International Journal of Pharmaceutics 469 (2014) 3139

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Transdermal delivery of the in situ hydrogels of curcumin and its

inclusion complexes of hydroxypropyl-b-cyclodextrin for melanoma
treatment
Yunbo Sun a,1, Lina Du a,1, Yangpu Liu a , Xin Li b , Miao Li a , Yiguang Jin a, *,
Xiaohong Qian a
a
b

Beijing Institute of Radiation Medicine, Beijing 100850, China

Chinese PLA General Hospital, Beijing 100853, China

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 26 January 2014
Received in revised form 5 April 2014
Accepted 15 April 2014
Available online 18 April 2014

Curcumin (Cur) is a hydrophobic polyphenol with diverse pharmacological effects, especially for cancer
treatment. However, its weak water solubility and stability was the major obstacle for the formulation
research of Cur. The complexation of Cur and hydroxypropyl-b-cyclodextrin (HP-b-CD) was done by
grinding. The increasing solubility of Cur was achieved due to complexation and the photochemical
stability of Cur was improved. The inclusion of Cur could happen when two ends of Cur were embedded
into the cavity of the HP-b-CD rings. The in situ hydrogels (ISGs) of Cur and its inclusion complexes were
prepared using poloxamers 407 and 188 as the matrix. The extent of drugs in vitro release from the ISGs
depended on the dissolution of drugs. Both of the ISGs had transdermal effect and cytotoxicity on B16-F10
cells. However, the effects of the ISGs containing Cur inclusion complexes were much higher than those of
Cur ISGs because of the improved Cur solubility in the former. The cytotoxicity of Cur on melanoma cells
was related to blocking of cellular proliferation in the G2/M stage followed by cellular apoptosis. The ISGs
of Cur inclusion complexes are a promising formulation for melanoma treatment.
2014 Published by Elsevier B.V.

Keywords:
In situ hydrogels
Inclusion complexes
Melanoma
Erosion
Transdermal

1. Introduction
Transdermal drug delivery systems (TDDSs) could not only act
on the topical skin, but also deliver drugs to the blood circulation
through skin. TDDS offered many advantages over the conventional dosage forms that could enhance the patient compliance by
virtue of low dose frequency, less adverse effects and noninvasive
delivery of drugs.
Curcumin (Cur), [7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] a hydrophobic polyphenol, is a principal
component and main colorant of turmeric (Fang et al., 2003)
used for centuries in Asian countries as a spice and also as a herbal
anti-inammatory agent. Cur has proven to be assuring as it
exhibits promotion of wound healing (Mohanty et al., 2012b), anti-

* Corresponding author at: Beijing Institute of Radiation Medicine, Department of

Pharmaceutical Sciences, 27 Taiping Road, Haidian District, Beijing 100850, China.
Tel.: +86 10 68246767; fax: +86 10 68214653.
E-mail address: jinyg@bmi.ac.cn (Y. Jin).
1
The authors contributed equally.
http://dx.doi.org/10.1016/j.ijpharm.2014.04.039
0378-5173/ 2014 Published by Elsevier B.V.

microbial (Hegge et al., 2010), anti-inammatory (Ammon and

Wahl, 1991) and anti-cancer effects (Kunnumakkara et al., 2008).
Its pharmacological safety, combined with its dose-dependent
chemotherapeutic effect in several tumor-bearing animal models,
makes it an ideal agent for the transdermal delivery in the
treatment of melanoma. However, the solubility and stability
property of Cur was disadvantageous for its formulation preparation. Cur dissolved in methanol, ethanol and propylene glycol, and
slightly dissolved in water. Cur was not stable in neutral medium,
and it could produce ferulic acid. In addition, the aqueous solubility
of Cur is as low as 0.0004 mg/ml at pH 7.4 (Mohanty et al., 2012a).
To address the problems, some of the novel Cur formulations were
investigated, including nanocrystal (Rachmawati et al., 2013), solid
lipid nanoparticles (Tiyaboonchai et al., 2007), transdermal lm
(Vidyalakshmi et al., 2004), microspheres (Kumar et al., 2002),
nanospheres (Mukerjee and Vishwanatha, 2009), nanoemulsion
(Wang et al., 2008), phospholipid complexes (Liu et al., 2006; Maiti
et al., 2007), etc.
The incidence of melanoma is increasing worldwide, especially
in USA. Despite early detection, appropriate surgical resection and
adjuvant therapy, the number of patients dying from metastatic

