Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Copyright copy 2011 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomedical Nanotechnology

Vol 7 406ndash414 2011

Curcumin-Loaded Lipid and Polymeric NanocapsulesStabilized by Nonionic Surfactants An In Vitro andIn Vivo Antitumor Activity on B16-F10 Melanoma and

Macrophage Uptake Comparative Study

Letiacutecia Mazzarino1 Luiacutes F C Silva1 Juliana C Curta2 Marley A Liciacutenio2 Aline Costa2Letiacutecia K Pacheco3 Jarbas M Siqueira3 Jorge Montanari4 Eder Romero4 Jamil Assreuy3

Maria C Santos-Silva2 and Elenara Lemos-Senna1lowast1Departamento de Ciecircncias Farmacecircuticas Centro de Ciecircncias da Sauacutede Universidade Federal de Santa Catarina

Campus Trindade Florianoacutepolis 88040-970 Brazil2Departamento de Anaacutelises Cliacutenicas Centro de Ciecircncias da Sauacutede Universidade Federal de Santa Catarina

Florianoacutepolis 88040-970 Brazil3Departamento de Farmacologia Centro de Ciecircncias Bioloacutegicas Universidade Federal de Santa Catarina

Florianoacutepolis 88049-970 Brazil4Departamento de Ciencia y Tecnologiacutea Universidad Nacional de Quilmes Bernal B1876 BXD Buenos Aires Argentina

Curcumin is a polyphenol obtained from the plant Curcuma longa (called turmeric) that displaysseveral pharmacological activities including anti-inflammatory antioxidant antimicrobial and anti-tumoral activity but clinical use has been limited by its poor solubility in water and consequentlyminimal systemic bioavailability We have therefore formulated the drug into nanocarrier systems inan attempt to improve its therapeutic properties This study evaluates the effect of intraperitoneallyadministered nanocapsules containing curcumin on subcutaneous melanoma in mice inoculatedwith B16-F10 cells and on the cytotoxicity activity against B16-F10 cells in vitro Phagocytic uptakeof formulations was also evaluated upon incubation with macrophage J774 cells by fluorescencemicroscopy Lipid and polymeric nanocapsules were prepared by the phase inversion and nano-precipitation methods respectively The uptake of the lipid nanocapsules prepared using SolutolHS15 was significantly reduced in J774 cells Curcumin as free drug or as drug-loaded nanocap-sules was administrated at a dose of 6 mgkg twice a week for 21 days Free drug and curcumin-loaded nanocapsules significantly reduced tumor volume (P lt 005 vs control) but no differencewas found in the antitumor activity displayed by lipid and polymeric nanocapsules This assumptionwas supported by the in vitro study in which free curcumin as well as loaded into nanocapsulescaused significant reduction of cell viability in a concentration- and time-dependent manner

Keywords Curcumin Lipid Nanocapsules Polymeric Nanocapsules B16-F10 MelanomaAntitumor Activity Cytotoxicity Macrophage Uptake

1 INTRODUCTION

Melanoma is the most aggressive form of skin can-cer and is characterized by abnormal proliferation ofmelanocytes that invade the basement membrane1 Theincidence rate is increasing faster than any other tumorand the World Health Organization (WHO) estimates132000 new cases of cutaneous melanoma per year2 Sunexposure patient phenotype family history and previous

lowastAuthor to whom correspondence should be addressed

history of melanoma are the major risk factors related tomelanoma3

Chemoprevention has been described as anunder-explored approach that could significantly decreasemorbidity and mortality from this deadly cancer4 Chemo-prevention strategies mostly imply the use of substanceswhich are able to inhibit or reverse cellular processsuch as decreased apoptosis increased proliferationand cell maturation or differentiation5 Particularly awide variety of botanicals mostly dietary flavonoidsor phenolic substances including quercetin resveratrol

406 J Biomed Nanotechnol 2011 Vol 7 No 3 1550-703320117406009 doi101166jbn20111296

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

and curcumin have been reported to possess substantialchemopreventive properties because of their antioxidantand anti-inflammatory ability Concerning skin cancerslaboratory and epidemiological studies have evidencedthat routine consumption of these polyphenol com-pounds may favorably provide some antiphotocarcinogenicprotection against skin disorders caused by solar UVradiation6

Curcumin is a yellow polyphenol derived from the rhi-zome of turmeric (Curcuma longa L) Extensive researchhas pointed out its potential in the prevention and treat-ment of cancer and other diseases Several studies car-ried out over the past two decades have indicated thatcurcumin displays antimutagenic activity and inhibits theradiation- as well as chemical carcinogen-induced neoplas-tic lesions in many tumor models including skin cancer6

The anticancer potential of curcumin is related to its abil-ity to down-regulate transcription factors NF-B AP-1Erg-1 and MAPK to down-regulate the expression ofCOX-2 LOX NOS MMP TNF- IL-1 and cyclin D1to modulate cytochrome P450 function to down-regulategrowth factor receptors and to inhibit the activity of theoncogenes ras fos jun and myc7ndash9 In vivo studies havedemonstrated that curcumin inhibits lung tumor noduleformation in mice inoculated with B16-F10 melanomacells and increases life span by inhibiting the expressionof metalloproteinases1011 Moreover the combined treat-ment with this polyphenol and a prophylactic immunepreparation caused substantial growth inhibition of B16-Rmelanoma cells resistant to doxorubicin and significantlyincreased the median survival time of animals bearingmelanoma tumors12

Despite the potential application of curcumin in the pre-vention and treatment of skin cancer this drug exhibitsvery low bioavailability due to its extremely poor watersolubility which results in low drug absorption in the gas-trointestinal tract and fast metabolization in the liver13 Inaddition curcumin undergoes alkaline hydrolysis at neu-tral and basic pH being quickly decomposed in physio-logical conditions14 Among the alternatives to overcomethese limitations the association of curcumin with drugdelivery systems has been considered a promising strat-egy to improve its bioavailability13 Recently some stud-ies have demonstrated the potential of nanostructured drugdelivery systems to render hydrophobic agents like cur-cumin dispersible in aqueous media thus circumventingthe pitfalls of its poor water solubility while maintain-ing its cytotoxic activity against a number of cancer celllines15ndash21 However in vivo evaluation of anticancer prop-erties of curcumin-loaded nanocarriers is wanting despitethe great potential of these delivery systems in target-ing drugs directly to cancer tissues On other hand whenadministrated intravenously conventional nanoparticles arerapidly opsonized and removed from circulation by themononuclear phagocyte system (MPS) mainly represented

by the Kupffer cells of the liver and spleen macrophages22

Consequently the contribution of conventional nanoparti-cles towards increasing anticancer drug efficacy is limitedto targeting tumors at the MPS level Coating nanoparti-cle surface with hydrophilic polymers such as polyethy-lene glycol (PEG) so-called Stealth particles has beenshown to reduce clearance by the MPS As a result Stealthnanoparticles remained in the circulation for a longerperiod of time and were also able to extravasate in patho-logical sites such as solid tumors located outside the MPSregions2123

In previous studies we reported the development oflipid and polymeric nanocapsules containing curcuminwith high encapsulation efficiency24 Nanocapsules sus-pensions were prepared using Poloxamer 188 (PluronicF68) or polyethylene glycol 660-hydroxystearate (Solu-tol HS15) as hydrophilic surfactants It is possible thatthese nanocapsule suspensions exhibit different in vitroand in vivo antitumor activities related to the presence ofPEG chains of Solutol HS15 which are located on thesurface of the particles In fact the results described inthe literature concerning the ability of the nanocapsulesmade from Solutol HS15 in to escape from the uptakeby immune cells are contradictory In vivo biodistributionstudies of paclitaxel-loaded lipid nanocapsules shown thatthe blood distribution of the particles was equivalent tothose achieved by other long circulating carriers Similarresults were observed when comparing pharmacokineticsdata (MRT AUC and clearance) of lipid and PLA-PEGnanocapsules indicating stealth properties of the formerwhich were explained by the presence of a comparativelyhigher density of PEG chains on the lipid nanocapsulesurface25 On the other hand Beacuteduneau et al describedthat lipid systems prepared with PEG 660-hydroxystearateare rapidly removed of blood circulation In this studylong circulating properties of particles were observed usingPEG 1500-hydroxystearate in their production and thenstealth properties were considered to be dependent on bothchain length and surface density of surfactant26

Considering the above mentioned this study aims toevaluate and to compare the in vitro and in vivo activityof curcumin-loaded lipid and polymeric nanocapsules sta-bilized by nonionic surfactants on a B16-F10 melanomamurine model Also in vitro studies were carried out onJ774 murine macrophage-like cells to verify the abilityof the nanocapsules to escape from the uptake by themacrophages

2 EXPERIMENTAL DETAILS

21 Materials

Curcumin and poly (DL-lactide) (PLA MW 90000ndash120000) were purchased from Sigma-Aldrich (St LouisMO USA) Soybean hydrogenated lecithin (LIPOID S75-3N) was provided by Lipoid GmbH (Ludwigshafen

J Biomed Nanotechnol 7 406ndash414 2011 407

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Germany) and castor oil was obtained from Via FarmaImportadora Ltda (Satildeo Paulo Brazil) Polyethylene gly-col 660-12-hydroxystearate (Solutol HS15) and Poloxamer(Pluronic F68) were kindly donated by BASF ChemicalCompany (Ludwigshafen Germany) Sodium chloride wasobtained from Vetec (Rio de Janeiro Brazil) Except forthe acetonitrile of HPLC grade used in the analysis (CarloErba Milan Italy) all other reagents and solvents were ofanalytical grade

