International Journal of Pharmaceutics 469 (2014) 31–39

Transdermal delivery of the in situ hydrogels of curcumin and itsinclusion complexes of hydroxypropyl-b-cyclodextrin for melanomatreatment

Yunbo Sun a,1, Lina Du a,1, Yangpu Liu a, Xin Li b, Miao Li a, Yiguang Jin a,*,Xiaohong Qian a

aBeijing Institute of Radiation Medicine, Beijing 100850, ChinabChinese PLA General Hospital, Beijing 100853, China

A R T I C L E I N F O

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

Keywords:In situ hydrogelsInclusion complexesMelanomaErosionTransdermal

A B S T R A C T

Curcumin (Cur) is a hydrophobic polyphenol with diverse pharmacological effects, especially for cancertreatment. However, its weak water solubility and stability was the major obstacle for the formulationresearch of Cur. The complexation of Cur and hydroxypropyl-b-cyclodextrin (HP-b-CD) was done bygrinding. The increasing solubility of Cur was achieved due to complexation and the photochemicalstability of Cur was improved. The inclusion of Cur could happen when two ends of Cur were embeddedinto the cavity of the HP-b-CD rings. The in situ hydrogels (ISGs) of Cur and its inclusion complexes wereprepared using poloxamers 407 and 188 as the matrix. The extent of drug’s in vitro release from the ISGsdepended on the dissolution of drugs. Both of the ISGs had transdermal effect and cytotoxicity on B16-F10cells. However, the effects of the ISGs containing Cur inclusion complexes were much higher than those ofCur ISGs because of the improved Cur solubility in the former. The cytotoxicity of Cur on melanoma cellswas related to blocking of cellular proliferation in the G2/M stage followed by cellular apoptosis. The ISGsof Cur inclusion complexes are a promising formulation for melanoma treatment.

ã 2014 Published by Elsevier B.V.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsev ier .com/locate / i jpharm

1. Introduction

Transdermal drug delivery systems (TDDSs) could not only acton the topical skin, but also deliver drugs to the blood circulationthrough skin. TDDS offered many advantages over the convention-al dosage forms that could enhance the patient compliance byvirtue of low dose frequency, less adverse effects and noninvasivedelivery of drugs.

Curcumin (Cur), [7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hep-tadiene-3,5-dione] a hydrophobic polyphenol, is a principalcomponent and main colorant of turmeric (Fang et al., 2003)used for centuries in Asian countries as a spice and also as a herbalanti-inflammatory agent. Cur has proven to be assuring as itexhibits promotion of wound healing (Mohanty et al., 2012b), anti-

* Corresponding author at: Beijing Institute of Radiation Medicine, Department ofPharmaceutical 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.2014.04.0390378-5173/ã 2014 Published by Elsevier B.V.

microbial (Hegge et al., 2010), anti-inflammatory (Ammon andWahl, 1991) and anti-cancer effects (Kunnumakkara et al., 2008).Its pharmacological safety, combined with its dose-dependentchemotherapeutic effect in several tumor-bearing animal models,makes it an ideal agent for the transdermal delivery in thetreatment of melanoma. However, the solubility and stabilityproperty of Cur was disadvantageous for its formulation prepara-tion. Cur dissolved in methanol, ethanol and propylene glycol, andslightly dissolved in water. Cur was not stable in neutral medium,and it could produce ferulic acid. In addition, the aqueous solubilityof 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 wereinvestigated, including nanocrystal (Rachmawati et al., 2013), solidlipid nanoparticles (Tiyaboonchai et al., 2007), transdermal film(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; Maitiet al., 2007), etc.

The incidence of melanoma is increasing worldwide, especiallyin USA. Despite early detection, appropriate surgical resection andadjuvant therapy, the number of patients dying from metastatic

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

32 Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39

disease continues to rise. According to WHO, approximately 80% ofall skin cancer-related deaths are attributed to melanoma,although it comprised only 5% of all skin cancers. Despite extensiveclinical research, the treatment options for metastatic disease werelimited, with melanoma being considered as one of the mostchemotherapy-resistant malignancies. Until recently, and over thepast 30 years, only three drugs had gained FDA approval for thetreatment of melanoma, namely dacarbazine, hydroxyurea, andinterleukin-2 (IL-2) (Gogas et al., 2013).

