubation with primary antibody (antibodies used were against phosphor-Akt, IkBa, NFkB P65, p21, c-Myc, cyclin-D1 and b actin in 1:1000 dilutions) for 1 h and secondary antibody (1:5000 dilution) for 40 min. Antigeneantibody complex was 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). 2.11. In vivo pharmakokinetics Animal experiment studies were carried out to analyze the pharmacokinetic study of delivered curcumin (in native and NPs form). It was performed with the permission of Institutional Animal Ethics Committee of institute of life sciences, Bhubaneswar, India. For in vivo pharmacokinetic study, BALB/c mice weighing 20e25 g were used. These mice were divided into two groups (n ¼ 4), group 1, received native curcumin dissolved in distilled water with Tween 20 (1%, v/v) and group 2, received nanoparticulate curcumin. Each mice was injected via lateral tail vein with either native curcumin or nanoparticulate curcumin (30 mg/kg), the blood was collected from retroorbital plexus at different time intervals, serum was separated, and the concentration of delivered curcumin was determined by HPLC analysis [14]. 2.12. Cell culture The above studied cancer cell lines were purchased from American Type Culture Collection (Manassas, VA) and cultured using DMEM with 10% FBS, 1% L-glutamine and 1% penicillinestreptomycin at 37 C in a humidified, 5% CO2 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. 2.13. 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 was indicative of a very significant difference. 3. Results diametere192 6.59 nm as measured by DLS with narrow monodispersed unimodal size distribution pattern and TEM images showed discrete spherical outline with monodispersed size distribution (e185 nm). Further, AFM observation confirmed the resultant particles were spherical in shape. The mean size distributions observed in DLS measurement were correlated to that observed in TEM (e185 nm) and AFM (e182 nm) (Fig. 1aec). The entrapment efficiency was around 90 2.55% as observed by HPLC. Besides demonstrating small size and high entrapment, it showed high zeta potential, i.e. 32 mV. Such higher zeta potential helps the formulation repel each other which ensure long term stability and avoid particle aggregation [25,34]. Further to confirms the presence of drug in nanoparticulate curcumin formulation, FTIR analysis was taken in to consideration (Fig. 2a) show the FTIR spectra’s of native curcumin, nanoparticulate curcumin and void NP. A band at 3490 cm1 has been previously attributed to eOH group stretching vibration in native curcumin [13,35]. In nanoparticulate curcumin a shift from 3490 cm1 to 3392 cm1 is shown, and the peak of 3392 cm1 becomes wider, this indicates hydrogen bonding is enhanced. The strong peaks at 2925 cm1 and 2855 cm1 and a weak peak at 1375 cm1 in all the case could be due to stretching and deformation of methyl groups. Similarly, a weak peak at 1465 cm1 has observed in all the three cases could be due to eCH2 bending vibration. The strong peak at 1740 cm1 in void NP and nanoparticulate curcumin is due to C]O adsorption. The signature peaks at 1627 cm1 and 1602 cm1 are found in native curcumin and nanoparticulate curcumin were due to C]C double bonds and aromatic C]C double bonds respectively. As these marker peaks were not found in void NPs, suggesting curcumin exists inside the nanoparticulate curcumin. Further, no shifting of peaks at 1627 cm1 and 1602 cm1 found in nanoparticulate curcumin compare to native curcumin, attributing curcumin could be present in dispersed condition in case of nanoparticulate curcumin formulation. Furthermore, the physical state of drug in the polymeric matrix of NPs, reported to influence the drug release characteristics [24]. In this view, DSC analysis was performed on native curcumin, void NPs and nanoparticulate curcumin (Fig. 2b). The result showed an endothermic peak of pluronic at 64 C, as pluronic exhibited a glass transition temperature (Tg) at 64 C. The endothermic peak of native curcumin was found approximately at 176 C. This characteristic peak was not observed in nanoparticulate curcumin. Similarly, the XRD study was further carried out to understand the nature of curcumin in our NP formulation (Fig. 2c). The characteristics peaks of native curcumin exhibited as shown in Fig. 2c(i), demonstrated the traits of high crystalline structure. However, there were no characteristics