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Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

disease continues to rise. According to WHO, approximately 80% of

all skin cancer-related deaths are attributed to melanoma,
although it comprised only 5% of all skin cancers. Despite extensive
clinical research, the treatment options for metastatic disease were
limited, with melanoma being considered as one of the most
chemotherapy-resistant malignancies. Until recently, and over the
past 30 years, only three drugs had gained FDA approval for the
treatment of melanoma, namely dacarbazine, hydroxyurea, and
interleukin-2 (IL-2) (Gogas et al., 2013).
In situ-forming hydrogels (ISGs) are liquid aqueous solutions
before administration, but gels under physiological conditions.
Gelation can occur in situ by ionic cross-linking or after a change in
pH or temperature (Eve and Leroux, 2004). ISGs as one of the most
optimal transdermal formulations offer several advantages, such
as simple mixing, easy application, long adhesion time on the skin
surface, and good permeation ability of therapeutic agents.
Therefore, ISGs are the best choice to prepare Cur transdermal
formulation for melanoma treatment.
Poloxamer block copolymers were one of the most important
thermosensitive hydrogel materials. This copolymer consists of
ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a
triblock structure EOxPOyEOx. Poloxamer has been presented by
FDA guide as an inactive ingredient for different types of
preparations, such as intravenous injection, inhalation, oral
solution, suspension, ophthalmic or topical formulation (Gilles
et al., 2006). The thermo-responsive solutiongelation transformation is attributed to the interaction between the segments of the
temperature-sensitive copolymers. Poloxamer 407 molecules in
solutions could likely aggregate into micelles with the increase in
temperature, resulting from dehydration of hydrophobic polypropylene oxide (PPO) blocks of the copolymer. The spherical micelles
may have a hydrophobic PPO core and a hydrophilic shell of
hydrated swollen polyethylene oxide (PEO) chains. The micelles
would pack to form a hydrogel network. At the temperature lower
than the solgel transition temperature (Tsolgel), there was no
intermolecular interaction between poloxamer molecules. As the
temperature increased to Tsolgel, hydrophilic PEO chain entanglement and hydrophobic PPO dehydration led to the formation of
micelles. At the temperature higher than Tsolgel, the outer PEO
chain of each micelle was interacted due to hydrogen bonding,
resulting in gel phenomena (Gaisford et al., 1998). The higher the
concentration of the copolymer, the higher are the amounts of
micelles and the easier is the formation of hydrogels. In general,
poloxamer is the most common and appropriate excipient as the
thermo-sensitive ISGs matrix.
In this study, the solubility and stability of Cur was improved by
preparing cyclodextrin complexes. High transdermal efciency
and good melanoma therapy of the Cur-loaded ISGs were achieved.
The physicochemical properties of the complexes, erosion of ISGs
matrix, drug release, cytotoxicity and the inhibition mechanism on
B16-F10 cells were also investigated.

2.2. Preparation of inclusion complexes

The complexation of Cur/HP-b-CD (1:1, 1:2, 1:3, molar ratio,
respectively) was prepared by the grinding method described
previously (Mura et al., 1999). In detail, Cur and HP-b-CD (Fig. 1)
with different molar ratios were grinded with 50% (v/v) ethanol for
0.5 h. The inclusion complexes were washed with methanol three
times to remove free Cur and then dried at 50
C to obtain a dry
powder.
Inclusion complexes of Cur (20 mg) and HP-b-CD were
dissolved in 50% ethanol (v/v) and ltered through a 0.45-mm
lter membrane. Filtrated solution (0.1 ml) was metered to 10 ml
with ethanol in a volume ask (10 ml). Then the Cur content was
determined by high-performance liquid chromatography (HPLC).
The inclusion efciency (%) was calculated as the following
formula.
Inclusion efficiency %

