Evaluation of a nanotechnology-based carrier for delivery of curcumin in prostate cancer cells

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Evaluation of a nanotechnology-based carrier for delivery
of curcumin in prostate cancer cells
RAJESH L. THANGAPAZHAM1,2, ANU PURI3, SHRIKANT TELE3,
ROBERT BLUMENTHAL3 and RADHA K. MAHESHWARI1
1Center for Combat Casualty and Life Sustainment Research, Department of Pathology, Uniformed Services University
of the Health Sciences (USUHS), Bethesda, MD 20814, USA; 2Birla Institute of Technology and Science,
Pilani 333031, India; 3CCR Nanobiology Program, National Cancer Institute at Fredrick, NIH, MD, USA
Received December 3, 2007; Accepted January 25, 2008
Abstract. We have initiated studies to enhance targeted
cancer chemopreventive in several different animal tumor bio-
delivery of an anticancer agent, curcumin, for prostate cancer
assay systems including colon (2,3), duodenal (4), stomach
treatment by incorporating this agent into the liposomes
(5), prostate (6) and breast (7) carcinogenesis both in vitro
(nanodelivery vehicles primarily composed of phospholipids)
and in vivo. Absence of dose limiting toxicity, when curcumin
coated with prostate membrane specific antigen specific
is administered up to 8 g/day in human clinical trials, reveals
antibodies. We prepared curcumin-loaded liposomes of
the possibility of curcumin in the prevention and treatment
various lipid compositions by sonication at an average size
of cancer (8,9).
of 100-150 nm. Un-entrapped curcumin was removed by size
In spite of its promising therapeutic index, the biological
exclusion chromatography. Data show that curcumin prefer-
activity of curcumin is severely limited due to its poor bio-
entially partitioned into liposomes prepared from dimyristoyl
availability (10). Effective methods to deliver chemo-
phosphatidyl choline (DMPC) and cholesterol among the
therapeutics to solid tumors and/or to increase their bio-
various compositions tested. The anti-proliferative activity of
availability have been a major challenge in current biomedical
liposomal curcumin was studied using two human prostate
research. Nanotechnology-based tools and techniques are
cancer cell lines (LNCaP and C4-2B) by a tetrazolium dye-
rapidly emerging in the fields of medical imaging and targeted
based (MTT) assay. Treatment of cells with liposomal
drug delivery (11). Among various drug delivery vehicles,
curcumin (5-10 ?M) for 24-48 h at 37?C resulted in at least
liposomes have been explored for decades due to their bio-
70-80% inhibition of cellular proliferation without affecting
degradability and the potential to load large concentrations of
their viability. On the other hand, free curcumin exhibited
therapeutic agents. Liposomes have the capacity to alter the
similar inhibition only at 10-fold higher doses (>50 ?M).
biodistribution of drugs they encapsulate through delayed
We also observed that LNCaP cells were relatively more
clearance and longer intravascular circulation time (12). The
sensitive to liposomal curcumin mediated block of cellular
currently undergoing trials for various liposome formulations
proliferation than C4-2B cells. We are currently developing
and the large number of commercially available therapeutics
liposome formulations with targeting ability to further
appear promising (13). Since curcumin is hydrophobic, it is
improve the efficacy of curcumin in vivo.
considered to be a good candidate for liposome incorporation
as it can be encapsulated in the lipid layer of the liposomes
Introduction
(14). Recent studies have shown that the incorporation of
curcumin into liposomes has increased the bio-availability
Turmeric (Curcuma longa Linn), is a crystalline compound
of curcumin significantly (14,15). In a study conducted on
which has been traditionally used in medicine and cuisine
human pancreatic carcinoma cells, the activity of liposomal
in India. Curcumin (diferuloylmethane) is the major active
curcumin was equal to or better than that of free curcumin
component of turmeric (1). Curcumin has been shown to be
at equimolar concentrations. Liposomal curcumin down-
regulated NF-?B machinery, suppressed growth, and induced
apoptosis of human pancreatic cells in vitro. Antitumor
_________________________________________
and anti-angiogenic effects were also observed in vivo (15).
In another study, both liposomal and HSA (human serum
Correspondence to: Dr Radha K. Maheshwari, Department of
albumin) vehicles were examined for the transfer of curcumin
Pathology, Uniformed Services University of the Health Sciences,
to spleen lymphocyte cells of the EL4 cell line. From these
4301 Jones Bridge Road, Bethesda, MD 20814, USA
studies, it was found that the liposomal vehicle was capable
E-mail: rmaheshwari@usuhs.mil
of loading more curcumin into cells than the human serum
albumin (HSA) or the aqueous-DMSO vehicles (14).