curcumin peaks were observed when entrapped in NP (Fig. 2c(ii)). This absence of detectable crystalline domains of curcumin in nanoparticulate curcumin clearly indicates that curcumin encapsulated in NPs is in the amorphous or disordered-crystalline phase or in the solid-state solubilized form in the polymeric matrix [24]. This disordered-crystalline phase of curcumin inside the polymeric matrix helps in sustained release of the drug from the NPs. Presence of drug in crystalline form inside NPs hampers its release as such large sized molecules cannot diffuse from the small pores of the NPs. However, if the drug is in amorphous or in disordered-crystalline phase easy diffusion of drug molecules can occur through the polymeric matrix, leading to a sustained release of the encapsulated drug. 3.1. Physicochemical characterization of nanoparticulate curcumin In a quest of developing an ideal formulation for achieving small size, maximum entrapment and for enhanced bioavailability of curcumin, we have prepared nanoparticulate curcumin (based on GMO) in a view to get maximum solubility and bioavailability of entrapped curcumin. Nanoparticulate curcumin showed an average 3.2. Analysis of photophysical properties of curcumin encapsulated in nanoparticulate curcumin and native curcumin To confirm the encapsulation as well as binding of curcumin in the hydrophobic core of our nanoparticulate curcumin formulation, the photophysical property of curcumin was taken into Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 5 Fig. 1. (a) Mean particle size of nanoparticulate curcumin measured by particle size analyzer (hydrodynamic diameter ¼ 192 6.59 nm, n ¼ 3). (b) Transmission electron micrograph of nanoparticulate curcumin (bar ¼ 0.185 mm). (c) Size distribution of nanoparticulate curcumin as measured by AFM. (d) The three dimensional view of (c) (as taken by AFM). consideration. Native curcumin is in methanolic solution showed a distinct high absorbance peak at around 425 nm. The absorbance intensity of our nanoparticulate curcumin formulation showed its peaks close to the absorbance peak of native curcumin (Fig. 3a). This result confirmed the successful entrapment of curcumin within nanoparticulate curcumin. Similarly, consistent to curcumins absorbance spectra, the fluorescence spectra also exhibited similar trends. While observing the fluorescence spectra of curcumin (from 450 to 700 nm) with excitation wavelength 420 nm, we found that native curcumin in methanolic solution showed a sharp fluorescence peak at 522 nm (Fig. 3a, inset), but the fluorescence spectrum of nanoparticulate curcumin was shifted towards blue spectrum and showed a well defined peak at 491 nm. This blue shift could be due to binding of curcumin with in hydrophobic domain of GMO present in nanoparticulate curcumin formulation. 3.3. In vitro release kinetics While observing the in vitro release profile, we observed a biphasic release pattern of entrapped curcumin from our nanoparticulate curcumin formulation. Herein, we found a rapid release of about 46% in 24 h followed by a sustained drug release of about 66% over 10 days of our observation (Fig. 3b). The observed initial burst release might be due to the dissociation of surface absorbed drugs present in the polymeric matrix. Subsequently, sustained release activity of the drugs was due to the slow release of drugs entrapped inside the polymer matrix [24]. 3.4. Solubility and stability study of curcumin To confirm the solubility of our formulation, we found nanoparticulate curcumin dissolved in aqueous solution gave a clear, well dispersed formulation with curcumins natural color (Fig. 4a (ii)), in contrast native curcumin is poorly soluble in aqueous media with microscopic undissolved flakes of the compound visible in the solution (Fig. 4a(i)). One of the major challenges of drug delivery to cancerous tissue is its instability and biodegradation in physiological pH [1,18,36]. In an attempt to study the biodegradation and instability properties of curcumin, we incubated curcumin (native and nanoparticulate curcumin) in PBS (0.01 M, pH ¼ 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, nanoparticulate curcumin was stable under the same condition (e90%) (Fig. 4b). Thus it is noteworthy that our formulation increased the stability of curcumin in PBS by protecting the encapsulated curcumin against hydrolysis and biotransformation. 