Encapsulation of Curcumin in Diblock Copolymer Micelles for Cancer Therapy

Research ArticleEncapsulation of Curcumin in Diblock Copolymer Micelles forCancer Therapy

Ali Mohammad Alizadeh1 Majid Sadeghizadeh2 Farhood Najafi3

Sussan K Ardestani4 Vahid Erfani-Moghadam5 Mahmood Khaniki6

Arezou Rezaei7 Mina Zamani2 Saeed Khodayari8

Hamid Khodayari8 and Mohammad Ali Mohagheghi1

1Cancer Research Center Tehran University of Medical Sciences Tehran 14197-33141 Iran2Department of Genetics School of Biological Sciences Tarbiat Modares University Tehran 14115-137 Iran3Department of Resin and Additives Institute for Color Science and Technology Tehran 16765-654 Iran4Immunology Lab Institute of Biochemistry and Biophysics University of Tehran Tehran 13145-1384 Iran5Department of Biotechnology Faculty of Advanced Medical Technology Golestan University of Medical SciencesGorgan 49175-553 Iran6Department of Pathology School of Medicine Tehran University of Medical Sciences Tehran 14197-33141 Iran7School of Biological Science Damghan University Damghan 36716-41167 Iran8Cancer Model Research Center Tehran University of Medical Sciences Tehran 14197-33141 Iran

Correspondence should be addressed to Ali Mohammad Alizadeh aalizadehrazitumsacir

Received 9 June 2014 Revised 22 October 2014 Accepted 23 October 2014

Academic Editor Bruno C Cavalcanti

Copyright copy 2015 Ali Mohammad Alizadeh et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Application of nanoparticles has recently promising results for water insoluble agents like curcumin In this study we synthesizedpolymeric nanoparticle-curcumin (PNPC) and then showed its efficiency drug loading stability and safety Therapeutic effectsof PNPC were also assessed on two cell lines and in an animal model of breast cancer PNPC remarkably suppressed mammaryand hepatocellular carcinoma cells proliferation (119875 lt 005) Under the dosing procedure PNPC was safe at 3125mgkg and lowerdoses Higher doses demonstrated minimal hepatocellular and renal toxicity in paraclinical and histopathological examinationsTumor take rate in PNPC-treated group was 375 compared with 875 in control (119875 lt 005) Average tumor size and weightwere significantly lower in PNPC group than control (119875 lt 005) PNPC increased proapoptotic Bax protein expression (119875 lt 005)Antiapoptotic Bcl-2 protein expression however was lower in PNPC-treated animals than the control ones (119875 lt 005) In additionproliferative and angiogenic parameters were statistically decreased in PNPC-treated animals (119875 lt 005) These results highlightthe suppressing role for PNPC in in vitro and in vivo tumor growth models Our findings provide credible evidence for superiorbiocompatibility of the polymeric nanocarrier in pharmacological arena together with an excellent tumor-suppressing response

1 Introduction

Curcumin (17-bis (4-hydroxy-3-methoxy-phenyl)-16-hep-tadiene-35-dione diferuloylmethane) is a natural yellowand lipid-soluble compound extracted from Curcuma longaplant with no discernible toxicity Recent studies have shownthat curcumin either alone or in combination with otheranticancer agents has potent anticancer effects [1] Curcumin

is water insoluble and is eliminated mostly unchanged andpartly altered in the gut In preclinical and clinical studiesoral administration of curcumin yields low plasma andtissue concentrations This may be due to low absorptionrapid metabolism and elimination and limited systemicbioavailability [2] To prevail over these weak points differentways were tested such as piperine [3] liposomal curcumin[4 5] and nanoparticle curcumin use [6 7] Application of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 824746 14 pageshttpdxdoiorg1011552015824746

2 BioMed Research International

nanoparticles has promising results for the poor water solu-ble hydrophobic agents like curcumin Feasible preparationand use size fitness environmental sustainability and shellviability are all important factors for a suitable carrier [8]According to some studies encapsulated form of curcuminimproves its medical properties and solubility [9] Previousstudies showed that dendrosome a diblock nanostructuremade by oleic acid (OA) and polyethylene glycol (PEG 400Dalton) with anticancer and proapoptosis effects is a suitableoption for curcumin encapsulation [10ndash12] In the presentresearch we used modified 2000 Dalton molecular weightmonomethoxy-PEG (mPEG) for curcumin encapsulationRecently our research group evaluated morphology andphysical behavior of mPEG