In situ-forming hydrogels (ISGs) are liquid aqueous solutionsbefore administration, but gels under physiological conditions.Gelation can occur in situ by ionic cross-linking or after a change inpH or temperature (Eve and Leroux, 2004). ISGs as one of the mostoptimal transdermal formulations offer several advantages, suchas simple mixing, easy application, long adhesion time on the skinsurface, and good permeation ability of therapeutic agents.Therefore, ISGs are the best choice to prepare Cur transdermalformulation for melanoma treatment.

Poloxamer block copolymers were one of the most importantthermosensitive hydrogel materials. This copolymer consists ofethylene oxide (EO) and propylene oxide (PO) blocks arranged in atriblock structure EOx–POy–EOx. Poloxamer has been presented byFDA guide as an “inactive” ingredient for different types ofpreparations, such as intravenous injection, inhalation, oralsolution, suspension, ophthalmic or topical formulation (Gilleset al., 2006). The thermo-responsive solution–gelation transfor-mation is attributed to the interaction between the segments of thetemperature-sensitive copolymers. Poloxamer 407 molecules insolutions could likely aggregate into micelles with the increase intemperature, resulting from dehydration of hydrophobic polypro-pylene oxide (PPO) blocks of the copolymer. The spherical micellesmay have a hydrophobic PPO core and a hydrophilic shell ofhydrated swollen polyethylene oxide (PEO) chains. The micelleswould pack to form a hydrogel network. At the temperature lowerthan the sol–gel transition temperature (Tsol–gel), there was nointermolecular interaction between poloxamer molecules. As thetemperature increased to Tsol–gel, hydrophilic PEO chain entangle-ment and hydrophobic PPO dehydration led to the formation ofmicelles. At the temperature higher than Tsol–gel, the outer PEOchain of each micelle was interacted due to hydrogen bonding,resulting in gel phenomena (Gaisford et al., 1998). The higher theconcentration of the copolymer, the higher are the amounts ofmicelles and the easier is the formation of hydrogels. In general,poloxamer is the most common and appropriate excipient as thethermo-sensitive ISGs matrix.

In this study, the solubility and stability of Cur was improved bypreparing cyclodextrin complexes. High transdermal efficiencyand good melanoma therapy of the Cur-loaded ISGs were achieved.The physicochemical properties of the complexes, erosion of ISGsmatrix, drug release, cytotoxicity and the inhibition mechanism onB16-F10 cells were also investigated.

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 (Ludwig-shafen, Germany). 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD)was supplied by Zhongqi Pharmaceutical Technology Co., Ltd.,Shijiazhuang, China. Propidium iodide (PI) were purchased fromSigma Chemical Co. (St. Louis, Missouri, USA). Paclitaxel injectionwas supplied by Beijing Huasu Pharmaceutical Co., Ltd., Beijing,China. Pure water was used for all solutions and dilution. All otherchemicals used were of analytical grade.

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 describedpreviously (Mura et al., 1999). In detail, Cur and HP-b-CD (Fig. 1)with different molar ratios were grinded with 50% (v/v) ethanol for0.5 h. The inclusion complexes were washed with methanol threetimes to remove free Cur and then dried at 50 �C to obtain a drypowder.

Inclusion complexes of Cur (20 mg) and HP-b-CD weredissolved in 50% ethanol (v/v) and filtered through a 0.45-mmfilter membrane. Filtrated solution (0.1 ml) was metered to 10 mlwith ethanol in a volume flask (10 ml). Then the Cur content wasdetermined by high-performance liquid chromatography (HPLC).The inclusion efficiency (%) was calculated as the followingformula.

Inclusion efficiency ð%Þ ¼ ðDetermined Cur contentsÞðTheoretical Cur contentsÞ � 100%

The encapsulation of Cur in HP-b-CD was confirmed bydifferential scanning calorimeter (DSC) analysis. In order to verifyencapsulation of Cur in HP-b-CD, DSC curves of four types ofsamples were obtained: (a) pure HP-b-CD, (b) pure Cur, (c)physical mixture of HP-b-CD/Cur, (d) inclusion complexes withHP-b-CD and Cur. A TA DSC (Q20, New Castle, DE, USA) wasemployed 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 flow rate of 20 ml/min between 50 and 380 �C at 20 �C/min.

Cur and its inclusion complexes were analyzed by the Fouriertransform infrared spectroscopy (FTIR, FTS-65A, Bio-Rad) in aregion ranging from 400 to 4000 cm�1. The samples (ca. 0.1 g) weremixed with KBr (0.1 g) and pressed to a tablet. The FTIR spectrumwas then recorded.