3.5. Toxicity studies of void polymeric nanoparticle In order to exclude the possibility of lethality shown from majority of polymeric constituents of NPs, we have supplementary evaluated the toxicity profile of void NPs in PANC-1 cell line [19]. The noncytotoxicity test confirmed the treated cell did not show any obstruction in cell proliferation and illustrated the similar trend Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 6 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 Fig. 2. (a) FTIR spectra of (i) native curcumin, (ii) nanoparticulate curcumin and (iii) void nanoparticles. (b) DSC thermogram of (i) native curcumin, (ii) void nanoparticles and (iii) nanoparticulate curcumin. (c) XRD pattern of (i) native curcumin, (ii) nanoparticulate curcumin and (iii) void nanoparticles. of cell population as observed by control cell (Fig. 5a). 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 nontoxicity feature was further confirmed by TNF-a assay (Fig. 5b). The result explained same trend release of TNF-a from void treated cell as well as control cell. It showed the formulated void NPs were not able to elicit the production of pro-inflammatory cytokines TNF-a, indicating noncytotoxic feature of the void NPs. 3.6. 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 nanoparticulate curcumin on pancreatic cell line [13]. Herein, PANC-1 pancreatic cell line was treated with curcumin (both native curcumin and nanoparticulate curcumin) at a dose of 15 mM for 7 days. The result showed that nanoparticulate curcumin profoundly inhibited the pancreatic colony formation compared to the colony observed from native curcumin treated cell (Fig. 6). This suggests that curcumin entrapped in nanoparticulate curcumin has comparative better antiproliferative activity as it was effectively blocked the clonogenicity of PANC-1 cell compared to native treated cell. 3.7. Cellular uptake studies Taking the advantage of photochemical properties of curcumin, the intracellular uptake of nanoparticulate curcumin was compared with native curcumin by fluorescence spectroscopy. The result of quantitative cellular uptake demonstrated nanoparticulate curcumin was internalized more efficiently by PANC-1 cell as compared to native curcumin (Fig. 7a). By measuring the fluorescence intensity of curcumin, a concentration dependent increase in cellular uptake of nanoparticulate curcumin and native curcumin was observed. However, cellular uptake of nanoparticulate curcumin at lower concentration, i.e. at dose 5 and 10 mM was 5.9 and 7.7 times Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 7 Fig. 3. (a) UltravioleteVisible absorbance spectra of native curcumin and nanoparticulate curcumin at a fixed concentration of 3 mg/ml. (i) Native curcumin in methanolic solution, (ii) nanoparticulate curcumin in aqueous solution. The inset corresponds to the fluorescence emission spectra of methanolic solution of native curcumin and aqueous solution of nanoparticulate curcumin when excited at 420 nm. (b) In vitro release kinetics of curcumin from nanoparticulate curcumin formulation in PBS (0.01 M, pH 7.4) at 37 C. Data as mean s.e.m, n ¼ 3. more than native curcumin while at higher concentrations (30 mM) 4.08 times increase in uptake values was observed in comparison to native curcumin. This result shows that at lower concentration the nanoparticulate curcumin uptake is more effective. Similar results were also observed by Sahu et al. where they have reported a concentration dependent increase in cellular uptake of native curcumin and curcumin encapsulated in bovine casein micelle in HeLa cell line [37]. Further, we have observed the intracellular uptake of native curcumin and nanoparticulate curcumin after 1 h of incubation qualitatively by microscopic (fluorescence and confocal microscope) observation in the same cell line at 10 mM concentration (Fig. 7b). The microscopic studies demonstrated the cells treated with nanoparticulate curcumin showed profound fluorescence intensity compared to the cells treated with native, indicating NPs internalized more efficiently by the cells than native curcumin. The time dependant cellular uptake studies of curcumin (both native and nanoparticulate curcumin) observed from confocal image studies, demonstrated the fluorescence intensity was initially restricted to cell membrane as shown by 30 min treated cell, while with time it enhanced and extended to cytoplasm (Fig. 7c). Cell treated with native curcumin showed maximum fluorescence 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 can slowly released from NP for a longer period of time. 3.8. In vitro mitogenic assay Therapeutic efficacy of curcumin (native and nanoparticulate curcumin) was investigated in different cell line by mitogenic assay [24]. All the studied cell line showed a typical dose dependant sigmoidal antiproliferative effect (Fig. 8). The in vitro half maximal inhibitory concentration (IC50) is the quantitative measurement for the cell toxicity induced by chemotherapeutic drug. This IC50 values were calculated from the obtained sigmoidal curves of all the studied cell line and the result demonstrated nanoparticulate curcumin has higher antiproliferative activity than native (Table 1). Nanoparticulate curcumin ise1.52, 3.4, 1.23, 2.05, 1.87 and 2.48 times more effective than native curcumin as observed in PANC-1, MIA PaCa-2, K562, MCF 7, HCT-116 and A549 cell line respectively. The obtained results demonstrated comparable inhibition of cell proliferation, where nanoparticulate curcumin was more effective than native in solution by controlling the tumor cell growth. Fig. 4. (a) Solubility study of curcumin (native and in formulation) in PBS. (I) Native curcumin (10 mg) dissolved in PBS (0.01 M, pH 7.4) was insoluble in aqueous solution. (II) Equivalent quantity of nanoparticulate curcumin was fully soluble in aqueous solution. (b) Stability of curcumin (native and nanoparticulate curcumin) in PBS (0.01 M, pH 7.4) at 37 C. Data as mean s.e.m, n ¼ 6. Native curcumin, nanoparticulate curcumin. 3.9. Apoptosis analysis by flow cytometry Available evidence suggests that apoptosis may represent a mechanism to counteract neoplastic development, which is Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 8 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 Fig. 5. (a) In vitro toxicity study of void nanoparticle, showing no morphological changes of its treated cell (PANC-1) compared to control cell as observed under confocal microscopy. (b) Toxicity study of void nanoparticle by measuring inflammatory response in TNF-a assay. (i) Anti-inflammatory response of different concentration of void nanoparticle (0.1e0.5 mg/ml) in PANC-1 cells after 24 h of incubation. (ii) Anti-inflammatory response of 0.5 mg/ml of void nanoparticle in PANC-1 cells incubated for different time periods. essential for cancer therapy [38,39]. 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 [36,39]. To determine the ability of curcumin for promoting apoptosis in PANC-1 cell line, we have investigated its apoptosis inducing efficiency by staining the cells with annexin V-FITC [24,36]. On treated cells, we have reported the presence of early apoptotic, advanced apoptotic and necrotic cell population (Fig. 9). However, the nanoparticulate curcumin treated cell showed more number of cell, i.e. 22.37% in apoptosis as compared to 5.81% of cell found in native curcumin treated cells. This result suggests that nanoparticulate curcumin treated PANC-1 cell showed 3.8 times more apoptosis compared to native curcumin. 3.10. Western blot analysis Curcumin induced apoptosis activity has been well studied before and it is mostly due to inhibition of the AkteNFkB signaling pathway (Fig. 10) [40e42]. To substantiate our finding, we have studied the molecular basis of apoptosis by accessing the AkteNFkB pathway. Our western blot results as shown in Fig. 11 demonstrated a decrease in phosphorylation of Akt (band at 60 kDa) in curcumin treated cell compared to control cell. It was further observed that the band intensity was decreased more in nanoparticulate curcumin treated cell compared to native curcumin treated cell, indicating more inhibition of Akt phosphorylation in cell treated with nanoparticulate curcumin. Similarly, our result demonstrated high intense NFkB (at 64 kDa) and IkBa (at 34 kDa) band in curcumin treated cell compared to untreated cells. Nanoparticulate curcumin treated cell further intensified the NFkB and IkBa bands compared to native curcumin treated cell suggesting the nanoparticulate curcumin 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 antiapoptosis respectively in tumor cells. In this view, we observed less intense cyclin-D (at 36 kDa) and c-Myc (at 65 kDa) bands in nanoparticulate curcumin treated cell compared to control Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 