22 Cell Lines

A highly metastatic B16-F10 mouse epithelial-likemelanoma cell line was donated from Bio-Rio (Rio deJaneiro Brazil) The cells were cultured in a Dulbeccorsquosmodified Eaglersquos medium (DMEM Sigma-Aldrich) pH74 supplemented with 10 fetal calf serum 100 Umlpenicillin 100 gml streptomycin and 10 mM HEPESin a humidified atmosphere containing 5 CO2 at 37 CFor in vivo experiments cells were harvested with atrypsinEDTA (005003 wv) solution washed and inocu-lated into mice in phosphate-buffered saline (PBS pH 74)For uptake studies the murine macrophage-like

J774 cells were cultured in RMPI 1640 medium (Sigma-Aldrich) supplemented with 10 fetal calf serum100 Uml penicillin 100 gml streptomycin and025 gml amphotericin B in a humidified atmospherecontaining 5 CO2 at 37 C

23 Preparation of the Nanocapsule Suspensions

231 Lipid Nanocapsules Suspensions

Lipid nanocapsule suspensions (LNC) were prepared usingthe phase inversion method27 An aqueous phase contain-ing 840 g distilled water 075 g NaCl and 420 g SolutolHS15 was added to the oil phase containing 140 g castoroil 014 g lecithin and 10 mg curcumin both previouslyheated to 90 C under magnetic stirring Three temper-ature cycles alternating from 60 to 95 C were appliedto reach the inversion process During the last coolingthe suspensions were rapidly diluted with 250 ml coldwater (approximately 0 C) and continuously stirred for30 minutes

232 Polymeric Nanocapsules Suspensions

Polymeric nanocapsule suspensions (PNC) were preparedusing the interfacial deposition process after solventdisplacement28 Briefly 600 mg PLA 0250 ml castoroil and 25 mg curcumin were dissolved in 2 ml ofacetone The resulting solution was added to a 10 mlacetoneethanol (6040 vv) mixture containing 250 mglecithin and the final volume was adjusted to 120 ml Thisorganic phase was poured into an aqueous phase (260 ml)

containing 0375 (wv) of Pluronic F68 (PNCP or Solu-tol HS15 (PNCS maintained under magnetic stirringThe organic solvents were then eliminated by evaporationunder reduced pressure and the final volume of the col-loidal suspension was adjusted to 100 mlLipid and polymeric nanocapsules suspensions were fil-

tered through 8 m pore size of filter paper (J-ProlabSatildeo Joseacute dos Pinhais Brazil) The samples were storedprotected from light at room temperature before useUnloaded lipid and polymeric nanocapsule suspensionswere prepared and treated in the same manner as thesamples

24 Determination of Encapsulation Efficiency andDrug Content

Entrapment efficiency and drug content were estimatedafter determination of the curcumin concentration in thenanocapsule suspensions by fluorescence spectrophotom-etry using a previously validated method29 The analysiswas carried out using a Hitachi F4500 Spectrofluorimeterequipped with a thermostated cell holder set at 250 CThe samples were continuously stirred in a standard1 cm quartz cell Both slits of excitation and emis-sion monochromators were adjusted to 50 nm The sam-ples were excited at 397 nm and the emission spectrawere recorded from 440 to 600 nm The relative flu-orescence intensities of the samples were measured atemi = 508 nm and compared with that obtained by acurcumin standard solution A calibration curve for cur-cumin in acetonitrile was constructed and was linear overthe range of 01 to 07 gml (R2 = 09957) The entrap-ment efficiency () was estimated as being the differencebetween the total concentration of curcumin found in thenanocapsule suspensions after their complete dissolutionin acetonitrile and the concentration of drug in the super-natant obtained by suspension ultrafiltrationcentrifugationprocedure using Microcon Centrifugal Filter Deviceswith Ultracel YM-100 membrane (nominal molecularweight limit of 100000 Da Millipore Corp USA) Thedrug content was expressed in g of curcuminml ofsuspension

25 Determination of Particle Size and Zeta Potential

The mean particle diameter and zeta potential were deter-mined by photon correlation spectroscopy and laser-Doppler anemometry respectively using a Zetasizer NanoSeries (Malvern Instruments Worcestershire UK) Themeasurements were taken at 25 C after appropriate dilu-tion of the samples in distilled water Each size analysislasted 120 s and was performed with an angle detectionof 173 For measurements of zeta potential ( nanocap-sule samples were placed in an electrophoretic cell wherea potential of plusmn150 mV was established The potential

408 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

values were calculated from mean electrophoretic mobilityvalues using Smoluchowskirsquos equation

26 pH Measurements

After preparation the pH of the nanocapsule suspensionswere determined using a pH meter (PHTEK pH100 SatildeoPaulo Brazil) previously calibrated with buffer solutionspH 40 and 70

27 Evaluation of In Vitro Cytotoxicity

In vitro cell viability was assessed using MTT(3-(45-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide)assay30 Free curcumin or curcumin-loaded lipid andpolymeric nanocapsule suspensions were added to B16-F10 cells at different concentrations Cells were incubatedat 37 C for 24 48 or 72 hours After incubation MTTwas added to each well followed by 3 h incubationCells were centrifugated the supernatant was discardedand the formazan precipitated was dissolved with 100 L004 N isopropyl alcoholHCl solution The absorbancewas determined at 540 nm using a microplate reader Con-trol groups were only plated with cell culture medium andMTT reagent All assays were performed in triplicate Thestatistical analysis was performed using analysis of vari-ance (ANOVA) followed by Bonferonirsquos post-hoc usingthe Graph-Pad Prism (San Diego CA USA) software

28 Morphological Assessment of Apoptosis

Apoptosis was verified as described by Geng et al31

B16-F10 cells were incubated with free curcumin andcurcumin-loaded lipid and polymeric nanocapsule suspen-sions for 48 hours Then acridine orange and ethidiumbromide dyes at 10 gml were mixed with cells andthe slide was examined under a fluorescence microscope(Olympus BX41) using 40 x objective Viable cells exhib-ited green fluorescence (acridine orange staining) whereasapoptotic cells exhibited an orange-red nuclear fluores-cence (ethidium bromide staining)

29 Macrophage Uptake Studies

Phagocytic uptake and intracellular fate of the fluores-cent free curcumin or curcumin-loaded nanocapsules weremonitored by fluorescence microscopy upon incubationwith J774 cells Cell uptake was determined in cells grownnear to confluence on rounded coverslips in 24-well platesUpon 60 minutes incubation with free curcumin (previ-ously dissolved in DMSO) or curcumin-loaded lipid orpolymeric nanocapsules at 37 C in the dark the nanocap-sules suspensions were removed cells were washed threetimes with phosphate buffered saline (PBS) and cover-slips were mounted on a fluorescence microscope Cell-associated fluorescence of curcumin was monitored with a

Nikon Alphaphot 2 YS2 fluorescence microscope (NikonCorporation Instruments Japan) Images of the cells wereacquired in the emission mode with 40 x objective afterexciting the sample at 450 to 490 nm using barrier fil-ter Fluorescence imaging in the wavelength of 505 nmwas collected In order to quantify the macrophage uptakeimage analysis was performed using computer programScion Image and the mean pixel density of cells was deter-mined by the program Sacci

210 Evaluation of In Vivo Antitumor Activity

Male specific pathogen-free C57BL6 3-month-old micewere used The animals were kept in a light controlledroom (12-hour-light-dark cycle) at a room temperatureof 23plusmn 2 C and 60plusmn 10 humidity Food and waterwere given ad libitum All animal studies were carriedout in accordance with the procedures outlined in proto-col number PP00161CEUA for the care and ethical useof animals in research (CEUAUFSC Florianoacutepolis SCBrazil)B16-F10 melanoma cells (1times 106 cells suspended in

100 l of DMEM medium) were subcutaneously inoc-ulated into the back of male C57BL6 mice (n = 8)Eleven days after inoculation of B16-F10 cells free cur-cumin unloaded or curcumin-loaded nanocapsule suspen-sions were administered intraperitoneally at a dose of6 mgkg twice a week corresponding to a total of 4 dosesaccording to the experimental schedule tested In orderto improve the dispersion of curcumin in the vehicle thedrug was suspended in PBS pH 60 containing 03 (wv)of carboxymethylcellulose and 005 (wv) of Tween 80The negative control received the cells and PBS while thepositive control received the cells and cisplatin which wasgiven at 1 mgkg by intraperitoneal route once a weekTumor volumes were measured in the days of treat-

ment throughout 21 days of experiment using a precisioncalipter and estimated according to the following stan-dard formula tumor volume (mm3 = width2 times lengthtimes052 according to Lee et al32 The animal body weightwas verified in the begging and the end of the experi-ment Twenty-one days after inoculation mice were sac-rificed by cervical dislocation and tumors were excisedand weighted The statistical analysis was performed usinganalysis of variance followed by Dunnet post-hoc

3 RESULTS AND DISCUSSION

31 Characterization of the Lipid and PolymericNanocapsule Suspensions

A summary of the physicochemical properties ofthe nanocapsule suspensions is presented in Table ICurcumin-loaded polymeric nanocapsule suspensions(Cur-PNCP and Cur-PNCS prepared by the nanoprecip-itation method displayed a mean particle diameter of

J Biomed Nanotechnol 7 406ndash414 2011 409

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Table I Summary of the physicochemical properties of curcumin-loaded lipid and polymeric nanocapsule suspensions

Mean diameter (nm) Zeta potential Drug contentSample (polydispersity index) (mV) (gml) pH