Determined Cur contents

 100%
Theoretical Cur contents

The encapsulation of Cur in HP-b-CD was conrmed by

differential scanning calorimeter (DSC) analysis. In order to verify
encapsulation of Cur in HP-b-CD, DSC curves of four types of
samples were obtained: (a) pure HP-b-CD, (b) pure Cur, (c)
physical mixture of HP-b-CD/Cur, (d) inclusion complexes with
HP-b-CD and Cur. A TA DSC (Q20, New Castle, DE, USA) was
employed and air was used as the reference.
Cur (2 mg) and the inclusion complexes that contained Cur
(2 mg) were weighed and placed in a 50-ml closed aluminum pan.
The scans were conducted under nitrogen at a ow rate of 20 ml/
min between 50 and 380
C at 20
C/min.
Cur and its inclusion complexes were analyzed by the Fourier
transform infrared spectroscopy (FTIR, FTS-65A, Bio-Rad) in a
region ranging from 400 to 4000 cm1. The samples (ca. 0.1 g) were
mixed with KBr (0.1 g) and pressed to a tablet. The FTIR spectrum
was then recorded.
2.3. HPLC analysis
HPLC experiments were performed on a Shimadzu 10Avp
HPLC system (Japan) consisting of a LC-10Avp pump, an SPD-

2. Materials and methods

2.1. Materials
Cur was provided by Guangfu Fine Chemical Institute of Tianjin.
Poloxamers 188 and 407 were purchased from BASF (Ludwigshafen, Germany). 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD)
was supplied by Zhongqi Pharmaceutical Technology Co., Ltd.,
Shijiazhuang, China. Propidium iodide (PI) were purchased from
Sigma Chemical Co. (St. Louis, Missouri, USA). Paclitaxel injection
was supplied by Beijing Huasu Pharmaceutical Co., Ltd., Beijing,
China. Pure water was used for all solutions and dilution. All other
chemicals used were of analytical grade.

Fig. 1. Chemical structures of Cur (A) and HP-b-CD (B).

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

10Avp UV detector, an SCL-10Avp controller, and Shimadzu

CLASS-VP 6.12 chromatographic workstation software. The
Diamonsil C18-ODS HPLC guard column (5 mm, 250 mm  4.6
mm) was purchased from Dikma Co., Ltd., China. A manual
injection valve and a 20 ml loop (7725i, Rheodyne, USA) were
used. The UV detector was set at 425 nm, and the HPLC column
temperature was maintained at 30
C with an AT-950 heater
and cooler (Tianjin Automatic Science Instrument Co., Ltd.). The
mobile phase was acetonitrile:water:acetic acid (50:49:1, v/v/v)
with a ow rate of 1.0 ml/min.
2.4. Solubility measurement
A series of HP-b-CD water solutions (105, 104, 103, 102 mol/l,
respectively) were added with excessive Cur and oscillated at 25
C
for 72 h. The supernatant was ltrated by a 0.45-mm lter membrane
and diluted with ethanol. The absorbance value of Cur by a UV
spectrometry (TU-1901, Beijing PURKINJE General Instrument Co.,
Ltd.) at 425 nm was determined, and the solubility of Cur in HP-b-CD
solution of different concentration was calculated.
2.5. Preparation of in situ hydrogels
Poloxamer 188 (4 g) and poloxamer 407 (24 g) were added
with water (60 ml) and stored at 4
C for 12 h. After totally
dissolving, water was added to 100 ml in the mixed solution of
poloxamers 188 and 407 mentioned above. Cur (55 mg) and
inclusion complexes of Cur (495 mg containing 55 mg Cur, Cur:
HP-b-CD = 1:2, m/m) were added to the thermo-sensitive hydrogels composed of poloxamers 188 and 407 (30 ml) mentioned
above.
2.6. Photochemical stability
Cur (55 mg) and its inclusion complexes (495 mg containing
55 mg Cur, Cur:HP-b-CD = 1:2, m/m) were exposed to light of
4500 lx for one month in an open glass dish (Aziz et al., 2007). After
exposure for 5, 10, and 30 days, samples were collected and the
residual ratio of Cur (%) (the ratio of the content of Cur retained to
the original one in the sample) was measured as follows: each
sample (1 ml) were dissolved in 1 ml of ethanol:water (4:1, v/v)
solution, vortexed vigorously for 10 min and left at room
temperature for 4 h, followed by centrifugation at 6000 rpm for
10 min. The supernatant was ltered through 0.45-mm ltration
membrane. After diluting 1000 times with ethanol:water (4:1, v/v)
solution, Cur content was measured by HPLC.
2.7. Measurement of erosion of ISG matrix and release of Cur
The sample tubes were weighed (W0) and added with Curloaded ISGs (2 ml, W1). And then the samples were placed into
water bath of constant 37
C. After 10 min, ISGs turned into semiliquid hydrogels and were added with acetic acid buffered solution
(pH 4.5, 2.4 ml) as the release medium. The sample tubes were
placed into an oscillation instrument of 37
C and agitated with the
speed of 100 rpm. The entire release medium was taken out every
20 min for 12 times in total (i.e., 240 min), and the sample tubes
were weighed (W2). After that, the fresh release medium with the
same temperature and the same volume was supplemented. The
triplicate samples were repeated. The release medium taken out at
the different time points was determined with HPLC for Cur
content.
The erosion ratio (%) was calculated as the following:
Erosion ratio %