Key words: nanotechnology, curcumin, prostate cancer
The aim of this study was to evaluate curcumin partitioning
potential into the liposomes composed of phospholipids with

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Figure 1. Lipid and curcumin structures. Egg phosphatidylcholine (EGG PC) cholesterol, dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl
phosphatidylcholine (DMPC).
a wide range of melting transition temperatures (Tm) and
curcumin film was then formed by removing the solvent using
optimize conditions for encapsulating curcumin and examine
a rotary evaporator. Any residual chloroform was removed
the efficacy of curcumin-loaded liposome formulations on
by placing the films overnight in a vacuum desiccator. Multi-
prostate cancer cells. We have chosen LNCaP and its isogenic
lamellar vesicles were formed by reconstituting the lipid film
more resistant derivative C4-2B as the in vitro model system.
with HBSE buffer (10 mM HEPES, 150 mM NaCl, 9.1 mM
The anti-proliferative effects of free curcumin and liposomal
EDTA, pH 7.5) with vigorous vortexing. Unilamellar vesicles
curcumin were studied using a tetrazolium dye-based (MTT)
were then formed using a probe sonicator W-375 (Heat
assay. Our studies show that curcumin selectively partitions
Systems-Ultrasonics, New York, USA) for 15-20 min on ice
with high efficiency into DMPC liposomes as compared to
(2-min pulse with 30-sec interval between each pulse). After
DPPC or egg PC. The high therapeutic index of liposomal
sonication, the liposomes were centrifuged at 2,000 x g to
curcumin when compared with free curcumin shows promise
pellet any curcumin not intercalated into the liposomes. The
for future experiments on targeted delivery.
liposomes were then passed through a size exclusion gel
chromatography column (Bio-Rad Biogel-A5M, Bio-Rad,
Materials and methods
Hercules, CA), equilibrated with HBSE buffer, pH 7.4, in
order to separate any residual curcumin loosely associated
Materials. Curcumin was obtained from LKT Laboratories
with the liposomes. Fractions containing liposomes were
(St. Paul, MN), dimyristoyl phosphatidylcholine (DMPC),
pooled and filtered through 22 ?m filter, stored at 4?C and
dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidyl-
used within 48 h for quantitation, sizing and further analysis.
choline (EGG PC) cholesterol (Fig. 1) and cholesterol were
obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Other
Quantification of curcumin. Liposomes containing curcumin
reagents were for Sigma-Aldrich Co. (St. Louis, MO).
were prepared as described above. Liposomal curcumin
was quantified using a simple colorimetric assay measured
Cell lines. Prostate cancer cell lines LNCaP and C4-2B cells
at 450 nm. A standard curve was formulated from known
were kind gifts from Dr Shiv Srivastava, CPDR, Rockville,
concentrations of curcumin HBSE-TX100 (10 mM HEPES,
MD, USA. Cultures of cells were maintained in RPMI-1640
140 mM NaCl, 4 mM EDTA, 1% TX-100) and was used to
supplemented with 10% (v/v) heat inactivated fetal bovine
determine curcumin concentration in liposomes subsequent
serum and 1X PSN Antibiotic Mixture (VWR, Bridgeport,
to lysis with 1% TX-100 (Fig. 2).
NJ). Cells were cultured at 37?C in a humidified atmosphere
Measurement of liposome size and charge was conducted
of 5% CO and 95% air.
2
by dynamic light scattering techniques by the National
Characterization Laboratory (Frederick, MD, USA) using
Liposome preparation and curcumin encapsulation. Lipo-
a Zeta nanosizer (Malvern, UK) (data not shown).
some [small unilamellar vesicles (SUV)] was prepared by
probe sonication. The lipids and curcumin were mixed at a
Cell proliferation assay. LNCaP and C4-2B cell proliferation
phospholipids:cholesterol:curcumin at a ratio of 90:10:10,
in the presence of various concentrations of liposomal and
wt:wt in chloroform in a round bottom flask and the lipid/
free curcumin was determined using the MTT (3-[4,5-

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on measurement of optical density (absorbance) of curcumin
at 450 nm in HBSE-TX100 (10 mM HEPES, 140 mM NaCl,
4 mM EDTA, 1% TX-100).