2.3. HPLC analysis

HPLC experiments were performed on a Shimadzu 10AvpHPLC system (Japan) consisting of a LC-10Avp pump, an SPD-

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39 33

10Avp UV detector, an SCL-10Avp controller, and ShimadzuCLASS-VP 6.12 chromatographic workstation software. TheDiamonsil C18-ODS HPLC guard column (5 mm, 250 mm � 4.6mm) was purchased from Dikma Co., Ltd., China. A manualinjection valve and a 20 ml loop (7725i, Rheodyne, USA) wereused. The UV detector was set at 425 nm, and the HPLC columntemperature was maintained at 30 �C with an AT-950 heaterand cooler (Tianjin Automatic Science Instrument Co., Ltd.). Themobile phase was acetonitrile:water:acetic acid (50:49:1, v/v/v)with a flow rate of 1.0 ml/min.

2.4. Solubility measurement

A series of HP-b-CD water solutions (10�5, 10�4,10�3,10�2mol/l,respectively) were added with excessive Cur and oscillated at 25 �Cfor 72 h. The supernatant was filtrated bya 0.45-mm filter membraneand diluted with ethanol. The absorbance value of Cur by a UVspectrometry (TU-1901, Beijing PURKINJE General Instrument Co.,Ltd.) at 425 nmwas determined, and the solubility of Cur in HP-b-CDsolution of different concentration was calculated.

2.5. Preparation of in situ hydrogels

Poloxamer 188 (4 g) and poloxamer 407 (24 g) were addedwith water (60 ml) and stored at 4 �C for 12 h. After totallydissolving, water was added to 100 ml in the mixed solution ofpoloxamers 188 and 407 mentioned above. Cur (55 mg) andinclusion complexes of Cur (495 mg containing 55 mg Cur, Cur:HP-b-CD = 1:2, m/m) were added to the thermo-sensitive hydro-gels composed of poloxamers 188 and 407 (30 ml) mentionedabove.

2.6. Photochemical stability

Cur (55 mg) and its inclusion complexes (495 mg containing55 mg Cur, Cur:HP-b-CD = 1:2, m/m) were exposed to light of4500 lx for one month in an open glass dish (Aziz et al., 2007). Afterexposure for 5, 10, and 30 days, samples were collected and theresidual ratio of Cur (%) (the ratio of the content of Cur retained tothe original one in the sample) was measured as follows: eachsample (1 ml) were dissolved in 1 ml of ethanol:water (4:1, v/v)solution, vortexed vigorously for 10 min and left at roomtemperature for 4 h, followed by centrifugation at 6000 rpm for10 min. The supernatant was filtered through 0.45-mm filtrationmembrane. 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 Cur-loaded ISGs (2 ml, W1). And then the samples were placed intowater bath of constant 37 �C. After 10 min, ISGs turned into semi-liquid hydrogels and were added with acetic acid buffered solution(pH 4.5, 2.4 ml) as the release medium. The sample tubes wereplaced into an oscillation instrument of 37 �C and agitated with thespeed of 100 rpm. The entire release medium was taken out every20 min for 12 times in total (i.e., 240 min), and the sample tubeswere weighed (W2). After that, the fresh release medium with thesame temperature and the same volume was supplemented. Thetriplicate samples were repeated. The release medium taken out atthe different time points was determined with HPLC for Curcontent.

The erosion ratio (%) was calculated as the following:

Erosion ratio ð%Þ ¼ ðW1 � W2ÞðW1 � W0Þ � 100%

2.8. Transdermal experiments

2.8.1. Preparation of full thickness skinsThe back skin of sacrificed 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 wasdefrosted at room temperature and then soaked in phosphate buffersaline (PBS, pH 7.4) for 1 h before transdermal experiments.

2.8.2. Permeation studyIn vitro transdermal experiments were performed with a

transdermal permeation instrument (Tianjin Xinzhou TechnicalCo., Ltd., Tianjin, China) with vertical Franz-type diffusion cells ofdiffusion area 1.96 cm2. The condition was set at 32 �C for 8 h (Huet al., 2011). Aliquots (17 ml) of PBS solutions (pH 7.4) were addedinto the receptor compartments and stirred at 200 rpm. The skinswere sandwiched between the donor and receptor compartmentswith the epidermal side upward. Aliquots (3 ml) of Cur-loadedhydrogels were added to the donor compartments. At thepredetermined time intervals of 1, 2, 3, 4, 6, 8 h, the samples(3 ml) were withdrawn from the receptor compartments for HPLCanalysis and then quickly filled with the same volume of PBS at32 �C. The donor compartments were covered with Parafilm (BemisCompany, Oshkosh, USA) to prevent water evaporation. Eachexperiment was repeated three times.