W 1  W 2
 100%
W 1  W 0

33

2.8. Transdermal experiments

2.8.1. Preparation of full thickness skins
The back skin of sacriced Sprague-Dawley rats was obtained by
surgical blade, washed with distilled water and physiological saline.
The skin was cut into the pieces of 2.5 cm2 area and stored at 80
C,
and the pieces were used within less than 3 months. The skin was
defrosted at room temperature and then soaked in phosphate buffer
saline (PBS, pH 7.4) for 1 h before transdermal experiments.

2.8.2. Permeation study

In vitro transdermal experiments were performed with a
transdermal permeation instrument (Tianjin Xinzhou Technical
Co., Ltd., Tianjin, China) with vertical Franz-type diffusion cells of
diffusion area 1.96 cm2. The condition was set at 32
C for 8 h (Hu
et al., 2011). Aliquots (17 ml) of PBS solutions (pH 7.4) were added
into the receptor compartments and stirred at 200 rpm. The skins
were sandwiched between the donor and receptor compartments
with the epidermal side upward. Aliquots (3 ml) of Cur-loaded
hydrogels were added to the donor compartments. At the
predetermined time intervals of 1, 2, 3, 4, 6, 8 h, the samples
(3 ml) were withdrawn from the receptor compartments for HPLC
analysis and then quickly lled with the same volume of PBS at
32
C. The donor compartments were covered with Paralm (Bemis
Company, Oshkosh, USA) to prevent water evaporation. Each
experiment was repeated three times.
Steady state ux (Jss, mg cm2 h1) at t time represented the
slope of the linear plots of cumulative drug permeation amount
(Qn, mg cm2) vs. time (t, h). Qn was the cumulative drug
permeation amount in the receptor compartments, and calculated
according to following equation:
"
#
n1
X
Cn  V V 0
Ci
Qn

i1

In the equation, V (ml) was the volume of PBS in the receptor

compartment. Cn (mg/ml) was the concentration of sample n, and
Ci was the concentration of sample i (mg/ml). V0 (ml) was the
withdrawn volume. A (cm2) was the permeation area.

2.9. In vitro drug release simulation

The drug release mechanisms of Cur ISGs in vitro were based on
the erosion of ISGs matrix and the release proles of Cur. And its
release mechanisms were tted to some mathematical models,
including zero order, rst order, Higuchi equation, and Ritger
Peppas exponent equation. The equation tting was calculated
with Excel 2003 Software (Microsoft Corporation, Washington,
USA) and the best model was with the highest correlation
coefcient (r). The drug release mechanisms could be deduced
based on the equations, possibly involving diffusion and degradation, or the combined mechanisms. The model equations were as
follows:
Zero-order model
F t kr0 t
In the equation above, Ft was the cumulative release percentage,
kr0 zero release rate constant, and t time.
First-order model
ln1  F t kr1 t ln M
In the equation above, Ft was the cumulative release percentage,
kr1 rst release rate constant, t time and M a constant.