Cur-LNC 347 (0247) minus45 25890plusmn054 595Cur-PNCP 1377 (0208) minus329 24999plusmn111 465Cur-PNCS 1433 (0191) minus264 25616plusmn075 460

around 140 nm while curcumin-loaded lipid nanocapsule(Cur-LNC) produced by the phase inversion process showna mean size of around 40 nm Zeta potential valueswere minus329 and minus264 mV for Cur-PNCP and Cur-PNCS respectively and minus45 mV for Cur-LNC The highzeta potential values observed for PLA nanocapsules havebeen attributed to the carboxyl end groups of the poly-mer which constitute the polymeric wall of the particlesThe negative zeta potential of lipid nanocapsules may becaused by the presence of soybean lecithin phospholipidswhich are preferentially located on the nanocapsule sur-face However negative charge was lower than expectedprobably due to the masking effect produced by the pres-ence of PEG chains of the Solutol HS15 on the surface ofLNC as recently described by Mazzarino et al24 Encap-sulation efficiency was above 99 for all formulationsThese high values can be explained by the poor watersolubility of curcumin in the acidic external phase of thenanosuspensions Drug contents were very close in allpreparations with values around 250 g middotmlminus1

32 Cytotoxic Effect of Free and EncapsulatedCurcumin on B16-F10 Melanoma

Data obtained in the in vitro cytotoxic study with B16-F10 melanoma cells are shown in Figure 1 Incuba-tion of B16-F10 melanoma cells with free curcuminand curcumin-loaded nanocapsules caused significantreduction of cell viability in a concentration- andtime-dependent manner After 24 hours of incubation sta-tistically significant reduction in the number of viablecells was observed for free curcumin at a concentration ofge25 M when compared to the control group (P lt 005)At 100 M concentration the incubation of cells with freecurcumin caused a 75 reduction in the number of viablecells while a 25 reduction occurred after incubation ofCur-LNC and Cur-PNCS at same time On the contraryafter 48 and 72 hours of incubation Cur-LNC induced asignificantly higher cytotoxic effect than free drug at thelowest tested concentration (10 M) producing a 75reduction in the number of viable cells (P lt 005) At10 M and 72 hours of incubation the cytotoxic effectin increasing order was Cur-LNCgt free curcumingt Cur-PNCS Cur-PNCP only exhibited cytotoxic effect at con-centration of ge25 M after 72 hours of incubation when

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

24 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

48 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

72 h

Fig 1 Cytotoxic effect of free curcumin and curcumin-loaded nanocap-sules suspensions on B16-F10 melanoma cells after 24 48 and 72hours of incubation Free curcumin (circel) Cur-LNC (square) Cur-PNCP (triangle) and Cur-PNCS (inversed triangle) Optical density ofcontrol groups was taken as 100 cell viability was confirmed byTrypan Blue exclusion method lowastP lt 005 compared with the controlgroup P lt 005 compared with free curcumin at the same concentrationn= 3

410 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

and curcumin have been reported to possess substantialchemopreventive properties because of their antioxidantand anti-inflammatory ability Concerning skin cancerslaboratory and epidemiological studies have evidencedthat routine consumption of these polyphenol com-pounds may favorably provide some antiphotocarcinogenicprotection against skin disorders caused by solar UVradiation6

Curcumin is a yellow polyphenol derived from the rhi-zome of turmeric (Curcuma longa L) Extensive researchhas pointed out its potential in the prevention and treat-ment of cancer and other diseases Several studies car-ried out over the past two decades have indicated thatcurcumin displays antimutagenic activity and inhibits theradiation- as well as chemical carcinogen-induced neoplas-tic lesions in many tumor models including skin cancer6

The anticancer potential of curcumin is related to its abil-ity to down-regulate transcription factors NF-B AP-1Erg-1 and MAPK to down-regulate the expression ofCOX-2 LOX NOS MMP TNF- IL-1 and cyclin D1to modulate cytochrome P450 function to down-regulategrowth factor receptors and to inhibit the activity of theoncogenes ras fos jun and myc7ndash9 In vivo studies havedemonstrated that curcumin inhibits lung tumor noduleformation in mice inoculated with B16-F10 melanomacells and increases life span by inhibiting the expressionof metalloproteinases1011 Moreover the combined treat-ment with this polyphenol and a prophylactic immunepreparation caused substantial growth inhibition of B16-Rmelanoma cells resistant to doxorubicin and significantlyincreased the median survival time of animals bearingmelanoma tumors12

Despite the potential application of curcumin in the pre-vention and treatment of skin cancer this drug exhibitsvery low bioavailability due to its extremely poor watersolubility which results in low drug absorption in the gas-trointestinal tract and fast metabolization in the liver13 Inaddition curcumin undergoes alkaline hydrolysis at neu-tral and basic pH being quickly decomposed in physio-logical conditions14 Among the alternatives to overcomethese limitations the association of curcumin with drugdelivery systems has been considered a promising strat-egy to improve its bioavailability13 Recently some stud-ies have demonstrated the potential of nanostructured drugdelivery systems to render hydrophobic agents like cur-cumin dispersible in aqueous media thus circumventingthe pitfalls of its poor water solubility while maintain-ing its cytotoxic activity against a number of cancer celllines15ndash21 However in vivo evaluation of anticancer prop-erties of curcumin-loaded nanocarriers is wanting despitethe great potential of these delivery systems in target-ing drugs directly to cancer tissues On other hand whenadministrated intravenously conventional nanoparticles arerapidly opsonized and removed from circulation by themononuclear phagocyte system (MPS) mainly represented

by the Kupffer cells of the liver and spleen macrophages22

Consequently the contribution of conventional nanoparti-cles towards increasing anticancer drug efficacy is limitedto targeting tumors at the MPS level Coating nanoparti-cle surface with hydrophilic polymers such as polyethy-lene glycol (PEG) so-called Stealth particles has beenshown to reduce clearance by the MPS As a result Stealthnanoparticles remained in the circulation for a longerperiod of time and were also able to extravasate in patho-logical sites such as solid tumors located outside the MPSregions2123

In previous studies we reported the development oflipid and polymeric nanocapsules containing curcuminwith high encapsulation efficiency24 Nanocapsules sus-pensions were prepared using Poloxamer 188 (PluronicF68) or polyethylene glycol 660-hydroxystearate (Solu-tol HS15) as hydrophilic surfactants It is possible thatthese nanocapsule suspensions exhibit different in vitroand in vivo antitumor activities related to the presence ofPEG chains of Solutol HS15 which are located on thesurface of the particles In fact the results described inthe literature concerning the ability of the nanocapsulesmade from Solutol HS15 in to escape from the uptakeby immune cells are contradictory In vivo biodistributionstudies of paclitaxel-loaded lipid nanocapsules shown thatthe blood distribution of the particles was equivalent tothose achieved by other long circulating carriers Similarresults were observed when comparing pharmacokineticsdata (MRT AUC and clearance) of lipid and PLA-PEGnanocapsules indicating stealth properties of the formerwhich were explained by the presence of a comparativelyhigher density of PEG chains on the lipid nanocapsulesurface25 On the other hand Beacuteduneau et al describedthat lipid systems prepared with PEG 660-hydroxystearateare rapidly removed of blood circulation In this studylong circulating properties of particles were observed usingPEG 1500-hydroxystearate in their production and thenstealth properties were considered to be dependent on bothchain length and surface density of surfactant26

Considering the above mentioned this study aims toevaluate and to compare the in vitro and in vivo activityof curcumin-loaded lipid and polymeric nanocapsules sta-bilized by nonionic surfactants on a B16-F10 melanomamurine model Also in vitro studies were carried out onJ774 murine macrophage-like cells to verify the abilityof the nanocapsules to escape from the uptake by themacrophages

2 EXPERIMENTAL DETAILS

21 Materials

Curcumin and poly (DL-lactide) (PLA MW 90000ndash120000) were purchased from Sigma-Aldrich (St LouisMO USA) Soybean hydrogenated lecithin (LIPOID S75-3N) was provided by Lipoid GmbH (Ludwigshafen

J Biomed Nanotechnol 7 406ndash414 2011 407

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Germany) and castor oil was obtained from Via FarmaImportadora Ltda (Satildeo Paulo Brazil) Polyethylene gly-col 660-12-hydroxystearate (Solutol HS15) and Poloxamer(Pluronic F68) were kindly donated by BASF ChemicalCompany (Ludwigshafen Germany) Sodium chloride wasobtained from Vetec (Rio de Janeiro Brazil) Except forthe acetonitrile of HPLC grade used in the analysis (CarloErba Milan Italy) all other reagents and solvents were ofanalytical grade

22 Cell Lines

A highly metastatic B16-F10 mouse epithelial-likemelanoma cell line was donated from Bio-Rio (Rio deJaneiro Brazil) The cells were cultured in a Dulbeccorsquosmodified Eaglersquos medium (DMEM Sigma-Aldrich) pH74 supplemented with 10 fetal calf serum 100 Umlpenicillin 100 gml streptomycin and 10 mM HEPESin a humidified atmosphere containing 5 CO2 at 37 CFor in vivo experiments cells were harvested with atrypsinEDTA (005003 wv) solution washed and inocu-lated into mice in phosphate-buffered saline (PBS pH 74)For uptake studies the murine macrophage-like

J774 cells were cultured in RMPI 1640 medium (Sigma-Aldrich) supplemented with 10 fetal calf serum100 Uml penicillin 100 gml streptomycin and025 gml amphotericin B in a humidified atmospherecontaining 5 CO2 at 37 C

23 Preparation of the Nanocapsule Suspensions

231 Lipid Nanocapsules Suspensions

Lipid nanocapsule suspensions (LNC) were prepared usingthe phase inversion method27 An aqueous phase contain-ing 840 g distilled water 075 g NaCl and 420 g SolutolHS15 was added to the oil phase containing 140 g castoroil 014 g lecithin and 10 mg curcumin both previouslyheated to 90 C under magnetic stirring Three temper-ature cycles alternating from 60 to 95 C were appliedto reach the inversion process During the last coolingthe suspensions were rapidly diluted with 250 ml coldwater (approximately 0 C) and continuously stirred for30 minutes