9 Fig. 6. Nanoparticulate curcumin inhibits the clonogenic potential of pancreatic cancer cell lines. Colony assays in soft agar were performed comparing the effects of native and nanoparticulate curcumin in inhibiting the clonogenicity of the pancreatic cancer cell line, PANC-1. Representative plates are illustrated for (i) control cells, (ii) void nanoparticletreated cells, (iii) native curcumin-treated cells and (iv) nanoparticulate curcumin-treated cells. The last two plates were treated at the equivalent of 15 mM curcumin dosage. Data as mean s.e.m, n ¼ 3. and native curcumin treated cell. These results supported our postulate that nanoparticulate curcumin more efficiently blocks the NFkB activation and NFkB regulated gene expression through inhibition of IkBa and Akt activation compared to native curcumin. 3.11. In vivo pharmacokinetic study Nanoparticulate curcumin was designed with a notion to improve the systemic bioavailability of delivered curcumin. In this view, the nanoparticulate curcumin 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 Fig. 12. Result showed maximum serum availability of 25 mg/ml curcumin was observed after 1 h of nanoparticulate curcumin administration where as at same duration the availability was 0.02 mg/ml in native curcumin administered case. Furthermore, it was observed that unlike native curcumin, curcumin was detected for a long duration time period as observed after 24 h of its administration in our formulation. Comparatively Anand et al. demonstrated e0.28 mg/ml curcumin bioavailability with PLGA NP ande0.008 mg/ml was reported by Shaikh et al. after 1 h administration of same polymeric NP compared to the native curcumin Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 10 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 Cellular uptake ng/ 50000 cells) a 35 ** 30 ** 25 ** 20 15 10 ** 5 0 5 10 20 Curcumin (µM) Native curcumin b 30 Nanoparticulate curcumin Nanoparticulate curcumin Native Curcumin Fluorescence microscopic image Confocal microscopic image c 0.5 h 8h 24 h Nanoparticulate curcumin Native Curcumin Fig. 7. (a) Cellular uptake study of native curcumin ( ) and nanoparticulate curcumin (-) on PANC-1 cells. Data as mean s.e.m., n ¼ 3. (**) p < 0.005. (b) Microscopic observation of PANC-1 cell treated with 10 mM curcumin (both native and nanoparticulate curcumin) and after 1 h of incubation showing maximum fluorescence intensity in nanoparticulate curcumin treated cell. (c) Confocal image showing time dependent increase of intracellular fluorescence intensity in nanoparticulate curcumin treated PANC-1 cell due to 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. Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 100 PANC-1 b ** % Viability 80 ** ** 60 ** ** 40 ** 40 0 ** ** 5 10 15 20 Conc. (µM/ml) 25 0 30 d ** ** ** ** 60 40 ** 0 5 10 15 20 25 30 35 40 Conc. (µM/ml) f ** % Viability ** 40 ** 20 ** 5 5 10 15 20 25 30 35 40 Conc. (µM/ml) ** 10 15 20 25 30 35 40 Conc. (µM/ml) HCT-116 100 ** 60 ** ** 40 20 0 0 0 ** ** 80 ** 60 ** 0 A549 80 MCF 7 40 0 ** ** ** 30 60 20 100 10 15 20 25 Conc. (µM/ml) 100 20 0 5 80 % Viability ** 80 % Viability ** 60 0 K562 % Viability ** 80 20 100 e MIA PaCa-2 ** 100 20 0 c % Viability a ** 11 0 5 ** ** ** ** 10 15 20 25 30 35 40 Conc. (µM/ml) Fig. 8. Dose dependent cytotoxicity of void nanoparticle (-), native curcumin (), and nanoparticulate curcumin (:) 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 5th day by the MTT assay. The inhibition was calculated with respect to respective controls. Data as mean s.e.m., n ¼ 6. (**) p < 0.005, native curcumin in solution versus nanoparticulate curcumin. [12,18]. Our result suggested the slow release of curcumin from nanoparticulate curcumin formulation increased the bioavailability of delivered curcumin with time as observed for 24 h of our observation. Whereas, in native case the level was subsequently Table 1 IC50 values of native curcumin and nanoparticulate curcumin in different tumor cells as assayed by MTT cytotoxicity assay. Tumor cells Native curcumin IC50 values (mM/ml) Nanoparticulate curcumin IC50 values (mM/ml) PANC-1 MIA PaCa-2 K562 MCF 7 HCT-116 A549 26.36 18.6 28.12 30.31 10.83 26.89 17.32 5.46 22.82 14.76 5.79 10.81 decreased with time and not detectable beyond 1 h, indicating rapid metabolism of native curcumin in physiological pH. 4. Discussion Nanoparticulate