34

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

Higuchi model
F t kH t1=2
In the equation above, Ft was the cumulative release percentage,
kH Higuchi release rate constant, and t time.
RitgerPeppas exponent model
ln F t nln t ln k
In the equation above, Ft was the cumulative release percentage
at a certain time point, k a constant, t time and n the diffusion
index.
2.10. Cytotoxic study
2.10.1. Cytotoxicity on mouse melanoma cells
The cytotoxicity of Cur and its inclusion complexes were
evaluated in vitro in mouse melanoma cell line B16-F10 (gifted
by Li Tong in Beijing Normal University). B16-F10 cells were
cultured in 1640 medium (Hyclone, Waltham, MA) with 100 U/ml
penicillin and 100 mg/ml streptomycin, and supplemented with
10% fetal bovine serum. The cells were incubated in 96-well plates
at 37
C in 5% CO2 for 24 h. The culture medium was removed, and
samples (100 ml) were added into the wells with concentrations of
1, 5, 10, 50, 300 mg/ml, respectively by the addition of culture
medium (100 ml) to each well and incubation for 48 h. The control
well received no treatment and added with culture medium
(200 ml). An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (20 ml, 5 mg/ml) was added to each well.
4 h later, the culture medium was removed. Formazan crystals
were dissolved in dimethyl sulfoxide (DMSO, 150 ml) for each well,
and the absorbance of the converted dye was measured at 570 nm
using a microplate reader (Multiskan MK3, Thermo Scientic). The
relative growth rate (%, RGR) was equal to the average ratio of

absorbance between the experimental and untreated groups

(n = 3).
2.10.2. Morphology of cells
The inuence of Cur and its inclusion complexes on cell
morphology was further explored by treating B16-F10 cells in the
6-well plate with the culture medium, Taxol (103 mol/l), Cur
(103 mol/l) and its inclusion complexes samples (103 mol/l),
respectively. The cells were incubated for 12 h and then xed in 10%
formalin and embedded in parafn. Sections of 5 mm thickness
were cut and stained with hematoxylin and eosin (H&E). Cells were
visualized using an epiuorescence microscope (BDS200-FL,
Chongqing Optec Instrument Co., Ltd., China), and images were
acquired with a 3.3-megapixel cooled charge-coupled-device
camera (Tucsen, Fuzhou, Fujian, China).
2.10.3. Flow cytometry
Single staining with PI enables the identication of apoptotic
cells and the distribution of the whole population in different cell
cycle phases. Cells (1 104/ml) were cultured for 12 h in the culture
asks. Culture medium, Taxol and Cur samples were added and
cultured for 48 h in 37
C with the nal concentration of samples
being 103 mol/l. The cells were digested with 0.25% trypsin (w/v)
and washed with PBS (pH 7.4, 3 ml). After resuspended with PBS
(4
C, 0.5 ml), the cells were added drop by drop into 75% ethanol
solution of 20
C (3 ml) and immobilized in 4
C for 12 h. The
supernatant was removed, and the cells were treated with RNAse
(Sigma Chemical Co., St. Louis, Missouri, USA) (50 mg/ml) at 37
C
for 30 min. And then the cells were permealized and stained with
1 ml PI (50 mg/ml) at 4
C for 30 min protecting from light. Finally,
10,000 cells were acquired in linear mode with a FASCalibur ow
cytometry equipped with an argon laser at the 488 nm excitation
wavelength on CELLQuest software (Becton Dickinson, New Jersey,

Fig. 2. DSC validation of the formation of the inclusion complexes with Cur and HP-b-CD. (A) Pure HP-b-CD, (B) pure Cur, (C) physical mixture of HP-b-CD/Cur, (D) inclusion
complexes with HP-b-CD and Cur.

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

USA). The percentage of cells in different cycle phases was

evaluated by CellFit2 software (Becton Dickinson).
2.11. Statistical analysis
The results were treated statistically using SPSS software
(Version 17, USA) and data were expressed as means  standard
deviations (S.D.). One way analysis of variance (ANOVA) with post
hoc Tukey (HSD) test was employed to identify signicant
differences (p < 0.05) between data sets.