A stock solution of curcumin standard (10 mg/ml) was
prepared in ethanol (>99.9%). Immediately prior to measu-
rement, a working dilution of standard curcumin solution
(0.5 mg/ml) was prepared in HBSE-TX100. This working
dilution was used to generate a standard curve using a 96-well
plate format as follows: in a flat-bottom clear 96-well plate,
various aliquots (0-10 ?l) in duplicate were added to the
wells. Volumes were adjusted to 0.2 ml per well by addition
of HBSE-TX100 buffer. Curcumin absorbance was measured
at 450 nm in an ELISA plate reader (BioTek Instruments,
Inc., Winooski, VT). To determine curcumin entrapment in
liposomes, known volume(s) of curcumin-loaded liposomes
Figure 2. A standard curve for curcumin concentration was formed via a
were added to the wells, final volumes adjusted to 0.2 ml
96-well format based colorimetric assay. Known concentrations of free
using HBSE-TX100 and measurements conducted at 450 nm
curcumin or liposome-entrapped curcumin were measured at 450 nm in
as desribed above. Using the standard curve, we determined
HBSE-TX-100 (10 mM HEPES, 140 mM NaCl, 4 mM EDTA, 1% TX-100)
0.125 mg/ml curcumin in the liposomes. The liposomes used
as described in Materials and methods. Using the standard curve, 20 ?l
liposomes correlates to 2.5 ?g of curcumin to a concentration of 125 mg/ml
in this study were found to have an average particle diameter
curcumin in the liposomes.
of 100 nm as measured by dynamic light scattering.
Cytotoxicity induced by curcumin on LNCaP and C4-2B
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell
cells. DMPC liposomal curcumin (5-10 ?M) treatment for
proliferation kit (Boehringer Mannheim, Indianapolis, IN)
24-48 h at 37?C resulted in 70-80% inhibition of cellular
as per manufacturer's protocol. Briefly, cells were plated
proliferation. On the other hand, free curcumin exhibited
in 96-well tissue culture plates in a range of 3,000 cells/well
similar inhibition only at 10-fold higher doses (>50 ?M).
in a final volume of 100 ?l of medium and were allowed to
It was also observed that LNCaP cells were relatively
attach overnight. The cells were then treated once with varying
more sensitive to liposomal curcumin mediated block of
doses of liposomal and free curcumin with appropriate media
cellular proliferation than C4-2B cells. Of LNCaP cells, 31
control and were observed after 24 and 48 h. After completion
and 70% survived 10 ?M of liposomal and free curcumin
of the treatment, the cells were incubated with MTT for
treatment, whereas C4-2B cells were more resistant to
3-4 h at 37?C. Cells were lysed and the reduced intracellular
liposomal treatment, surviving around 36 and 75% with 10 ?M
formazan product was dissolved in the solubilization buffer
of liposomal and free curcumin treatment, respectively
provided in the kit. MTT is reduced to a colored water
(Fig. 3).
insoluble formazan salt only by metabolically active cells
DPPC and DMPC liposomal curcumin had improved
which is quantitated in a conventional ELISA plate reader
efficacy compared to free curcumin in inhibiting the
at 570 nm.
proliferation of prostate cancer cells (Fig. 4). However,
among the liposome tested, DMPC liposomal curcumin was
Results
found to be the most effective. Control liposomes showed
toxic effects (10-15%) at higher doses in our experiments
Curcumin encapsulation efficiency. We prepared curcumin-
which was consistent with earlier studies.
loaded liposomes of various lipid compositions by sonication
to an average size of about 100-150 nm. Our initial efforts
Discussion
to partition curcumin into EGG PC liposomes were not suc-
cessful. It was found that curcumin partitioned favorably
Chemoprevention is a promising preventive measure because
into DMPC based liposomes. Encapsulation efficiency
of its overall availability and affordability. It is conceivable
refers to the relative amounts of curcumin that intercalated
that in the future patients might only need to take specifically
into the liposomes. DMPC-based liposomes allowed the
formulated pills that prevent cancer or delay its onset.
greatest amount of curcumin to be intercalated into the
Although prostate cancer mortality has recently begun to
lipid membrane, whereas Egg PC-based liposomes had
decline in the US (2003), the cumulative costs of various treat-
the lowest amount of curcumin intercalation. The curcumin
ments for early stage disease, treatment-related morbidity
intercalation efficiency into DPPC liposomes was moderate.
and treatment of biochemical failures remains substantial
All the formulations were made at a 1:10 curcumin:lipid
(16). Chemoprevention by natural products such as edible
ratio (w/w basis). We conclude that curcumin partition
phytochemicals is a suitable alternative as an inexpensive,
into liposome is dependent on the type of lipid used.
readily applicable, acceptable and accessible approach to
cancer control and management (16). Hence, developing a
Curcumin quantification. Although fluorescence-based
phytochemical which has shown tremendous promise in vitro
quantitation methods are available for determining curcumin
and preclinical animal testing in a pharmaceutically acceptable
concentration, we have developed a colorimetric assay based
dosage form becomes pivotal.