curcumin was developed to overcome major obstacles associated with curcumin delivery like poor solubility, rapid degradation and poor bioavailability. After successful formulation the physicochemical characterization of drug delivery system was taken in consideration, as it influences the physical stability, cellular uptake, biodistribution and release of encapsulated drug [24]. In this regard, the size distribution and surface charge of NPs were taken for observation. As we know small size of particles are advantageous for passive targeting to tumor tissue by enhanced permeability and retention effect [1,37] and higher zeta potential influence the particle stability, cellular uptake and Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 12 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 Fig. 9. Induction of apoptosis in PANC-1 cell line treated with a concentration of 6 mM/ml of native curcumin and nanoparticulate curcumin. 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. intracellular trafficking [34]. Hence, we can anticipate that the small size and high surface charge of our formulated nanoparticulate curcumin could have enhanced circulation half lives as well as evades the reticuloendothelial system (RES). Furthermore, the FTIR, DSC and XRD analysis clearly demonstrated the chemical integrity of curcumin and its interaction with the polymer, the result further confirmed the encapsulated curcumin retained its properties even inside the NPs. The amount of entrapped drug in the NP is an important factor for determining the therapeutic efficacy of drug delivery system. In this view, our spectroscopic results demonstrated the successful entrapment of curcumin in our nanoparticulate curcumin formulation. The blue shifted fluorescence spectrum of nanoparticulate curcumin further explained the successful binding of curcumin to the hydrophobic domain of GMO in nanoparticulate curcumin formulation. The studies conducted by Sahu et al. also reported, curcumin encapsulated bovine casein micelle showed similar type of shift in fluorescence spectra from 540 nm to 500 nm due to binding of curcumin in hydrophobic domain of protein molecule in bovine micelle [37]. This binding can be feasible by certain molecular interaction for example, it was reported that hydrophobic fluorescence probe like 8-Anilino-1naphthalenesulfonate (ANS), 2-p-toluidinylnaphthalene-6-sulfonate (TNS), Nile Red and Pyrene strongly bind to the bovine casein micelle through hydrophobic and electrostatic interaction [37]. Here, we attributed same type of interaction could be exist with curcumin and hydrophobic domain of GMO in our curcumin delivery system. The major challenges of curcumin delivery in therapeutics grounds involves while defining its stability. In this regard, while demonstrating the stability of entrapped curcumin, our results showed unlike native curcumin the curcumin encapsulated within nanoparticulate curcumin was dramatically retained its stability in PBS (0.01 M, pH ¼ 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 ¼ 7.4) and approximately 30.41% of native curcumin remained intact after 20 min of incubation [36]. Similarly, Wang et al. reported 90% of native curcumin degraded after 30 min in phosphate buffer (0.01 M, pH 7.4) [43]. 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 [4,5,11,36]. Hence, it can be said our formulated carrier system can efficiently increased the stability of curcumin even in PBS by protecting the encapsulated curcumin against hydrolysis and biotransformation for a longer time. In spite of several formulation challenges some formulation strategy with this carrier system has already been developed to deliver anticancer drug. As example, GMO/polyxamer 407 cubic NPs 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 h [44]. While assessing the in vitro release profile of our formulation, we observed a biphasic release pattern of encapsulated curcumin from nanoparticulate curcumin. The initial burst release is due to discharge of surface bound drug molecule present in the polymer matrix of nanoparticulate curcumin [18,25]. At later stages the slow and sustained release of the drug can be attributed due to partitioning of drug in the hydrophobic core of GMO and subsequently diffusion/erosion of polymeric matrix made the drug released from nanoparticulate curcumin to the aqueous medium. Studies conducted by Trickler et al. in