232 Polymeric Nanocapsules Suspensions

Polymeric nanocapsule suspensions (PNC) were preparedusing the interfacial deposition process after solventdisplacement28 Briefly 600 mg PLA 0250 ml castoroil and 25 mg curcumin were dissolved in 2 ml ofacetone The resulting solution was added to a 10 mlacetoneethanol (6040 vv) mixture containing 250 mglecithin and the final volume was adjusted to 120 ml Thisorganic phase was poured into an aqueous phase (260 ml)

containing 0375 (wv) of Pluronic F68 (PNCP or Solu-tol HS15 (PNCS maintained under magnetic stirringThe organic solvents were then eliminated by evaporationunder reduced pressure and the final volume of the col-loidal suspension was adjusted to 100 mlLipid and polymeric nanocapsules suspensions were fil-

tered through 8 m pore size of filter paper (J-ProlabSatildeo Joseacute dos Pinhais Brazil) The samples were storedprotected from light at room temperature before useUnloaded lipid and polymeric nanocapsule suspensionswere prepared and treated in the same manner as thesamples

24 Determination of Encapsulation Efficiency andDrug Content

Entrapment efficiency and drug content were estimatedafter determination of the curcumin concentration in thenanocapsule suspensions by fluorescence spectrophotom-etry using a previously validated method29 The analysiswas carried out using a Hitachi F4500 Spectrofluorimeterequipped with a thermostated cell holder set at 250 CThe samples were continuously stirred in a standard1 cm quartz cell Both slits of excitation and emis-sion monochromators were adjusted to 50 nm The sam-ples were excited at 397 nm and the emission spectrawere recorded from 440 to 600 nm The relative flu-orescence intensities of the samples were measured atemi = 508 nm and compared with that obtained by acurcumin standard solution A calibration curve for cur-cumin in acetonitrile was constructed and was linear overthe range of 01 to 07 gml (R2 = 09957) The entrap-ment efficiency () was estimated as being the differencebetween the total concentration of curcumin found in thenanocapsule suspensions after their complete dissolutionin acetonitrile and the concentration of drug in the super-natant obtained by suspension ultrafiltrationcentrifugationprocedure using Microcon Centrifugal Filter Deviceswith Ultracel YM-100 membrane (nominal molecularweight limit of 100000 Da Millipore Corp USA) Thedrug content was expressed in g of curcuminml ofsuspension

25 Determination of Particle Size and Zeta Potential

The mean particle diameter and zeta potential were deter-mined by photon correlation spectroscopy and laser-Doppler anemometry respectively using a Zetasizer NanoSeries (Malvern Instruments Worcestershire UK) Themeasurements were taken at 25 C after appropriate dilu-tion of the samples in distilled water Each size analysislasted 120 s and was performed with an angle detectionof 173 For measurements of zeta potential ( nanocap-sule samples were placed in an electrophoretic cell wherea potential of plusmn150 mV was established The potential

408 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

values were calculated from mean electrophoretic mobilityvalues using Smoluchowskirsquos equation

26 pH Measurements

After preparation the pH of the nanocapsule suspensionswere determined using a pH meter (PHTEK pH100 SatildeoPaulo Brazil) previously calibrated with buffer solutionspH 40 and 70

27 Evaluation of In Vitro Cytotoxicity

In vitro cell viability was assessed using MTT(3-(45-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide)assay30 Free curcumin or curcumin-loaded lipid andpolymeric nanocapsule suspensions were added to B16-F10 cells at different concentrations Cells were incubatedat 37 C for 24 48 or 72 hours After incubation MTTwas added to each well followed by 3 h incubationCells were centrifugated the supernatant was discardedand the formazan precipitated was dissolved with 100 L004 N isopropyl alcoholHCl solution The absorbancewas determined at 540 nm using a microplate reader Con-trol groups were only plated with cell culture medium andMTT reagent All assays were performed in triplicate Thestatistical analysis was performed using analysis of vari-ance (ANOVA) followed by Bonferonirsquos post-hoc usingthe Graph-Pad Prism (San Diego CA USA) software

28 Morphological Assessment of Apoptosis

Apoptosis was verified as described by Geng et al31

B16-F10 cells were incubated with free curcumin andcurcumin-loaded lipid and polymeric nanocapsule suspen-sions for 48 hours Then acridine orange and ethidiumbromide dyes at 10 gml were mixed with cells andthe slide was examined under a fluorescence microscope(Olympus BX41) using 40 x objective Viable cells exhib-ited green fluorescence (acridine orange staining) whereasapoptotic cells exhibited an orange-red nuclear fluores-cence (ethidium bromide staining)

29 Macrophage Uptake Studies

Phagocytic uptake and intracellular fate of the fluores-cent free curcumin or curcumin-loaded nanocapsules weremonitored by fluorescence microscopy upon incubationwith J774 cells Cell uptake was determined in cells grownnear to confluence on rounded coverslips in 24-well platesUpon 60 minutes incubation with free curcumin (previ-ously dissolved in DMSO) or curcumin-loaded lipid orpolymeric nanocapsules at 37 C in the dark the nanocap-sules suspensions were removed cells were washed threetimes with phosphate buffered saline (PBS) and cover-slips were mounted on a fluorescence microscope Cell-associated fluorescence of curcumin was monitored with a

Nikon Alphaphot 2 YS2 fluorescence microscope (NikonCorporation Instruments Japan) Images of the cells wereacquired in the emission mode with 40 x objective afterexciting the sample at 450 to 490 nm using barrier fil-ter Fluorescence imaging in the wavelength of 505 nmwas collected In order to quantify the macrophage uptakeimage analysis was performed using computer programScion Image and the mean pixel density of cells was deter-mined by the program Sacci

210 Evaluation of In Vivo Antitumor Activity

Male specific pathogen-free C57BL6 3-month-old micewere used The animals were kept in a light controlledroom (12-hour-light-dark cycle) at a room temperatureof 23plusmn 2 C and 60plusmn 10 humidity Food and waterwere given ad libitum All animal studies were carriedout in accordance with the procedures outlined in proto-col number PP00161CEUA for the care and ethical useof animals in research (CEUAUFSC Florianoacutepolis SCBrazil)B16-F10 melanoma cells (1times 106 cells suspended in

100 l of DMEM medium) were subcutaneously inoc-ulated into the back of male C57BL6 mice (n = 8)Eleven days after inoculation of B16-F10 cells free cur-cumin unloaded or curcumin-loaded nanocapsule suspen-sions were administered intraperitoneally at a dose of6 mgkg twice a week corresponding to a total of 4 dosesaccording to the experimental schedule tested In orderto improve the dispersion of curcumin in the vehicle thedrug was suspended in PBS pH 60 containing 03 (wv)of carboxymethylcellulose and 005 (wv) of Tween 80The negative control received the cells and PBS while thepositive control received the cells and cisplatin which wasgiven at 1 mgkg by intraperitoneal route once a weekTumor volumes were measured in the days of treat-

ment throughout 21 days of experiment using a precisioncalipter and estimated according to the following stan-dard formula tumor volume (mm3 = width2 times lengthtimes052 according to Lee et al32 The animal body weightwas verified in the begging and the end of the experi-ment Twenty-one days after inoculation mice were sac-rificed by cervical dislocation and tumors were excisedand weighted The statistical analysis was performed usinganalysis of variance followed by Dunnet post-hoc

3 RESULTS AND DISCUSSION

31 Characterization of the Lipid and PolymericNanocapsule Suspensions

A summary of the physicochemical properties ofthe nanocapsule suspensions is presented in Table ICurcumin-loaded polymeric nanocapsule suspensions(Cur-PNCP and Cur-PNCS prepared by the nanoprecip-itation method displayed a mean particle diameter of

J Biomed Nanotechnol 7 406ndash414 2011 409

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Table I Summary of the physicochemical properties of curcumin-loaded lipid and polymeric nanocapsule suspensions

Mean diameter (nm) Zeta potential Drug contentSample (polydispersity index) (mV) (gml) pH

Cur-LNC 347 (0247) minus45 25890plusmn054 595Cur-PNCP 1377 (0208) minus329 24999plusmn111 465Cur-PNCS 1433 (0191) minus264 25616plusmn075 460

around 140 nm while curcumin-loaded lipid nanocapsule(Cur-LNC) produced by the phase inversion process showna mean size of around 40 nm Zeta potential valueswere minus329 and minus264 mV for Cur-PNCP and Cur-PNCS respectively and minus45 mV for Cur-LNC The highzeta potential values observed for PLA nanocapsules havebeen attributed to the carboxyl end groups of the poly-mer which constitute the polymeric wall of the particlesThe negative zeta potential of lipid nanocapsules may becaused by the presence of soybean lecithin phospholipidswhich are preferentially located on the nanocapsule sur-face However negative charge was lower than expectedprobably due to the masking effect produced by the pres-ence of PEG chains of the Solutol HS15 on the surface ofLNC as recently described by Mazzarino et al24 Encap-sulation efficiency was above 99 for all formulationsThese high values can be explained by the poor watersolubility of curcumin in the acidic external phase of thenanosuspensions Drug contents were very close in allpreparations with values around 250 g middotmlminus1

32 Cytotoxic Effect of Free and EncapsulatedCurcumin on B16-F10 Melanoma

Data obtained in the in vitro cytotoxic study with B16-F10 melanoma cells are shown in Figure 1 Incuba-tion of B16-F10 melanoma cells with free curcuminand curcumin-loaded nanocapsules caused significantreduction of cell viability in a concentration- andtime-dependent manner After 24 hours of incubation sta-tistically significant reduction in the number of viablecells was observed for free curcumin at a concentration ofge25 M when compared to the control group (P lt 005)At 100 M concentration the incubation of cells with freecurcumin caused a 75 reduction in the number of viablecells while a 25 reduction occurred after incubation ofCur-LNC and Cur-PNCS at same time On the contraryafter 48 and 72 hours of incubation Cur-LNC induced asignificantly higher cytotoxic effect than free drug at thelowest tested concentration (10 M) producing a 75reduction in the number of viable cells (P lt 005) At10 M and 72 hours of incubation the cytotoxic effectin increasing order was Cur-LNCgt free curcumingt Cur-PNCS Cur-PNCP only exhibited cytotoxic effect at con-centration of ge25 M after 72 hours of incubation when

