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Biomaterials xxx (2010) 1e15
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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.
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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.,
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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).
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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.
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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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References
[1] Das M, Mohanty C, Sahoo SK. Ligand-based targeted therapy for cancer tissue.
Expert Opin Drug Deliv 2009;6(3):285e304.
[2] Sahoo SK, Labhasetwar V. Nanotech approaches to drug delivery and imaging.
Drug Discov Today 2003;8(24):1112e20.
[3] Sahoo SK, Parveen S, Panda J. The present and future of nanotechnology in
human health care. Nanomedicine 2007;3(1):20e31.
[4] Anand P, Sundaram C, Jhurani S, Kunnumakkara AB, Aggarwal BB. Curcumin
and cancer: an “old-age” disease with an “age-old” solution. Cancer Lett
2008;267(1):133e64.
[5] Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 2003;23(1A):363e98.
[6] Fryer RA, Galustian C, Dalgelish AG. Recent advances and developments in
treatment strategies against pancreatic cancer. Curr Clin Pharmacol 2009;4
(2):102e12.
[7] Aggarwal BB, Sung B. Pharmacological basis for the role of curcumin in chronic
diseases: an age-old spice with modern targets. Trends Pharmacol Sci 2009;30
(2):85e94.
[8] Ammon HP, Wahl MA. Pharmacology of Curcuma longa. Planta Med 1991;57
(1):1e7.
[9] Hsu CH, Cheng AL. Clinical studies with curcumin. Adv Exp Med Biol
2007;595:471e80.
[10] Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, et al. Phase I clinical trial of
curcumin, a chemopreventive agent, in patients with high-risk or premalignant lesions. Anticancer Res 2001;21(4B):2895e900.
[11] Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of
curcumin: problems and promises. Mol Pharm 2007;4(6):807e18.
[12] Anand P, Nair HB, Sung B, Kunnumakkara AB, Yadav VR, Tekmal RR, et al.
Design of curcumin-loaded PLGA nanoparticles formulation with enhanced
cellular uptake, and increased bioactivity in vitro and superior bioavailability
in vivo. Biochem Pharmacol 2010;79(3):330e8.
[13] Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, et al. Polymeric
nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for
human cancer therapy. J Nanobiotechnol 2007;5:3.
[14] Maiti K, Mukherjee K, Gantait A, Saha BP, Mukherjee PK. Curcumin-phospholipid complex: preparation, therapeutic evaluation and pharmacokinetic
study in rats. Int J Pharm 2007;330(1e2):155e63.
[15] Preetha A, Banerjee R, Huilgol N. Tensiometric profiles and their modulation
by cholesterol: implications in cervical cancer. Cancer Invest 2007;25
(3):172e81.
[16] Suresh D, Srinivasan K. Studies on the in vitro absorption of spice principles e
curcumin, capsaicin and piperine in rat intestines. Food Chem Toxicol 2007;45
(8):1437e42.
[17] Tiyaboonchai W, Tungpradit W, Plianbangchang P. Formulation and characterization of curcuminoids loaded solid lipid nanoparticles. Int J Pharm
2007;337(1e2):299e306.
[18] Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MN. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when
compared to curcumin administered with piperine as absorption enhancer.
Eur J Pharm Sci 2009;37(3e4):223e30.
[19] Mohanty C, Acharya S, Mohanty AK, Dilnawaz F, Sahoo SK. Curcumin encapsulated MePEG/ PCL block copolymeric micelles: A novel controlled delivery
vehicle for cancer therapy. Nanomedicine (Lond) 2010;5(3):433e49.
[20] Trickler WJ, Nagvekar AA, Dash AK. A novel nanoparticle formulation for
sustained paclitaxel delivery. AAPS PharmSciTech 2008;9(2):486e93.
[21] Trickler WJ, Nagvekar AA, Dash AK. The in vitro sub-cellular localization and
in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.
Pharm Res 2009;26(8):1963e73.
[22] Wyatt DM, Dorschel D. A cubic-phase delivery system composed of glyceryl
monooleate and water for sustained release of water-soluble drugs. Pharm
Tech 1992;October:116.
[23] Barakat NS. Magnetically modulated nanosystems: a unique drug-delivery
platform. Nanomedicine 2009;4(7):799e812.
[24] Acharya S, Dilnawaz F, Sahoo SK. Targeted epidermal growth factor receptor
nanoparticle bioconjugates for breast cancer therapy. Biomaterials 2009;30
(29):5737e50.
[25] Misra R, Acharya S, Dilnawaz F, Sahoo SK. Sustained antibacterial activity of
doxycycline-loaded poly(D,L-lactide-co-glycolide) and poly(epsilon-caprolactone) nanoparticles. Nanomedicine 2009;4(5):519e30.
[26] Mu L, Seow P, Ngo A, Shen F. Study on surfactant coating of polymeric
nanoparticles for controlled delivery of anticancer drug. Colloid Polym Sci
2004;283(1):58e65.
[27] Kabanov AV, Batrakova EV, Alakhov VY. Pluronic block copolymers for
overcoming drug resistance in cancer. Adv Drug Deliv Rev 2002;54
(5):759e79.
15
[28] Sahu A, Bora U, Kasoju N, Goswami P. Synthesis of novel biodegradable and
self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for
curcumin delivery to cancer cells. Acta Biomater 2008;4(6):1752e61.