3. Results and discussion

3.1. Characterization of inclusion complexes
The formation of the inclusion complexes with Cur and HP-

b-CD were validated by DSC and FTIR. DSC is the most classical
technique to characterize the inclusion complexes. When guest
molecules are included in CD cavities or crystal lattice, their

35

melting, boiling, and sublimation points shift to different

temperatures or disappear. The thermograms of Cur, HP-b-CD,
physical mixture, and inclusion complexes were compared. The
DSC diagram of HP-b-CD showed a broader endothermic effect at
135
C (Fig. 2A). The curve of Cur exhibited a sharp endothermic
peak at 172
C (Fig. 2B), indicating the melting point of Cur. For the
physical mixture of Cur and HP-b-CD, there were a broader peak
at about 110
C, and a smaller peak around 176
C (Fig. 2C). Since
the thermogram of the physical mixture was similar to the
superimposition of the thermograms of individual Cur and HPb-CD, there could be an absence of interaction between Cur and
HP-b-CD in the physical mixture (Yan et al., 2008). The
disappearance of the melting peak of Cur from the thermogram
of the inclusion complexes (Fig. 2D) might be due to the
crystalline Cur being included within the central cavity of the HPb-CD ring molecule.
As for FTIR spectrum of Cur, the exible vibration of C
H bonds
in the chemical structure of Cur appeared at 2849.5 cm1 (Fig. 3A).
After the inclusion complexes of Cur and HP-b-CD formed, the
typical peak at 2849.5 cm1 disappeared, and the stretching

Fig. 3. FTIR spectrum of the inclusion complexes of Cur and HP-b-CD. (A) Pure Cur, (B) the inclusion complexes of Cur and HP-b-CD.

36

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

vibration of O
H bonds of HP-b-CD occurred at 3414.8 cm1
(Fig. 3B). It demonstrated that part of Cur was encapsulated
successfully in the cavity of HP-b-CD.
3.2. Increasing solubility and improved photochemical stability of Cur
based on complexation
Cyclodextrins have been successfully used to modify the
solubility, stability or diffusivity mainly due to their capability
of forming noncovalent complexes with drugs (Bibby et al., 2000).
The hydrophobic side chains of drugs may be embedded into the
cone-shaped cavity of cyclodextrins. HP-b-CD was the ether
derivatives of b-CD. Hydroxypropyl group of HP-b-CD destroyed
the intramolecular hydrogen bond of b-CD. And this modication
improved its aqueous solubility and safety, and is more appropriate
for the inclusion of hydrophobic drugs.
Solubility and instability under light were the two major
challenges for the formulation research of Cur. HP-b-CD could
improve the solubility and stability of Cur simultaneously. As the
concentration of HP-b-CD increased from 105 to 102 mol/l, the
solubility of Cur in water increased from 0.15  0.02 mg/ml to
3.09  0.15 mg/ml (Fig. 4A), more than 20 times. This result
corresponded with the previous study that cyclodextrins improved
the aqueous solubility of Cur (Tomren et al., 2007).
The light stability of Cur and its inclusion complexes were
carried out for 30 days under 4500 lx. In the 10th day, the residual
ratio of Cur was only 45.33  2.18%, and that of the inclusion
complexes were 73.57  3.54% (p < 0.05) (Fig. 4B). It demonstrated
that the inclusion complexes might protect Cur from degradation
under light. The labile phenol hydroxy group might be embedded
in the cavity of HP-b-CD ring molecules. The photostability of
avobenzone was also improved when the concentration of HPb-CD was 30% (w/w) (Yang et al., 2008).
3.3. Formation mechanism of Cur inclusion complexes
The inclusion efciencies changed with the different molar
ratio between Cur and HP-b-CD. When the molar ratio of Cur to
HP-b-CD were 1:1, 1:2, 1:3, respectively, the inclusion efciencies
were 60.3  0.4, 97.4  0.8, 62.7  2.6%, respectively (Fig. 5A). The
chemical structure of Cur was symmetrical with bulky side groups
on the phenyl moiety of both ends which seemed to t better into
the cavity of HP-b-CD (Fig. 5B). And this corresponded with the
spectrum of DSC and FTIR.

Fig. 5. The inclusion efciencies (A) and the inclusion mechanism (B) of Cur and
HP-b-CD with different molar ratio.

3.4. Erosion of hydrogels, in vitro release of Cur and transdermal delivery

Matrix erosion and release proles represented drug release
from ISGs onto the skin surface. And transdermal permeability
efciencies represented the transdermal absorption extent of
drugs. Overall, the release proles of Cur inclusion complexes were
much higher than those of Cur no matter for erosion (Fig. 6A),
release in vitro (Fig. 6B) or transdermal efciencies (Fig. 6C).
Improved solubility of Cur with the help of HP-b-CD played an
important role in these phenomena.
Another possible mechanism to enhance transdermal drug
delivery of Cur inclusion complexes may be HP-b-CD as the drug
reservoir to release Cur continuously onto the skin. At the same
time, HP-b-CD in the skin increased the partition of the drug into
the skin and created a high drug concentration within the upper
layers of the skin. This resulted in a higher concentration gradient
which is the driving force for transdermal drug delivery. This
possibility was supported by our results that a higher ux
accompanied with Cur inclusion complexes (0.626 mg cm2 h1).