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

24 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

48 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

72 h

Fig 1 Cytotoxic effect of free curcumin and curcumin-loaded nanocap-sules suspensions on B16-F10 melanoma cells after 24 48 and 72hours of incubation Free curcumin (circel) Cur-LNC (square) Cur-PNCP (triangle) and Cur-PNCS (inversed triangle) Optical density ofcontrol groups was taken as 100 cell viability was confirmed byTrypan Blue exclusion method lowastP lt 005 compared with the controlgroup P lt 005 compared with free curcumin at the same concentrationn= 3

410 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Germany) and castor oil was obtained from Via FarmaImportadora Ltda (Satildeo Paulo Brazil) Polyethylene gly-col 660-12-hydroxystearate (Solutol HS15) and Poloxamer(Pluronic F68) were kindly donated by BASF ChemicalCompany (Ludwigshafen Germany) Sodium chloride wasobtained from Vetec (Rio de Janeiro Brazil) Except forthe acetonitrile of HPLC grade used in the analysis (CarloErba Milan Italy) all other reagents and solvents were ofanalytical grade

22 Cell Lines

A highly metastatic B16-F10 mouse epithelial-likemelanoma cell line was donated from Bio-Rio (Rio deJaneiro Brazil) The cells were cultured in a Dulbeccorsquosmodified Eaglersquos medium (DMEM Sigma-Aldrich) pH74 supplemented with 10 fetal calf serum 100 Umlpenicillin 100 gml streptomycin and 10 mM HEPESin a humidified atmosphere containing 5 CO2 at 37 CFor in vivo experiments cells were harvested with atrypsinEDTA (005003 wv) solution washed and inocu-lated into mice in phosphate-buffered saline (PBS pH 74)For uptake studies the murine macrophage-like

J774 cells were cultured in RMPI 1640 medium (Sigma-Aldrich) supplemented with 10 fetal calf serum100 Uml penicillin 100 gml streptomycin and025 gml amphotericin B in a humidified atmospherecontaining 5 CO2 at 37 C

23 Preparation of the Nanocapsule Suspensions

231 Lipid Nanocapsules Suspensions

Lipid nanocapsule suspensions (LNC) were prepared usingthe phase inversion method27 An aqueous phase contain-ing 840 g distilled water 075 g NaCl and 420 g SolutolHS15 was added to the oil phase containing 140 g castoroil 014 g lecithin and 10 mg curcumin both previouslyheated to 90 C under magnetic stirring Three temper-ature cycles alternating from 60 to 95 C were appliedto reach the inversion process During the last coolingthe suspensions were rapidly diluted with 250 ml coldwater (approximately 0 C) and continuously stirred for30 minutes

232 Polymeric Nanocapsules Suspensions

Polymeric nanocapsule suspensions (PNC) were preparedusing the interfacial deposition process after solventdisplacement28 Briefly 600 mg PLA 0250 ml castoroil and 25 mg curcumin were dissolved in 2 ml ofacetone The resulting solution was added to a 10 mlacetoneethanol (6040 vv) mixture containing 250 mglecithin and the final volume was adjusted to 120 ml Thisorganic phase was poured into an aqueous phase (260 ml)

containing 0375 (wv) of Pluronic F68 (PNCP or Solu-tol HS15 (PNCS maintained under magnetic stirringThe organic solvents were then eliminated by evaporationunder reduced pressure and the final volume of the col-loidal suspension was adjusted to 100 mlLipid and polymeric nanocapsules suspensions were fil-

tered through 8 m pore size of filter paper (J-ProlabSatildeo Joseacute dos Pinhais Brazil) The samples were storedprotected from light at room temperature before useUnloaded lipid and polymeric nanocapsule suspensionswere prepared and treated in the same manner as thesamples

24 Determination of Encapsulation Efficiency andDrug Content

Entrapment efficiency and drug content were estimatedafter determination of the curcumin concentration in thenanocapsule suspensions by fluorescence spectrophotom-etry using a previously validated method29 The analysiswas carried out using a Hitachi F4500 Spectrofluorimeterequipped with a thermostated cell holder set at 250 CThe samples were continuously stirred in a standard1 cm quartz cell Both slits of excitation and emis-sion monochromators were adjusted to 50 nm The sam-ples were excited at 397 nm and the emission spectrawere recorded from 440 to 600 nm The relative flu-orescence intensities of the samples were measured atemi = 508 nm and compared with that obtained by acurcumin standard solution A calibration curve for cur-cumin in acetonitrile was constructed and was linear overthe range of 01 to 07 gml (R2 = 09957) The entrap-ment efficiency () was estimated as being the differencebetween the total concentration of curcumin found in thenanocapsule suspensions after their complete dissolutionin acetonitrile and the concentration of drug in the super-natant obtained by suspension ultrafiltrationcentrifugationprocedure using Microcon Centrifugal Filter Deviceswith Ultracel YM-100 membrane (nominal molecularweight limit of 100000 Da Millipore Corp USA) Thedrug content was expressed in g of curcuminml ofsuspension

25 Determination of Particle Size and Zeta Potential

The mean particle diameter and zeta potential were deter-mined by photon correlation spectroscopy and laser-Doppler anemometry respectively using a Zetasizer NanoSeries (Malvern Instruments Worcestershire UK) Themeasurements were taken at 25 C after appropriate dilu-tion of the samples in distilled water Each size analysislasted 120 s and was performed with an angle detectionof 173 For measurements of zeta potential ( nanocap-sule samples were placed in an electrophoretic cell wherea potential of plusmn150 mV was established The potential

408 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

values were calculated from mean electrophoretic mobilityvalues using Smoluchowskirsquos equation

26 pH Measurements

After preparation the pH of the nanocapsule suspensionswere determined using a pH meter (PHTEK pH100 SatildeoPaulo Brazil) previously calibrated with buffer solutionspH 40 and 70

27 Evaluation of In Vitro Cytotoxicity

In vitro cell viability was assessed using MTT(3-(45-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide)assay30 Free curcumin or curcumin-loaded lipid andpolymeric nanocapsule suspensions were added to B16-F10 cells at different concentrations Cells were incubatedat 37 C for 24 48 or 72 hours After incubation MTTwas added to each well followed by 3 h incubationCells were centrifugated the supernatant was discardedand the formazan precipitated was dissolved with 100 L004 N isopropyl alcoholHCl solution The absorbancewas determined at 540 nm using a microplate reader Con-trol groups were only plated with cell culture medium andMTT reagent All assays were performed in triplicate Thestatistical analysis was performed using analysis of vari-ance (ANOVA) followed by Bonferonirsquos post-hoc usingthe Graph-Pad Prism (San Diego CA USA) software

28 Morphological Assessment of Apoptosis

Apoptosis was verified as described by Geng et al31

B16-F10 cells were incubated with free curcumin andcurcumin-loaded lipid and polymeric nanocapsule suspen-sions for 48 hours Then acridine orange and ethidiumbromide dyes at 10 gml were mixed with cells andthe slide was examined under a fluorescence microscope(Olympus BX41) using 40 x objective Viable cells exhib-ited green fluorescence (acridine orange staining) whereasapoptotic cells exhibited an orange-red nuclear fluores-cence (ethidium bromide staining)

29 Macrophage Uptake Studies

Phagocytic uptake and intracellular fate of the fluores-cent free curcumin or curcumin-loaded nanocapsules weremonitored by fluorescence microscopy upon incubationwith J774 cells Cell uptake was determined in cells grownnear to confluence on rounded coverslips in 24-well platesUpon 60 minutes incubation with free curcumin (previ-ously dissolved in DMSO) or curcumin-loaded lipid orpolymeric nanocapsules at 37 C in the dark the nanocap-sules suspensions were removed cells were washed threetimes with phosphate buffered saline (PBS) and cover-slips were mounted on a fluorescence microscope Cell-associated fluorescence of curcumin was monitored with a

Nikon Alphaphot 2 YS2 fluorescence microscope (NikonCorporation Instruments Japan) Images of the cells wereacquired in the emission mode with 40 x objective afterexciting the sample at 450 to 490 nm using barrier fil-ter Fluorescence imaging in the wavelength of 505 nmwas collected In order to quantify the macrophage uptakeimage analysis was performed using computer programScion Image and the mean pixel density of cells was deter-mined by the program Sacci

210 Evaluation of In Vivo Antitumor Activity

Male specific pathogen-free C57BL6 3-month-old micewere used The animals were kept in a light controlledroom (12-hour-light-dark cycle) at a room temperatureof 23plusmn 2 C and 60plusmn 10 humidity Food and waterwere given ad libitum All animal studies were carriedout in accordance with the procedures outlined in proto-col number PP00161CEUA for the care and ethical useof animals in research (CEUAUFSC Florianoacutepolis SCBrazil)B16-F10 melanoma cells (1times 106 cells suspended in

100 l of DMEM medium) were subcutaneously inoc-ulated into the back of male C57BL6 mice (n = 8)Eleven days after inoculation of B16-F10 cells free cur-cumin unloaded or curcumin-loaded nanocapsule suspen-sions were administered intraperitoneally at a dose of6 mgkg twice a week corresponding to a total of 4 dosesaccording to the experimental schedule tested In orderto improve the dispersion of curcumin in the vehicle thedrug was suspended in PBS pH 60 containing 03 (wv)of carboxymethylcellulose and 005 (wv) of Tween 80The negative control received the cells and PBS while thepositive control received the cells and cisplatin which wasgiven at 1 mgkg by intraperitoneal route once a weekTumor volumes were measured in the days of treat-

ment throughout 21 days of experiment using a precisioncalipter and estimated according to the following stan-dard formula tumor volume (mm3 = width2 times lengthtimes052 according to Lee et al32 The animal body weightwas verified in the begging and the end of the experi-ment Twenty-one days after inoculation mice were sac-rificed by cervical dislocation and tumors were excisedand weighted The statistical analysis was performed usinganalysis of variance followed by Dunnet post-hoc