[29] Hafeli UO, Riffle JS, Harris-Shekhawat L, Carmichael-Baranauskas A, Mark F,
Dailey JP, et al. Cell uptake and in vitro toxicity of magnetic nanoparticles
suitable for drug delivery. Mol Pharm 2009;6(5):1417e28.
[30] Dilnawaz F, Singh A, Mohanty C, Sahoo SK. Dual drug loaded superparamagnetic iron oxide nanoparticles for targeted cancer therapy. Biomaterials 31(13):3694e706.
[31] Zhang S, Chen X, Gu C. The effect of iron oxide magnetic nanoparticles on
smooth muscle cells. Nanoscale Res Lett 2008;4(1):70e7.
[32] Kunwar A, Barik A, Mishra B, Rathinasamy K, Pandey R, Priyadarsini KI.
Quantitative cellular uptake, localization and cytotoxicity of curcumin in
normal and tumor cells. Biochim Biophys Acta 2008;1780(4):673e9.
[33] Sahoo SK, Ma W, Labhasetwar V. Efficacy of transferrin-conjugated paclitaxelloaded nanoparticles in a murine model of prostate cancer. Int J Cancer
2004;112(2):335e40.
[34] Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol
associated with poly(D,L-lactide-co-glycolide) nanoparticles affects their
physical properties and cellular uptake. J Control Release 2002;82(1):105e14.
[35] Pan CJ, Tang JJ, Shao ZY, Wang J, Huang N. Improved blood compatibility of
rapamycin-eluting stent by incorporating curcumin. Colloids Surf B Biointerfaces 2007;59(1):105e11.
[36] Ma Z, Haddadi A, Molavi O, Lavasanifar A, Lai R, Samuel J. Micelles of poly
(ethylene oxide)-b-poly(epsilon-caprolactone) as vehicles for the solubilization, stabilization, and controlled delivery of curcumin. J Biomed Mater Res A
2008;86(2):300e10.
[37] Sahu A, Kasoju N, Bora U. Fluorescence study of the curcuminecasein micelle
complexation and its application as a drug nanocarrier to cancer cells. Biomacromolecules 2008;9(10):2905e12.
[38] Lev-Ari S, Vexler A, Starr A, Ashkenazy-Voghera M, Greif J, Aderka D, et al.
Curcumin augments gemcitabine cytotoxic effect on pancreatic adenocarcinoma cell lines. Cancer Invest 2007;25(6):411e8.
[39] Lev-Ari S, Strier L, Kazanov D, Elkayam O, Lichtenberg D, Caspi D, et al. Curcumin synergistically potentiates the growth-inhibitory and pro-apoptotic
effects of celecoxib in osteoarthritis synovial adherent cells. Rheumatology
(Oxford) 2006;45(2):171e7.
[40] Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S, Aggarwal BB.
Curcumin (diferuloylmethane) down-regulates expression of cell proliferation
and antiapoptotic and metastatic gene products through suppression of
IkappaBalpha kinase and Akt activation. Mol Pharmacol 2006;69(1):195e206.
[41] Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, BuesoRamos CE, et al. Curcumin suppresses the paclitaxel-induced nuclear factorkappaB pathway in breast cancer cells and inhibits lung metastasis of human
breast cancer in nude mice. Clin Cancer Res 2005;11(20):7490e8.
[42] Siwak DR, Shishodia S, Aggarwal BB, Kurzrock R. Curcumin-induced antiproliferative and proapoptotic effects in melanoma cells are associated with
suppression of IkappaB kinase and nuclear factor kappaB activity and are
independent of the B-Raf/mitogen-activated/extracellular signal-regulated
protein kinase pathway and the Akt pathway. Cancer 2005;104(4):879e90.
[43] Wang YJ, Pan MH, Cheng AL, Lin LI, Ho YS, Hsieh CY, et al. Stability of curcumin
in buffer solutions and characterization of its degradation products. J Pharm
Biomed Anal 1997;15(12):1867e76.
[44] Lai J, Chen J, Lu Y, Sun J, Hu F, Yin Z, et al. Glyceryl monooleate/poloxamer 407
cubic nanoparticles as oral drug delivery systems: I. In vitro evaluation and
enhanced oral bioavailability of the poorly water-soluble drug simvastatin.
AAPS PharmSciTech 2009;10(3):960e6.
[45] Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev
Cytol 1980;68:251e306.
[46] Ganta S, Amiji M. Coadministration of Paclitaxel and curcumin in nanoemulsion formulations to overcome multidrug resistance in tumor cells. Mol
Pharm 2009;6(3):928e39.
[47] Goel A, Boland CR, Chauhan DP. Specific inhibition of cyclooxygenase-2 (COX2) expression by dietary curcumin in HT-29 human colon cancer cells. Cancer
Lett 2001;172(2):111e8.
[48] Schlieman MG, Fahy BN, Ramsamooj R, Beckett L, Bold RJ. Incidence, mechanism and prognostic value of activated AKT in pancreas cancer. Br J Cancer
2003;89(11):2110e5.
[49] Ma Z, Shayeganpour A, Brocks DR, Lavasanifar A, Samuel J. High-performance
liquid chromatography analysis of curcumin in rat plasma: application to
pharmacokinetics of polymeric micellar formulation of curcumin. Biomed
Chromatogr 2007;21(5):546e52.
[50] Marczylo TH, Verschoyle RD, Cooke DN, Morazzoni P, Steward WP, Gescher AJ.
Comparison of systemic availability of curcumin with that of curcumin
formulated with phosphatidylcholine. Cancer Chemother Pharmacol 2007;60
(2):171e7.
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