Fig. 4. The inuence of HP-b-CD on the solubility and stability of Cur. (A) Solubility of Cur increased with the increased concentration of HP-b-CD, (B) stability of Cur under
light increased after complexation (*, p < 0.05).

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

37

Fig. 6. ISG erosion proles (A), in vitro release proles (B) and transdermal permeability efciencies (C) of Cur and its inclusion complexes (*, p < 0.05).

When the inclusion complexes formed, the drug release kinetics

changed to independent of the drug concentration, and the drug
release rate was constant. This was more ideal and appropriate for
continuous release of drugs to exert therapeutic effects.

To ascertain the release kinetics, the in vitro drug release data

were applied to zero order, rst order, and Higuchi kinetics models.
RitgerPeppas equation was used to characterize the drug release
mechanism (Chaudhary et al., 2011; Nasira et al., 2012). The
different values of n in the RitgerPeppas exponent model
represented the different release mechanisms. n < 0.45 stands
for Ficks diffusion, and n > 0.89 stands for the dissolution
controlling mechanism. The values of n between 0.45 and 0.89
showed that the release mechanism was the cooperation of Ficks
diffusion and dissolution. As for the ISGs of Cur and its inclusion
complexes, n was 1.248, 1.219, respectively (Table 1). It implied that
the dissolution was the major mechanism. Dissolution is the
process of drug molecules from solid phase into the liquid phase
and it is the rate-limited step in the drug release and absorption for
the hard soluble drugs, such as Cur.
The best t with highest regression coefcient values (r) was
predicted by rst-order model and zero-order model (r = 0.9920
and r = 0.9960 for Cur and its inclusion complexes, respectively)
rather than for Higuchi models (r = 0.9497 and r = 0.9550 for Cur
and its inclusion complexes, respectively). This clearly indicated
that Cur release from ISGs depend on the residual drug content.

3.5. Cytotoxicity of Cur and its inclusion complexes

The inhibition ratios of Cur and its inclusion complexes
(300 mg/ml) on B16-F10 cells were 83.1  5.71, 47.1 15.12%,
respectively (Fig. 7A). And the 50% inhibiting concentration
(IC50) were 702.27, 281.88 mg/ml, respectively. The inhibition
effect of Cur inclusion complexes was much higher than that of Cur
(p < 0.05). The improved solubility of Cur by HP-b-CD might be the
predominant reason.
The mode of cell death in response to cytotoxic drugs may be
necrosis, apoptosis or autophagy (Shao et al., 2004). Flow
cytometry was used to investigate the action mode of Cur with
taxol as the reference in this study. The proliferative activity
evaluated on the treated cells was expressed as G2/M percentage of
total cycling cells. The strong apoptosis induction in taxol-treated
cells was 12.50% as for G2/M blockage. G2/M percentages of total

Table 1
Release kinetic parameters and correlation coefcients of Cur and its inclusion complexes.
ISGs of Cur

Zero-order
First-order
Higuchi
RitgerPeppas

ISGs of Cur inclusion complexes

Equation

Equation

Ft = 5.390t  0.630
ln (1  Ft) = 16.41t + 75.78
Ft = 11.52t0.5  4.820
ln Ft = 1.248ln t + 1.388

0.9910
0.9920
0.9497
0.9975

Ft = 26.58t  2.277
ln (1  Ft) = 0.676t + 4.960
Ft = 55.96t0.5  22.12
ln Ft = 1.219ln t + 3.040

0.9960
0.9381
0.9550
0.9879

38

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

Fig. 7. The cytotoxicity of Cur and its inclusion complexes on B16-F10 cells. (A) The relative growth ratio of B16-F10 cells, (B) B16-F10 cells treated with different samples
evaluated by the ow cytometry assay, (C) the morphology of B16-F10 cells treated with different samples.