3 RESULTS AND DISCUSSION

31 Characterization of the Lipid and PolymericNanocapsule Suspensions

A summary of the physicochemical properties ofthe nanocapsule suspensions is presented in Table ICurcumin-loaded polymeric nanocapsule suspensions(Cur-PNCP and Cur-PNCS prepared by the nanoprecip-itation method displayed a mean particle diameter of

J Biomed Nanotechnol 7 406ndash414 2011 409

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Table I Summary of the physicochemical properties of curcumin-loaded lipid and polymeric nanocapsule suspensions

Mean diameter (nm) Zeta potential Drug contentSample (polydispersity index) (mV) (gml) pH

Cur-LNC 347 (0247) minus45 25890plusmn054 595Cur-PNCP 1377 (0208) minus329 24999plusmn111 465Cur-PNCS 1433 (0191) minus264 25616plusmn075 460

around 140 nm while curcumin-loaded lipid nanocapsule(Cur-LNC) produced by the phase inversion process showna mean size of around 40 nm Zeta potential valueswere minus329 and minus264 mV for Cur-PNCP and Cur-PNCS respectively and minus45 mV for Cur-LNC The highzeta potential values observed for PLA nanocapsules havebeen attributed to the carboxyl end groups of the poly-mer which constitute the polymeric wall of the particlesThe negative zeta potential of lipid nanocapsules may becaused by the presence of soybean lecithin phospholipidswhich are preferentially located on the nanocapsule sur-face However negative charge was lower than expectedprobably due to the masking effect produced by the pres-ence of PEG chains of the Solutol HS15 on the surface ofLNC as recently described by Mazzarino et al24 Encap-sulation efficiency was above 99 for all formulationsThese high values can be explained by the poor watersolubility of curcumin in the acidic external phase of thenanosuspensions Drug contents were very close in allpreparations with values around 250 g middotmlminus1

32 Cytotoxic Effect of Free and EncapsulatedCurcumin on B16-F10 Melanoma

Data obtained in the in vitro cytotoxic study with B16-F10 melanoma cells are shown in Figure 1 Incuba-tion of B16-F10 melanoma cells with free curcuminand curcumin-loaded nanocapsules caused significantreduction of cell viability in a concentration- andtime-dependent manner After 24 hours of incubation sta-tistically significant reduction in the number of viablecells was observed for free curcumin at a concentration ofge25 M when compared to the control group (P lt 005)At 100 M concentration the incubation of cells with freecurcumin caused a 75 reduction in the number of viablecells while a 25 reduction occurred after incubation ofCur-LNC and Cur-PNCS at same time On the contraryafter 48 and 72 hours of incubation Cur-LNC induced asignificantly higher cytotoxic effect than free drug at thelowest tested concentration (10 M) producing a 75reduction in the number of viable cells (P lt 005) At10 M and 72 hours of incubation the cytotoxic effectin increasing order was Cur-LNCgt free curcumingt Cur-PNCS Cur-PNCP only exhibited cytotoxic effect at con-centration of ge25 M after 72 hours of incubation when

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

24 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

48 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

72 h

Fig 1 Cytotoxic effect of free curcumin and curcumin-loaded nanocap-sules suspensions on B16-F10 melanoma cells after 24 48 and 72hours of incubation Free curcumin (circel) Cur-LNC (square) Cur-PNCP (triangle) and Cur-PNCS (inversed triangle) Optical density ofcontrol groups was taken as 100 cell viability was confirmed byTrypan Blue exclusion method lowastP lt 005 compared with the controlgroup P lt 005 compared with free curcumin at the same concentrationn= 3

410 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

values were calculated from mean electrophoretic mobilityvalues using Smoluchowskirsquos equation

26 pH Measurements

After preparation the pH of the nanocapsule suspensionswere determined using a pH meter (PHTEK pH100 SatildeoPaulo Brazil) previously calibrated with buffer solutionspH 40 and 70

27 Evaluation of In Vitro Cytotoxicity

In vitro cell viability was assessed using MTT(3-(45-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide)assay30 Free curcumin or curcumin-loaded lipid andpolymeric nanocapsule suspensions were added to B16-F10 cells at different concentrations Cells were incubatedat 37 C for 24 48 or 72 hours After incubation MTTwas added to each well followed by 3 h incubationCells were centrifugated the supernatant was discardedand the formazan precipitated was dissolved with 100 L004 N isopropyl alcoholHCl solution The absorbancewas determined at 540 nm using a microplate reader Con-trol groups were only plated with cell culture medium andMTT reagent All assays were performed in triplicate Thestatistical analysis was performed using analysis of vari-ance (ANOVA) followed by Bonferonirsquos post-hoc usingthe Graph-Pad Prism (San Diego CA USA) software

28 Morphological Assessment of Apoptosis

Apoptosis was verified as described by Geng et al31

B16-F10 cells were incubated with free curcumin andcurcumin-loaded lipid and polymeric nanocapsule suspen-sions for 48 hours Then acridine orange and ethidiumbromide dyes at 10 gml were mixed with cells andthe slide was examined under a fluorescence microscope(Olympus BX41) using 40 x objective Viable cells exhib-ited green fluorescence (acridine orange staining) whereasapoptotic cells exhibited an orange-red nuclear fluores-cence (ethidium bromide staining)

29 Macrophage Uptake Studies

Phagocytic uptake and intracellular fate of the fluores-cent free curcumin or curcumin-loaded nanocapsules weremonitored by fluorescence microscopy upon incubationwith J774 cells Cell uptake was determined in cells grownnear to confluence on rounded coverslips in 24-well platesUpon 60 minutes incubation with free curcumin (previ-ously dissolved in DMSO) or curcumin-loaded lipid orpolymeric nanocapsules at 37 C in the dark the nanocap-sules suspensions were removed cells were washed threetimes with phosphate buffered saline (PBS) and cover-slips were mounted on a fluorescence microscope Cell-associated fluorescence of curcumin was monitored with a

Nikon Alphaphot 2 YS2 fluorescence microscope (NikonCorporation Instruments Japan) Images of the cells wereacquired in the emission mode with 40 x objective afterexciting the sample at 450 to 490 nm using barrier fil-ter Fluorescence imaging in the wavelength of 505 nmwas collected In order to quantify the macrophage uptakeimage analysis was performed using computer programScion Image and the mean pixel density of cells was deter-mined by the program Sacci

210 Evaluation of In Vivo Antitumor Activity

Male specific pathogen-free C57BL6 3-month-old micewere used The animals were kept in a light controlledroom (12-hour-light-dark cycle) at a room temperatureof 23plusmn 2 C and 60plusmn 10 humidity Food and waterwere given ad libitum All animal studies were carriedout in accordance with the procedures outlined in proto-col number PP00161CEUA for the care and ethical useof animals in research (CEUAUFSC Florianoacutepolis SCBrazil)B16-F10 melanoma cells (1times 106 cells suspended in

100 l of DMEM medium) were subcutaneously inoc-ulated into the back of male C57BL6 mice (n = 8)Eleven days after inoculation of B16-F10 cells free cur-cumin unloaded or curcumin-loaded nanocapsule suspen-sions were administered intraperitoneally at a dose of6 mgkg twice a week corresponding to a total of 4 dosesaccording to the experimental schedule tested In orderto improve the dispersion of curcumin in the vehicle thedrug was suspended in PBS pH 60 containing 03 (wv)of carboxymethylcellulose and 005 (wv) of Tween 80The negative control received the cells and PBS while thepositive control received the cells and cisplatin which wasgiven at 1 mgkg by intraperitoneal route once a weekTumor volumes were measured in the days of treat-

ment throughout 21 days of experiment using a precisioncalipter and estimated according to the following stan-dard formula tumor volume (mm3 = width2 times lengthtimes052 according to Lee et al32 The animal body weightwas verified in the begging and the end of the experi-ment Twenty-one days after inoculation mice were sac-rificed by cervical dislocation and tumors were excisedand weighted The statistical analysis was performed usinganalysis of variance followed by Dunnet post-hoc

3 RESULTS AND DISCUSSION

31 Characterization of the Lipid and PolymericNanocapsule Suspensions

A summary of the physicochemical properties ofthe nanocapsule suspensions is presented in Table ICurcumin-loaded polymeric nanocapsule suspensions(Cur-PNCP and Cur-PNCS prepared by the nanoprecip-itation method displayed a mean particle diameter of

J Biomed Nanotechnol 7 406ndash414 2011 409

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Table I Summary of the physicochemical properties of curcumin-loaded lipid and polymeric nanocapsule suspensions

Mean diameter (nm) Zeta potential Drug contentSample (polydispersity index) (mV) (gml) pH

Cur-LNC 347 (0247) minus45 25890plusmn054 595Cur-PNCP 1377 (0208) minus329 24999plusmn111 465Cur-PNCS 1433 (0191) minus264 25616plusmn075 460

around 140 nm while curcumin-loaded lipid nanocapsule(Cur-LNC) produced by the phase inversion process showna mean size of around 40 nm Zeta potential valueswere minus329 and minus264 mV for Cur-PNCP and Cur-PNCS respectively and minus45 mV for Cur-LNC The highzeta potential values observed for PLA nanocapsules havebeen attributed to the carboxyl end groups of the poly-mer which constitute the polymeric wall of the particlesThe negative zeta potential of lipid nanocapsules may becaused by the presence of soybean lecithin phospholipidswhich are preferentially located on the nanocapsule sur-face However negative charge was lower than expectedprobably due to the masking effect produced by the pres-ence of PEG chains of the Solutol HS15 on the surface ofLNC as recently described by Mazzarino et al24 Encap-sulation efficiency was above 99 for all formulationsThese high values can be explained by the poor watersolubility of curcumin in the acidic external phase of thenanosuspensions Drug contents were very close in allpreparations with values around 250 g middotmlminus1