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Figure 3. DMPC liposomal curcumin inhibits the proliferation of LNCaP
Figure 4. DPPC liposomal curcumin inhibits the proliferation of LNCaP
and C4-2B incubated for 24 and 48 h at 37?C. (A) Effect of different
and C4-2B incubated for 24 and 48 h at 37?C. (A) Effect of different
concentrations of liposomal curcumin on the proliferation of LNCaP. (B)
concentrations of liposomal curcumin on the proliferation of LNCaP. (B)
Effect of different concentrations of liposomal curcumin on the proliferation
Effect of different concentrations of liposomal curcumin on the proliferation
of C4-2B. The results were compared with equivalent amount of free
of C4-2B. The results were compared with equivalent amount of free
curcumin.
curcumin.
Although cancer chemoprevention studies and preclinical
those human tissues (20). It becomes critical for the drug
trials have shown curcumin as a favorable cancer chemo-
to reach the infection site and attain a pharmacologically
preventive agent in colon cancer and other gastrointestinal
desired concentration to be considered a potential drug
disorders, its efficacy in other organs is debatable due to its
candidate.
poor bioavailability. No curcumin was detected in the serum
Hence, it becomes pivotal to develop a suitable delivery
of human subjects administered up to 8 g/day of curcumin
agent and ways to increase the bioavailability of curcumin to
(17). Recent finding implicates rapid intestinal sulfation,
take advantage of curcumin's chemopreventive potential to
glucuronidation, and reduction, especially in humans to be a
various affected organs. Research is being pursued to increase
plausible explanation for the poor systemic availability of
the bioavailability of curcumin by various other means such
curcumin (18). This situation is probably analogous to the
as formulating curcumin with phosphatidylcholine and soy-
low bioavailability of drugs, such as the oral contraceptive
phospholipids (21,22).
ethinylestradiol, which is thought to be caused by extensive
Previous studies have reported successful use of various
sulfate conjugation. Another study from the same group
chemotherapeutic agents delivered in a liposome formulation
clearly indicates GIT as a more approvable target for curcumin
(23-25). In this study, we have considered the use of lipo-
as it is significantly exposed to unmetabolized curcumin
somes as a delivery model for curcumin. The results from the
and the metabolized byproducts of curcumin have reduced
cell proliferation assays provide strong evidence for liposomes
ability to inhibit COX-2 expression (19). Level of curcumin
as effective nanodelivery vehicles that increase the bio-
and its metabolites in portal and peripheral blood, bile and
availability of curcumin. Currently, strategies to conjugate
liver tissue as measured by HPLC was found to be so low
liposome formulations with prostate membrane specific
that it was unlikely to exert pharmacological activity in
antigen (PMSA) are being developed such that they can

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specifically target the prostate cancer cells to further improve
11. Dorai T, Cao YC, Dorai B, Buttyan R and Katz AE: Therapeutic
efficacy of curcumin in vivo.
potential of curcumin in human prostate cancer. III. Curcumin
inhibits proliferation, induces apoptosis and inhibits angio-
genesis of LNCaP prostate cancer cells in vivo. Prostate 47:
Acknowledgements
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12. Allen TM: Liposomes. Opportunities in drug delivery. Drugs 54
(Suppl 4): 8-14, 1997.
This work was supported by intramural grant from the
13. Goyal P, Goyal K, Vijaya Kumar SG, Singh A, Katare OP
Uniformed Services University of the Health Science, Bethesda
and Mishra DN: Liposomal drug delivery systems - clinical
applications. Acta Pharm 55: 1-25, 2005.
and US-INDIA Foreign Currency Fund from US Department
14. Kunwar A, Barik A, Pandey R and Priyadarsini KI: Transport
of State to USUHS. This research was supported (in part) by
of liposomal and albumin loaded curcumin to living cells:
the intramural research program of the NIH, NCI, Center for
an absorption and fluorescence spectroscopic study. Biochim
Biophys Acta 1760: 1513-1520, 2006.
Cancer Research. The authors are grateful to Mr. Anuj Sharma
15. Li L, Braiteh FS and Kurzrock R: Liposome-encapsulated
for his help and valuable suggestions. The opinions or
curcumin: in vitro and in vivo effects on proliferation, apoptosis,
assertions contained herein are the private views of the authors
signaling and angiogenesis. Cancer 104: 1322-1331, 2005.
16. Klein EA and Thompson IM: Update on chemoprevention
and should not be construed as official or necessarily reflecting
of prostate cancer. Curr Opin Urol 14: 143-149, 2004.
the views of the Uniformed Services University of the Health
17. Lao CD, Ruffin MT, Normolle D, Heath DD, Murray SI,
Sciences or the Department of Defense, USA.
Bailey JM, Boggs ME, Crowell J, Rock CL and Brenner DE:
Dose escalation of a curcuminoid formulation. BMC
Complement Altern Med 6: 10, 2006.
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