Steady state flux (Jss, mg cm�2 h�1) at t time represented theslope of the linear plots of cumulative drug permeation amount(Qn, mg cm�2) vs. time (t, h). Qn was the cumulative drugpermeation amount in the receptor compartments, and calculatedaccording to following equation:

Qn ¼Cn � V þ V0

Xn�1

i¼1

Ci

" #

A

In the equation, V (ml) was the volume of PBS in the receptorcompartment. Cn (mg/ml) was the concentration of sample n, andCi was the concentration of sample i (mg/ml). V0 (ml) was thewithdrawn 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 onthe erosion of ISGs matrix and the release profiles of Cur. And itsrelease mechanisms were fitted to some mathematical models,including zero order, first order, Higuchi equation, and Ritger–Peppas exponent equation. The equation fitting was calculatedwith Excel 2003 Software (Microsoft Corporation, Washington,USA) and the best model was with the highest correlationcoefficient (r). The drug release mechanisms could be deducedbased on the equations, possibly involving diffusion and degrada-tion, or the combined mechanisms. The model equations were asfollows:

Zero-order model

Ft ¼ kr0t

In the equation above, Ftwas the cumulative release percentage,kr0 zero release rate constant, and t time.

First-order model

lnð1 � FtÞ ¼ �kr1t þ ln M

In the equation above, Ftwas the cumulative release percentage,kr1 first release rate constant, t time and M a constant.

34 Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39

Higuchi model

Ft ¼ kHt1=2

In the equation above, Ftwas the cumulative release percentage,kH Higuchi release rate constant, and t time.

Ritger–Peppas exponent model

ln Ft ¼ nln t þ ln k

In the equation above, Ftwas the cumulative release percentageat a certain time point, k a constant, t time and n the diffusionindex.

2.10. Cytotoxic study

2.10.1. Cytotoxicity on mouse melanoma cellsThe cytotoxicity of Cur and its inclusion complexes were

evaluated in vitro in mouse melanoma cell line – B16-F10 (giftedby Li Tong in Beijing Normal University). B16-F10 cells werecultured in 1640 medium (Hyclone, Waltham, MA) with 100 U/mlpenicillin and 100 mg/ml streptomycin, and supplemented with10% fetal bovine serum. The cells were incubated in 96-well platesat 37 �C in 5% CO2 for 24 h. The culture medium was removed, andsamples (100 ml) were added into the wells with concentrations of1, 5, 10, 50, 300 mg/ml, respectively by the addition of culturemedium (100 ml) to each well and incubation for 48 h. The controlwell received no treatment and added with culture medium(200 ml). An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide) solution (20 ml, 5 mg/ml) was added to each well.4 h later, the culture medium was removed. Formazan crystalswere dissolved in dimethyl sulfoxide (DMSO, 150 ml) for each well,and the absorbance of the converted dye was measured at 570 nmusing a microplate reader (Multiskan MK3, Thermo Scientific). Therelative growth rate (%, RGR) was equal to the average ratio of

Fig. 2. DSC validation of the formation of the inclusion complexes with Cur and HP-b-CDcomplexes with HP-b-CD and Cur.

absorbance between the experimental and untreated groups(n = 3).

2.10.2. Morphology of cellsThe influence of Cur and its inclusion complexes on cell

morphology was further explored by treating B16-F10 cells in the6-well plate with the culture medium, Taxol (10�3mol/l), Cur(10�3mol/l) and its inclusion complexes samples (10�3mol/l),respectively. The cells were incubated for 12 h and then fixed in 10%formalin and embedded in paraffin. Sections of 5 mm thicknesswere cut and stained with hematoxylin and eosin (H&E). Cells werevisualized using an epifluorescence microscope (BDS200-FL,Chongqing Optec Instrument Co., Ltd., China), and images wereacquired with a 3.3-megapixel cooled charge-coupled-devicecamera (Tucsen, Fuzhou, Fujian, China).