chitosan/GMO NPs observed the similar trends of release for paclitaxel and dexamethasone Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 13 Fig. 10. Schematic representation of molecular mechanism of apoptosis induced by curcumin. Reports from earlier studies support the hypothesis that Akt/NFkB pathway contributes to tumor cell survival in PANC-1 cancer cell. Activated Akt mediates oncogenesis through phosphorylation/activation of NFkB, which in turn positively regulates cell growth and apoptosis. Internalization of nanoparticulate curcumin result in sustained release of drug curcumin resulting in blocking of NFkB pathway. showing an initial burst release ofe10% ande45% respectively in 1 h, followed by a slower and constant release thereafter [20]. Toxicity studies of void polymeric NPs were performed to evaluate the preliminary safety profile of our drug delivery carrier. The toxicity induced by polymeric NP, usually showed a typical signs of apoptosis like blebbing of the nucleus and condensation of the chromatin, etc. [45]. However, these aberrations were not at all observed in our void treated cell confirming its noncytotoxicity. Its nontoxicity was further confirmed by getting the same pattern of TNF-a released trend from treated as well as from control cell. Here, TNF-a was taken as a parameter to quantify the toxicity induced by void NPs. As TNF-a is released from cells when the cells are damaged so, here it is taken as a marker cytokines for inflammation, which promotes antitumor and immune responses [31]. Cellular uptake study is an important parameter that needs to be explained for justifying successful drug delivery of our formulation to cancer tissue. In vitro cellular uptake study demonstrated native curcumin treated cell showed maximum fluorescence initially (for few hour of treatment) but gradually the fluorescence intensity was decreased with time as observed after 8 and 24 h of incubation by PANC-1 cells. In contrast, we observed the subsequent enhanced uptake (as studied qualitative and quantitative experiments) of our formulation with time by PANC-1 cell. Though cellular uptake studies demonstrated a comparative better uptake of nanoparticulate curcumin over native curcumin but other factor like its cell cytotoxicity study is the vital parameter which needs to be investigated for evaluating the pharmacological activity of drug. Our MTT results confirmed that nanoparticulate curcumin demonstrated a lower IC50 values when compared to native curcumin, as studied in different cancer cell lines (Table 1). This could be due to difference in uptake profile which might be the reason for better antiproliferative activity of nanoparticulate curcumin. Further, it is well known that the antiproliferative effect of drug is very well correlated with the duration of its intracellular retention and drugs stability [36]. While observing the mode of internalization of native drug in to cell, it always diffuses across cell membrane (when used as a solution) [24]. So, after attaining saturation inside the cytoplasm further entry will be restricted. These small fractions of internalized native drug showed its antiproliferative effect for a short time of its existence. However, the mode of entry of nanoparticle to the cell through endocytosis make sufficient availability of nanoparticulate curcumin inside the cell and release the encapsulated curcumin 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 clearly confirms the fact that curcumin eliciting apoptosis signals in a varied number of tumor tissues including colorectal, lung breast, pancreatic and prostrate carcinoma [6,38,46]. Our result supported 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 5.57% of cell in necrotic stage and. 5.81 3.31% of cell in apoptosis stage. However, nanoparticulate curcumin treated cells showed less number of cells, i.e. 32.39 4.32% in necrosis and more number of cells, i.e. 22.37 3.92% 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 nanoparticulate curcumin 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 resulting in reduction of cell viability, as observed in MTT assay. Apoptosis induction properties of curcumin have been attributed to its ability to inhibit COX-2 Please cite this article in press as: Mohanty C, Sahoo SK, The in vitro stability and in vivo..., Biomaterials (2010), doi:10.1016/j. biomaterials.2010.04.062 ARTICLE IN PRESS 14 