32 Cytotoxic Effect of Free and EncapsulatedCurcumin on B16-F10 Melanoma

Data obtained in the in vitro cytotoxic study with B16-F10 melanoma cells are shown in Figure 1 Incuba-tion of B16-F10 melanoma cells with free curcuminand curcumin-loaded nanocapsules caused significantreduction of cell viability in a concentration- andtime-dependent manner After 24 hours of incubation sta-tistically significant reduction in the number of viablecells was observed for free curcumin at a concentration ofge25 M when compared to the control group (P lt 005)At 100 M concentration the incubation of cells with freecurcumin caused a 75 reduction in the number of viablecells while a 25 reduction occurred after incubation ofCur-LNC and Cur-PNCS at same time On the contraryafter 48 and 72 hours of incubation Cur-LNC induced asignificantly higher cytotoxic effect than free drug at thelowest tested concentration (10 M) producing a 75reduction in the number of viable cells (P lt 005) At10 M and 72 hours of incubation the cytotoxic effectin increasing order was Cur-LNCgt free curcumingt Cur-PNCS Cur-PNCP only exhibited cytotoxic effect at con-centration of ge25 M after 72 hours of incubation when

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

24 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

48 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

72 h

Fig 1 Cytotoxic effect of free curcumin and curcumin-loaded nanocap-sules suspensions on B16-F10 melanoma cells after 24 48 and 72hours of incubation Free curcumin (circel) Cur-LNC (square) Cur-PNCP (triangle) and Cur-PNCS (inversed triangle) Optical density ofcontrol groups was taken as 100 cell viability was confirmed byTrypan Blue exclusion method lowastP lt 005 compared with the controlgroup P lt 005 compared with free curcumin at the same concentrationn= 3

410 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Table I Summary of the physicochemical properties of curcumin-loaded lipid and polymeric nanocapsule suspensions

Mean diameter (nm) Zeta potential Drug contentSample (polydispersity index) (mV) (gml) pH

Cur-LNC 347 (0247) minus45 25890plusmn054 595Cur-PNCP 1377 (0208) minus329 24999plusmn111 465Cur-PNCS 1433 (0191) minus264 25616plusmn075 460

around 140 nm while curcumin-loaded lipid nanocapsule(Cur-LNC) produced by the phase inversion process showna mean size of around 40 nm Zeta potential valueswere minus329 and minus264 mV for Cur-PNCP and Cur-PNCS respectively and minus45 mV for Cur-LNC The highzeta potential values observed for PLA nanocapsules havebeen attributed to the carboxyl end groups of the poly-mer which constitute the polymeric wall of the particlesThe negative zeta potential of lipid nanocapsules may becaused by the presence of soybean lecithin phospholipidswhich are preferentially located on the nanocapsule sur-face However negative charge was lower than expectedprobably due to the masking effect produced by the pres-ence of PEG chains of the Solutol HS15 on the surface ofLNC as recently described by Mazzarino et al24 Encap-sulation efficiency was above 99 for all formulationsThese high values can be explained by the poor watersolubility of curcumin in the acidic external phase of thenanosuspensions Drug contents were very close in allpreparations with values around 250 g middotmlminus1

32 Cytotoxic Effect of Free and EncapsulatedCurcumin on B16-F10 Melanoma

Data obtained in the in vitro cytotoxic study with B16-F10 melanoma cells are shown in Figure 1 Incuba-tion of B16-F10 melanoma cells with free curcuminand curcumin-loaded nanocapsules caused significantreduction of cell viability in a concentration- andtime-dependent manner After 24 hours of incubation sta-tistically significant reduction in the number of viablecells was observed for free curcumin at a concentration ofge25 M when compared to the control group (P lt 005)At 100 M concentration the incubation of cells with freecurcumin caused a 75 reduction in the number of viablecells while a 25 reduction occurred after incubation ofCur-LNC and Cur-PNCS at same time On the contraryafter 48 and 72 hours of incubation Cur-LNC induced asignificantly higher cytotoxic effect than free drug at thelowest tested concentration (10 M) producing a 75reduction in the number of viable cells (P lt 005) At10 M and 72 hours of incubation the cytotoxic effectin increasing order was Cur-LNCgt free curcumingt Cur-PNCS Cur-PNCP only exhibited cytotoxic effect at con-centration of ge25 M after 72 hours of incubation when

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

24 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

48 h

0

25

50

75

100

125

0 10 25 50 100

Curcumin concentration (microM)

Via

ble

cells

(

)

72 h

Fig 1 Cytotoxic effect of free curcumin and curcumin-loaded nanocap-sules suspensions on B16-F10 melanoma cells after 24 48 and 72hours of incubation Free curcumin (circel) Cur-LNC (square) Cur-PNCP (triangle) and Cur-PNCS (inversed triangle) Optical density ofcontrol groups was taken as 100 cell viability was confirmed byTrypan Blue exclusion method lowastP lt 005 compared with the controlgroup P lt 005 compared with free curcumin at the same concentrationn= 3

410 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

compared to the control group (P lt 005) The experi-ments were also performed using the unloaded nanocap-sules suspensions (data not shown) The incubation of cellswith unloaded nanocapsules caused the reduction of thenumber of viable cells suggesting some cytotoxic activityHowever curcumin-loaded nanocapsules reduced the num-ber of viable cells in a significant manner (P lt 005) whencompared with the unloaded nanocapsules The highercytotoxicity displayed by Cur-LNC may be associated tosome extent with the small size and lipid structure whichseems favorable in delivering curcumin inside the cellsPrevious studies carried out by our research group (datanot shown) demonstrated that curcumin is released morequickly from LNC than PNC (77 and 48 for LNC andPNC respectively after 48 hours) due to smaller particlesize and thinner shell24 Perhaps this nanocapsule propertyhas contributed to the higher cytotoxic effect of LNC whencompared to PNCS and PNCP In addition the nanoencap-sulation may have protected the drug from inactivation inthe culture medium since free curcumin is more prone toundergo hydrolytic decomposition at pH 7433

The ability of free curcumin and curcumin-loaded nanocapsules to induce apoptosis on B16-F10 melanoma cells was initially screened by usingacridine orangeethidium bromide staining The treatedB16-F10 melanoma cells showed obvious nuclear conden-sation after 48 hours of treatment Control cells showedbright green nuclei with uniform intensity and had not

Fig 2 Cytomorphological changes of B16-F10 melanoma cells induced by free curcumin and curcumin-loaded nanocapsules suspensions upon48 hours incubation (objective 40 x) (A) Control (B) Free curcumin (C) Cur-PNCS and (D) Cur-PNCP Viable cells exhibited green fluorescence(acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining)

taken up ethidium bromide where the apoptotic cellsappeared orange in color (Fig 2) Based on the cytomor-phological changes and cell death described above theeffect of free curcumin or encapsulated curcumin on thesecells was indicative of apoptosis

33 Uptake Studies

Uptake of the nanocapsules by macrophages was mon-itored by morphologic observation of the cells after60 minutes of incubation (Fig 3) The yellow fluorescencein the micrographs confirmed the intracellular accumu-lation of curcumin Cur-LNC seemed scarcely capturedby cells as can be verified by the weak fluorescence inthe cytoplasm of the macrophages On the other handpolymeric nanocapsules especially Cur-PNCP were moreactively phagocyted and accumulated in the cells as wasrevealed by the bright fluorescence observed To quan-tify the macrophage uptake the fluorescence intensityper cell was related to the number of pixels obtainedafter image analysis Fluorescence intensities of Cur-PNCS Cur-PNCp and free curcumin were around 35126 and 5374 times higher than Cur-LNC respectivelyThese results suggest that the PEG chains of SolutolHS15 located on the surface of Cur-LNC and Cur-PNCS

reduce the interaction of the particles with J774 cellsIn addition the images showed substantial differencein phagocytic uptake between free and encapsulated

J Biomed Nanotechnol 7 406ndash414 2011 411

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

Fig 3 Fluorescence microscopy images of J774 cells upon 60 minutes incubation in the dark with (A) Cur-LNC (B) Cur-PNCS (C) Cur-PNCP and(D) free curcumin (objective 40 x) Yellow fluorescence indicates the distribution of curcumin in the macrophages

curcumin which indicates that drug uptake can be signif-icantly reduced with the use of colloidal systems

34 In Vitro and In Vivo Antitumor Activity

To evaluate the effect of free curcumin and curcumin-loaded nanocapsules on tumor growth C57BL6 micewere inoculated subcutaneously with B16-F10 melanomacells (1times106 in 100 lanimal) and treated with the sam-ples The results of these experiments are summarized inFigure 4 Given twice a week intraperitoneal injectionsof free curcumin or curcumin-loaded nanocapsule sus-pensions at a dose of 6 mgkg significantly decreasedtumor growth rate throughout the 21 days of experimentBy the end of the experiment tumor volume was signi-ficantly inhibited by about 596 (11284 mm3 614(10782 mm3 and 713 (8014 mm3 after treatmentwith free curcumin Cur-LNC and Cur-PNCS respectivelywhen compared to the control group treated with cell cul-ture medium only (27910 mm3 (P lt 005) Cisplatin ata dose of 1 mgkg once a week decreased the tumorvolume by 724 (7700 mm3 No significant differencewas found between the tumor volume of the mice thatreceived the free drug and those that received curcumin-loaded nanocapsules and between the tumor volume ofthe mice that received unloaded nanocapsules and thosethat received only saline (negative control group) In a pre-vious study we had demonstrated that the encapsulationof camptothecin in pegylated PLA-based nanocapsulesintraperitoneally administered improves the effectiveness

of the drug against the lung metastasis spread in mice inoc-ulated with B16-F10 melanoma when compared with theadministration of free drug and non pegylated nanocap-sules This result was attributed to the protection of thedrug by nanoencapsulation and to the reduced uptake ofparticles by macrophages located in the lymph nodes34 Inthe present study no effect of the nanoencapsulation ofcurcumin on antitumor activity was verified In additionin spite of the differences found in the cytotoxicity andcell uptake exhibited by the colloidal carrier formulationsno improvement in the therapeutic efficacy provided by a