2.10.3. Flow cytometrySingle staining with PI enables the identification of apoptotic

cells and the distribution of the whole population in different cellcycle phases. Cells (1 �104/ml) were cultured for 12 h in the cultureflasks. Culture medium, Taxol and Cur samples were added andcultured for 48 h in 37 �C with the final concentration of samplesbeing 10�3mol/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% ethanolsolution of �20 �C (3 ml) and immobilized in 4 �C for 12 h. Thesupernatant was removed, and the cells were treated with RNAse(Sigma Chemical Co., St. Louis, Missouri, USA) (50 mg/ml) at 37 �Cfor 30 min. And then the cells were permealized and stained with1 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 flowcytometry equipped with an argon laser at the 488 nm excitationwavelength on CELLQuest software (Becton Dickinson, New Jersey,

. (A) Pure HP-b-CD, (B) pure Cur, (C) physical mixture of HP-b-CD/Cur, (D) inclusion

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39 35

USA). The percentage of cells in different cycle phases wasevaluated 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 � standarddeviations (S.D.). One way analysis of variance (ANOVA) with posthoc Tukey (HSD) test was employed to identify significantdifferences (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 classicaltechnique to characterize the inclusion complexes. When guestmolecules are included in CD cavities or crystal lattice, their

Fig. 3. FTIR spectrum of the inclusion complexes of Cur and HP-b-CD

melting, boiling, and sublimation points shift to differenttemperatures or disappear. The thermograms of Cur, HP-b-CD,physical mixture, and inclusion complexes were compared. TheDSC diagram of HP-b-CD showed a broader endothermic effect at135 �C (Fig. 2A). The curve of Cur exhibited a sharp endothermicpeak at 172 �C (Fig. 2B), indicating the melting point of Cur. For thephysical mixture of Cur and HP-b-CD, there were a broader peakat about 110 �C, and a smaller peak around 176 �C (Fig. 2C). Sincethe thermogram of the physical mixture was similar to thesuperimposition of the thermograms of individual Cur and HP-b-CD, there could be an absence of interaction between Cur andHP-b-CD in the physical mixture (Yan et al., 2008). Thedisappearance of the melting peak of Cur from the thermogramof the inclusion complexes (Fig. 2D) might be due to thecrystalline Cur being included within the central cavity of the HP-b-CD ring molecule.

As for FTIR spectrum of Cur, the flexible vibration of C��H bondsin the chemical structure of Cur appeared at 2849.5 cm�1 (Fig. 3A).After the inclusion complexes of Cur and HP-b-CD formed, thetypical peak at 2849.5 cm�1 disappeared, and the stretching

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

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

36 Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39

vibration of O��H bonds of HP-b-CD occurred at 3414.8 cm�1

(Fig. 3B). It demonstrated that part of Cur was encapsulatedsuccessfully in the cavity of HP-b-CD.

3.2. Increasing solubility and improved photochemical stability of Curbased on complexation

Cyclodextrins have been successfully used to modify thesolubility, stability or diffusivity mainly due to their capabilityof forming noncovalent complexes with drugs (Bibby et al., 2000).The hydrophobic side chains of drugs may be embedded into thecone-shaped cavity of cyclodextrins. HP-b-CD was the etherderivatives of b-CD. Hydroxypropyl group of HP-b-CD destroyedthe intramolecular hydrogen bond of b-CD. And this modificationimproved its aqueous solubility and safety, and is more appropriatefor the inclusion of hydrophobic drugs.

Solubility and instability under light were the two majorchallenges for the formulation research of Cur. HP-b-CD couldimprove the solubility and stability of Cur simultaneously. As theconcentration of HP-b-CD increased from 10�5 to 10�2mol/l, thesolubility of Cur in water increased from 0.15 � 0.02 mg/ml to3.09 � 0.15 mg/ml (Fig. 4A), more than 20 times. This resultcorresponded with the previous study that cyclodextrins improvedthe aqueous solubility of Cur (Tomren et al., 2007).

The light stability of Cur and its inclusion complexes werecarried out for 30 days under 4500 lx. In the 10th day, the residualratio of Cur was only 45.33 � 2.18%, and that of the inclusioncomplexes were 73.57 � 3.54% (p < 0.05) (Fig. 4B). It demonstratedthat the inclusion complexes might protect Cur from degradationunder light. The labile phenol hydroxy group might be embeddedin the cavity of HP-b-CD ring molecules. The photostability ofavobenzone was also improved when the concentration of HP-b-CD was 30% (w/w) (Yang et al., 2008).