cycling cells for Cur and its inclusion complexes were 7.58 and
8.19%, respectively. This was much higher than that of the negative
control (4.32%) (Fig. 7B). It demonstrated that the possible
cytotoxic mechanism of Cur might be that it blocks the cells
proliferation in G2/M stage and therefore, induces cells apoptosis.
A cell that is undergoing apoptosis demonstrates morphological
changes that include cell shrinkage, membrane blebbing, nuclear
condensation and DNA fragmentation (Lane et al., 2005). The cells
of negative control group were normal fusiform shape and grew
well. There were some discrete particles in cytoplasm. As for the
positive controltaxol, most of B16-F10 cells dissolved and there
were no discrete particles in the rest cells. 50% and 80% cells
dissolved in Cur and its inclusion complexes groups, respectively.
Rest of the cells were spherical, and it demonstrated the powerful
cytotoxic effect. And the cytotoxicity of Cur inclusion complexes
was much higher than that of Cur (Fig. 7C). On one hand, the
solubility of Cur inclusion complexes was much higher. On the
other hand, it would not be therapeutically useful if the solubilized
and stable Cur was not taken up by cancer cells. Cur must be
bioavailable, or be able to enter a cell, to exert biological effects.
After Cur was prepared into inclusion complexes, a higher
permeability, a more soluble and bioavailable form of Cur may
be attained. Furthermore, the protection of Cur against degradation could be another important factor (Vandita et al., 2012).
Based on the results mentioned above and the previous study,
the therapeutic effect of Cur ISGs on melanoma may have two
sides. On one hand, Cur ISGs acted directly on carcinoma cells on
topical skin. On the other hand, Cur ISGs induced cellular apoptosis
by complicated mechanism indirectly. Anti-oxidant activity and
suppression of NF-kB activation were the major mechanisms. As all
know, oxidative stress plays a major role in the pathogenesis of
various diseases, such as myocardial ischemia, hemorrhage and

shock, nerve cell injury, cancer, etc. (Maheshwari et al., 2006). Cur
was shown to be a potent scavenger of a variety of reactive oxygen
species (ROS) including superoxide anion radicals, hydroxyl
radicals (Reddy and Aggarwal, 1994) and nitrogen dioxide radicals
(Sreejayan and Rao, 1997).
The transcription factor NF-kB is constitutively expressed in
almost all cancer types and suppresses apoptosis in a wide variety
of tumors. Cur is a potent blocker of NF-kB activation induced by
different inammatory stimuli, thus resulting in the suppression of
NF-kB-dependent gene products that suppress apoptosis and
mediate proliferation, invasion, and angiogenesis (Yallapu et al.,
2012). So the transdermal absorption of Cur might down-regulate
ROS, block the activation of NF-kB pathway, inhibit the melanoma
cells in G2/M phase and nally induce apoptosis.
For curcumin application in melanoma treatment, all of the
researches focused on the evaluation on melanoma cell lines to our
knowledge. For human melanoma A375 cells, it was reported that
the cell viability was 60% in a curcumin solution (10 mM) and about
30% in another curcumin solution (25 mM) (Chatterjee and Pandey,
2011). The curcumin concentration of the in situ hydrogels of
curcumin inclusion complexes was high to 4.96 mM that was
ensured to have the enough anti-melanoma effect.
4. Conclusions
Cur is a compound with diverse pharmacological effects, such
as antioxidant, anti-inammatory, antiproliferative and antiangiogenic activities. However, the lower aqueous solubility and
photochemical instability were the major obstacles for its
formulation research. This manuscript was the rst report to
investigate the therapeutic possibility of Cur on melanoma with
the improved solubility and photochemical stability to our

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 3139

knowledge. It focused on practical application of Cur inclusion

complexes formulation assisted by HP-b-CD. The solubility,
stability under light, erosion, release in vitro, transdermal
permeability efciency and cytotoxicity of Cur inclusion complexes
were all improved. However, future studies on the developed
formulation will focus on the validation of therapeutic effect in an
in vivo setting. Anyway, ISGs of Cur inclusion complexes in the near
future are likely to bring this promising formulation to the
forefront of therapeutic agents for the treatment of melanoma.
Acknowledgement
We are thankful for the nancial support from the National Key
Technologies R&D Program for New Drugs (No. 2012ZX09301003001-009).
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