Fig 4 Effect of free curcumin unloaded and curcumin-loadednanocapsules on tumor volume inhibition in mice inoculated with 1times106B16-F10 melanoma cells lowastP lt 005 and lowastlowastP lt 001 compared with con-trol (n= 8

412 J Biomed Nanotechnol 7 406ndash414 2011

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Mazzarino et al Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants

possible stealth property of LNC could be confirmed inthe in vivo studiesAlthough both free curcumin and curcumin-loaded

nanocapsules demonstrated significant inhibition ofmelanoma growth the association of the drug with col-loidal systems seems to be beneficial since it allowsthe administration of this hydrophobic drug by severalroutes as an aqueous dispersion which may result inan increased bioavailability In earlier studies we demon-strated that the encapsulation of curcumin into both lipidand polymeric nanocapsules increases the concentrationof the drug by a factor of up to 46000 times regardingits aqueous solubility and besides it slows down thehydrolytic and photochemical degradation of curcumin24

However pharmacokinetic studies are still needed to com-prehend the fate of the curcumin after intraperitonealadministration of the nanocapsules

Acknowledgments The authors are thankful to LarissaDuarte Christofoletti and Tuacutelio Vinicius Duarte Christofo-letti for the support with the image analysis in the uptakestudies

References and Notes

1 D R Siwak S Shishodia B B Aggarwal and R KurzrockCurcumin-induced antiproliferative and proapoptotic effects inmelanoma cells are associated with suppression of IB kinaseand nuclear factor B activity and are independent of the B-rafmitogenactivatedextracellular signal-regulated protein kinasepathway and the akt pathway Cancer 104 879 (2005)

2 A V Giblin and J M Thomas Incidence mortality and survivalin cutaneous melanoma J Plast Reconstr Aesthet Surg 60 32(2007)

3 F Braud D Khayat B B R Kroon R Valdagni P Bruzziand N Cascinelli Malignant melanoma Crit Rev Oncol Hematol47 35 (2003)

4 C D Lao M F Demierre and V K Sondak Targeting events inmelanoma carcinogenesis for the prevention of melanoma ExpertRev Anticancer Ther 6 1559 (2006)

5 P Fresco F Borges C Diniz and M P Marques New insightson the anticancer properties of dietary polyphenols Med Res Rev26 747 (2006)

6 M S Baliga and S K Katiyar Chemoprevention of photocarcino-genesis by selected dietary botanicals Photochem Photobiol Sci5 243 (2006)

7 B Aggarwal A Kumar and A Bharti Anticancer potential ofcurcumin Preclinical and clinical studies Anticancer Res 23 363(2003)

8 R Thangapazham A Sharma and R Maheshwari Multiple molec-ular targets in cancer chemoprevention by curcumin AAPS J8 E443 (2006)

9 M Loacutepez-Laacutezaro Anticancer and carcinogenic properties of cur-cumin Considerations for its clinical development as a cancerchemopreventive and chemotherapeutic agent Mol Nutr Food Res52 S103 (2008)

10 L Menon R Kuttan and G Kuttan Inhibition of lung metastasisin mice induced by B16F10 melanoma cells by polyphenolic com-pounds Cancer Lett 95 221 (1995)

11 L Menon R Kuttan and G Kuttan Anti-metastatic activity of cur-cumin and catechin Cancer Lett 141 159 (1999)

12 J Odot P Albert A Carlier M Tarpin J Devy and C Madoulet Invitro and in vivo anti-tumoral effect of curcumin against melanomacells Int J Cancer 111 381 (2004)

13 P Anand A B Kunnumakkara R A Newman and B B Aggar-wal Bioavailability of curcumin Problems and promises MolPharm 4 807 (2007)

14 Y Wang M Pan A Cheng L Lin Y Ho C Hsieh and J JLin Stability of curcumin in buffer solutions and characteriza-tion of its degradation product J Pharm Biomed Anal 15 1867(1997)

15 S Bisht G Feldmann S Soni R Ravi C Karikari A Maitra andA Maitra Polymeric nanoparticle-encapsulated curcumin (nanocur-cumin) A novel strategy for human cancer therapy J Nanobiotech-nology 5 3 (2007)

16 K Sou S Inenaga S Takeoka and E Tsuchida Loading ofcurcumin into macrophages using lipid-based nanoparticles IntJ Pharm 352 287 (2008)

17 W Tiyaboonchai W Tungpradit and P Plianbangchang Formula-tion and characterization of curcuminoids loaded solid lipid nanopar-ticles Int J Pharm 337 299 (2007)

18 A Sahu U Bora N Kasoju and P Goswami Synthesis of novelbiodegradable and self-assembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells ActaBiomater 4 1752 (2008)

19 D Wang M S Veena K Stevenson C Tang B Ho J DSuh V M Duarte K F Faull K Mehta E S Srivatsanand M B Wang Liposome-encapsulated curcumin suppressesgrowth of head and neck squamous cell carcinoma in vitroand in xenografts through the inhibition of nuclear factor IcircBby an AKT-independent pathway Clin Cancer Res 14 6228(2008)

20 C Chen T D Johnston H Jeon R Gedaly P P McHugh T GBurke and D Ranjan An in vitro study of liposomal curcuminStability toxicity and biological activity in human lymphocytesand Epstein-Barr virus-transformed human B-cells Int J Pharm366 133 (2009)

21 V Mohanraj and Y Chen NanoparticlesmdashA review Trop J PharmRes 5 561 (2006)

22 V C F Mosqueira P Legrand A Gulik O Bourdon R GrefD Labarre and G Barratt Relationship between complementactivation cellular uptake and surface physicochemical aspectsof novel PEG-modified nanocaacutepsulas Biomaterials 22 2967(2001)

23 I Brigger C Dubernet and P Couvreur Nanoparticles incancer therapy and diagnosis Adv Drug Deliv Rev 54 631(2002)

24 L Mazzarino C L Dora I C Bellettini E Minatti S GCardoso and E Lemos-Senna Curcumin-loaded polymeric and lipidnanocapsules Preparation characterization and chemical stabilityevaluation Lat Am J Pharm 29 933 (2010)

25 F Lacoeuille F Hindre F Moal J Rpux C Passirani O CouturierP Cales J J L Jeune A Lamprecht and J P Benoit In vivoevaluation of lipid nanocapsules as a promising colloidal carrier forpaclitaxel Int J Pharm 344 143 (2007)

26 A Beacuteduneau P Saulnier N Anton F Hindreacute C PassiraniH Rajerison N Noiret and J Benoit Pegylated nanocapsules pro-duced by an organic sovent-free method Evaluation of their stealthproperties Pharm Res 23 2190 (2006)

27 B Heurtault P Saulnier B Pech J E Proust and J P BenoitProcess for the preparation of lipid nanocarriers Pharm Res 19 876(2002)

28 H Fessi F Puisieux J P Devissaguet N Ammoury and S BenitaNanocapsule formation by interfacial deposition following solventdisplacement Int J Pharm 55 R1 (1989)

29 L Mazzarino I C Bellettini E Minatti and E Lemos-SennaDevelopment and validation of a fluorimetric method to determine

J Biomed Nanotechnol 7 406ndash414 2011 413

Delivered by Ingenta toElenara Senna

IP 15016212157Sun 07 Aug 2011 152359

RESEARCH

ARTIC

LE

Curcumin-Loaded Lipid and Polymeric Nanocapsules Stabilized by Nonionic Surfactants Mazzarino et al

curcumin in lipid and polymeric nanocapsule suspensions BJPS46 219 (2010)

30 A V Loosdrecht R Beelen G Ossenkoppele M Broekhoven andM Langenhuijsen A tetrazolium-based colorimetric MTT assay toquantitate human monocyte mediated cytotoxicity against leukemiccells from cell lines and patients with acute myeloid leukemiaJ Immunol Methods 174 311 (1994)

31 Y J Geng T Azuma J X Tang J H Hartwig M MuszynskiQ W P Libby and D J Kwiatkowski Caspases-3-induced gelsolinfragmentation contributes to actin cytoskeletal collapse nucleolysisand apoptosis of vascular smooth muscle cells exposed to proinflam-matory cytokines Eur J Cell Biol 77 294 (1998)

32 Y S Lee H O Yang K H Shin H S Choi S H Jung Y MKim D K Oh R J Linhardt and Y S Kim Suppression of tumorgrowth by a new glycosaminoglycan isolated from the African giantsnail Achtina fulica Eur J Pharmacol 465 191 (2003)

33 H H Tonnesen M Maacutesson and T Loftsson Studies of curcuminand curcuminoids XXVII Cyclodextrin complexation solubilitychemical and photochemical stability Int J Pharm 244 127 (2002)

34 G Loch-Neckel D Nemen A C Puhl D Fernandes M AStimamiglio M A Silva M Hangai M C S Silva andE Lemos-Senna Stealth and non-stealth nanocapsules containingcamptothecin In-vitro and in-vivo activity on B16-F10 melanomaJ Pharm Pharmac 59 1359 (2007)

Received 15 November 2010 RevisedAccepted 8 February 2011

414 J Biomed Nanotechnol 7 406ndash414 2011