3.3. Formation mechanism of Cur inclusion complexes

The inclusion efficiencies changed with the different molarratio between Cur and HP-b-CD. When the molar ratio of Cur toHP-b-CD were 1:1, 1:2, 1:3, respectively, the inclusion efficiencieswere 60.3 � 0.4, 97.4 � 0.8, 62.7 � 2.6%, respectively (Fig. 5A). Thechemical structure of Cur was symmetrical with bulky side groupson the phenyl moiety of both ends which seemed to fit better intothe cavity of HP-b-CD (Fig. 5B). And this corresponded with thespectrum of DSC and FTIR.

Fig. 4. The influence of HP-b-CD on the solubility and stability of Cur. (A) Solubility of Culight increased after complexation (*, p < 0.05).

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

Matrix erosion and release profiles represented drug releasefrom ISGs onto the skin surface. And transdermal permeabilityefficiencies represented the transdermal absorption extent ofdrugs. Overall, the release profiles of Cur inclusion complexes weremuch higher than those of Cur no matter for erosion (Fig. 6A),release in vitro (Fig. 6B) or transdermal efficiencies (Fig. 6C).Improved solubility of Cur with the help of HP-b-CD played animportant role in these phenomena.

Another possible mechanism to enhance transdermal drugdelivery of Cur inclusion complexes may be HP-b-CD as the drugreservoir to release Cur continuously onto the skin. At the sametime, HP-b-CD in the skin increased the partition of the drug intothe skin and created a high drug concentration within the upperlayers of the skin. This resulted in a higher concentration gradientwhich is the driving force for transdermal drug delivery. Thispossibility was supported by our results that a higher fluxaccompanied with Cur inclusion complexes (0.626 mg cm�2 h�1).

r increased with the increased concentration of HP-b-CD, (B) stability of Cur under

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

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39 37

To ascertain the release kinetics, the in vitro drug release datawere applied to zero order, first order, and Higuchi kinetics models.Ritger–Peppas equation was used to characterize the drug releasemechanism (Chaudhary et al., 2011; Nasira et al., 2012). Thedifferent values of n in the Ritger–Peppas exponent modelrepresented the different release mechanisms. n < 0.45 standsfor Fick’s diffusion, and n > 0.89 stands for the dissolutioncontrolling mechanism. The values of n between 0.45 and 0.89showed that the release mechanism was the cooperation of Fick’sdiffusion and dissolution. As for the ISGs of Cur and its inclusioncomplexes, n was 1.248,1.219, respectively (Table 1). It implied thatthe dissolution was the major mechanism. Dissolution is theprocess of drug molecules from solid phase into the liquid phaseand it is the rate-limited step in the drug release and absorption forthe hard soluble drugs, such as Cur.

The best fit with highest regression coefficient values (r) waspredicted by first-order model and zero-order model (r = 0.9920and r = 0.9960 for Cur and its inclusion complexes, respectively)rather than for Higuchi models (r = 0.9497 and r = 0.9550 for Curand its inclusion complexes, respectively). This clearly indicatedthat Cur release from ISGs depend on the residual drug content.

Table 1Release kinetic parameters and correlation coefficients of Cur and its inclusion comple

ISGs of Cur

Equation r

Zero-order Ft = 5.390t � 0.630 0.9First-order ln (1 � Ft) = �16.41t + 75.78 0.9Higuchi Ft = 11.52t0.5� 4.820 0.9Ritger–Peppas ln Ft= 1.248ln t + 1.388 0.9

When the inclusion complexes formed, the drug release kineticschanged to independent of the drug concentration, and the drugrelease rate was constant. This was more ideal and appropriate forcontinuous release of drugs to exert therapeutic effects.

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 inhibitioneffect 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 thepredominant reason.

The mode of cell death in response to cytotoxic drugs may benecrosis, apoptosis or autophagy (Shao et al., 2004). Flowcytometry was used to investigate the action mode of Cur withtaxol as the reference in this study. The proliferative activityevaluated on the treated cells was expressed as G2/M percentage oftotal cycling cells. The strong apoptosis induction in taxol-treatedcells was 12.50% as for G2/M blockage. G2/M percentages of total

xes.

ISGs of Cur inclusion complexes

Equation r

910 Ft= 26.58t � 2.277 0.9960920 ln (1 � Ft) = �0.676t + 4.960 0.9381497 Ft= 55.96t0.5� 22.12 0.9550975 ln Ft= 1.219ln t + 3.040 0.9879

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 samplesevaluated by the flow cytometry assay, (C) the morphology of B16-F10 cells treated with different samples.