C. Mohanty, S.K. Sahoo / Biomaterials xxx (2010) 1e15 Concentration of curcumin (µg/ml) 30 25 Native Nanoparticulate curcumin 20 15 10 5 0 0 5 10 15 Time (Hrs) 20 25 Fig. 12. In vivo bioavailability of native curcumin and nanoparticulate curcumin. The mice were divided in to two groups (n ¼ 4). Equivalent concentration of native curcumin and nanoparticulate curcumin (30 mg/kg) was given to group 1 and group 2 mice respectively. Curcumin and nanoparticulate curcumin were administered intravenously and blood was collected at different time intervals. Serum was separated and the concentration of curcumin was determined by HPLC analysis, as described in Materials and methods. Fig. 11. In vitro apoptosis inducing and cell cycle inhibiting activity of curcumin on pancreatic cancer cells. PANC-1 were exposed to 6 mM/ml curcumin (both in native and in formulation) for 24 h and the targets were detected by immunoblotting with specific antibodies mentioned in Materials and methods. because it is well known that curcumin is a natural COX-2 inhibitor [38,39,47]. Further, we hypothesized the cytoplasmic NFkB may induce apoptosis in curcumin treated tumor cell. Previously it was reported that curcumin showed potent antiproliferative activity by inhibiting NFkB DNA binding motion in tumor cell including pancreatic cancer cell [40,41]. In this regards, consistent to apoptosis result our western blot analysis confirmed our proposition that NFkB pathway was inhibited by curcumin in pancreatic tumor, as observed in PANC-1 cell. Schlieman et al. reported Akt (a target of PI3K) was phosphorylated and activated in variety of pancreatic cell line including PANC-1 cell [48]. Our finding also suggests curcumin dephosphorylated Akt, which consequently inhibited NFkB signaling pathway. Whereas, NFkB is a transcription factor present in the cytoplasm, as an inactive heterodimer consisting of p50, p65 and IkBa subunits. So, curcumin restrained Akt activation and consequently blocked phosphorylation of IkBa and p65. Which in turn inhibited the activation and translocation of NFkB in to nucleous and as well as transcription of NFkB regulated gene. Our results confirmed the presence of more cytosolic NFkB (in an inactive state) in curcumin treated cell. However, in untreated cell NFkB gets translocated in to nucleous and hence less intense band was observed. Similarly, high intense band of IkBa in nanoparticulate curcumin treated cell confirmed the presence of more IkBa in cytosol compared to native treated cell and thus more inhibition of NFkB pathway. Here we attribute the better uptake and stability of curcumin encapsulated in nanoparticulate curcumin, 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 nanoparticulate curcumin is to improve in vivo bioavailability of curcumin. Interestingly, our results showed, nanoparticulate curcumin ise32- and 1000-folds more bioavailable compared to native curcumin after 30 min and 60 min observation respectively. Similar type of observation related to In vivo pharmacokinetics has been reported by various groups [11,14,18,49,50]. As evidenced from our in vitro and in vivo studies, we further anticipated our formulation will be a suitable delivery system for curcumin to the animals. Hence, the discussed comprehensible results justified nanoparticulate curcumin hold better chemopreventive, chemotherapeutic properties than native curcumin due to its better bioavailability and it consequently exert induction of apoptosis in tumor cells, advocating their potential use as a better strategy for cancer control. 5. Conclusion 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 nanoparticulate curcumin 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 nanoparticulate curcumin was comparatively more effective than native curcumin under in vitro condition with time due to greater cellular uptake, sustained intercellular drug retention and enhanced antiproliferative effect by inducing apoptosis. Most importantly, the enhanced cellular internalization and sustained release of entrapped curcumin in our formulation results its enhanced systemic bioavailability. Thus, the nanoparticulate curcumin 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. Appendix Figures with essential color discrimination. Figs. 1, 2, 4e7 in this article are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2010.04.062. 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