38 Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39

cycling cells for Cur and its inclusion complexes were 7.58 and8.19%, respectively. This was much higher than that of the negativecontrol (4.32%) (Fig. 7B). It demonstrated that the possiblecytotoxic mechanism of Cur might be that it blocks the cellsproliferation in G2/M stage and therefore, induces cells apoptosis.

A cell that is undergoing apoptosis demonstrates morphologicalchanges that include cell shrinkage, membrane blebbing, nuclearcondensation and DNA fragmentation (Lane et al., 2005). The cellsof negative control group were normal fusiform shape and grewwell. There were some discrete particles in cytoplasm. As for thepositive control–taxol, most of B16-F10 cells dissolved and therewere no discrete particles in the rest cells. 50% and 80% cellsdissolved in Cur and its inclusion complexes groups, respectively.Rest of the cells were spherical, and it demonstrated the powerfulcytotoxic effect. And the cytotoxicity of Cur inclusion complexeswas much higher than that of Cur (Fig. 7C). On one hand, thesolubility of Cur inclusion complexes was much higher. On theother hand, it would not be therapeutically useful if the solubilizedand stable Cur was not taken up by cancer cells. Cur must bebioavailable, or be able to enter a cell, to exert biological effects.After Cur was prepared into inclusion complexes, a higherpermeability, a more soluble and bioavailable form of Cur maybe attained. Furthermore, the protection of Cur against degrada-tion 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 twosides. On one hand, Cur ISGs acted directly on carcinoma cells ontopical skin. On the other hand, Cur ISGs induced cellular apoptosisby complicated mechanism indirectly. Anti-oxidant activity andsuppression of NF-kB activation were the major mechanisms. As allknow, oxidative stress plays a major role in the pathogenesis ofvarious diseases, such as myocardial ischemia, hemorrhage and

shock, nerve cell injury, cancer, etc. (Maheshwari et al., 2006). Curwas shown to be a potent scavenger of a variety of reactive oxygenspecies (ROS) including superoxide anion radicals, hydroxylradicals (Reddy and Aggarwal, 1994) and nitrogen dioxide radicals(Sreejayan and Rao, 1997).

The transcription factor NF-kB is constitutively expressed inalmost all cancer types and suppresses apoptosis in a wide varietyof tumors. Cur is a potent blocker of NF-kB activation induced bydifferent inflammatory stimuli, thus resulting in the suppression ofNF-kB-dependent gene products that suppress apoptosis andmediate proliferation, invasion, and angiogenesis (Yallapu et al.,2012). So the transdermal absorption of Cur might down-regulateROS, block the activation of NF-kB pathway, inhibit the melanomacells in G2/M phase and finally induce apoptosis.

For curcumin application in melanoma treatment, all of theresearches focused on the evaluation on melanoma cell lines to ourknowledge. For human melanoma A375 cells, it was reported thatthe cell viability was 60% in a curcumin solution (10 mM) and about30% in another curcumin solution (25 mM) (Chatterjee and Pandey,2011). The curcumin concentration of the in situ hydrogels ofcurcumin inclusion complexes was high to 4.96 mM that wasensured to have the enough anti-melanoma effect.

4. Conclusions

Cur is a compound with diverse pharmacological effects, suchas antioxidant, anti-inflammatory, antiproliferative and antiangio-genic activities. However, the lower aqueous solubility andphotochemical instability were the major obstacles for itsformulation research. This manuscript was the first report toinvestigate the therapeutic possibility of Cur on melanoma withthe improved solubility and photochemical stability to our

Y. Sun et al. / International Journal of Pharmaceutics 469 (2014) 31–39 39

knowledge. It focused on practical application of Cur inclusioncomplexes formulation assisted by HP-b-CD. The solubility,stability under light, erosion, release in vitro, transdermalpermeability efficiency and cytotoxicity of Cur inclusion complexeswere all improved. However, future studies on the developedformulation will focus on the validation of therapeutic effect in anin vivo setting. Anyway, ISGs of Cur inclusion complexes in the nearfuture are likely to bring this promising formulation to theforefront of therapeutic agents for the treatment of melanoma.

Acknowledgement

We are thankful for the financial support from the National KeyTechnologies R&D Program for New Drugs (No. 2012ZX09301003-001-009).

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