2000-OA [13] Based on CMC

measurement this novel nanocarrier can be considered as anappropriate drug delivery system for curcumin delivery incancer cells A unique feature of mPEG

2000-OA is the ease

with which the structure of their monomers can be variedin order to provide suitable inert drug porters for targetcells In this study we exploited mPEG

2000-OA for curcumin

encapsulation as polymeric nanoparticle curcumin (PNPC)and investigated its efficiency drug loading and stability withmore focus on clinical observations hematologicalbloodchemistry tests and histological examinations In additionPNPCrsquos protective and therapeutic effects were also assessedin an animal model of breast cancer Our results shed newlight on PNPC potential biocompatibility in in vitro and invivo biological systems

2 Materials and Methods

21 Materials Curcumin (gt99) doxorubicin hydrochlo-ride cyclophosphamide ketamine and xylazine were pur-chased from Sigma Aldrich Co (St Louis MO USA) Mate-rials for the polymeric nanocarrier (OM2000) preparationincluding oleoyl chloride and methoxy polyethylene glycol2000 (mPEG 2000) were got from Sigma-Aldrich Co (StLouis MO USA) Chloroform and triethyl amine werealso purchased from Merck-Serono Company (Germany)The human hepatocellular (HuH-7) mouse mammary (4T1)carcinoma cell lines and normal human fibroblast cells wereprocured from Pasteur Institute of Iran (Pasteur InstituteTehran Iran) Polyclonal mouse anti-ratrabbit Bax Bcl-2and CD31 antibodies (DAKO Corporation USA) and rabbitmonoclonal antibody against Ki-67 (Thermo Fisher ScientificInc San Jose CA) were purchased

22 The Study Design Studies were conducted in five seriesof experiments including (i) PNPC synthesis (ii) PNPCstability and drug loading (iii) PNPC effects on cell viabilityof mammary and hepatocellular carcinoma cells (iv) PNPCacute and chronic toxicity and (v) PNPC protective andtherapeutic effects on animal model of breast cancer

23 Animals All animal studies have been conductedaccording to relevant national and international guidelines ofthe Weatherall report and Institutional Animal Care and Use

Committee (IACUC) of Tehran University of Medical Sci-ences Inbred female BALBcmice (6ndash8 weeks old purchasedfrom Iran Pasteur Institute) were maintained under 12-hourdark and light cycle with free access to food and water

24 Polymeric Nanocarrier Synthesis Polymeric nanoparticle(PNP OM2000) was synthesized by esterification of oleoylchloride (301 g 001mol) and methoxy polyethylene glycol2000 (20 g 001mol) in the presence of triethyl amine (12 g0012mol) and chloroform as solvent at 25∘C for 2 h (seeSupplemental Figure 1 in Supplementary Materials avail-able online at httpdxdoiorg1011552015824746) Triethylamine hydrochloride was filtered from organic phase Chlo-roform was then evaporated and OM2000 dried in a 40∘Cvacuum oven for 4 h with 96 purity Fourier transforminfrared (FT-IR) spectroscopic measurements in the potas-sium bromide (KBr) pellets were done using a Perkin-ElmerspectrometerThe 1HNMR spectrumswere carried out usingBruker 400MHZ in DMSO-d

5

25 Critical Micelle Concentration Determination Criticalmicelle concentration (CMC) of PNP was determined bydetecting shifts in the pyrene fluorescence absorbance spectra[14] In this regard 3mL of pyrene solution (6 times 10minus6M)in acetone was added to a glass test tube and evaporated toremove the solvent Then 5mL of PNP (0005 to 1mgmL)was conjugated with phosphate-buffered saline (PBS 001MpH 74) added to the glass test tubes to reach to 6 times 10minus7Mpyrene final concentration The solutions were vortexed andconditioned at 37∘C overnight Fluorescence excitation spec-tra of pyrene (300ndash350 nm) were measured at an emissionwavelength of 390 nm with slit widths of 25 and 50 nm(Perkin-Elmer Fluorimeter USA) for excitation and emis-sion respectively The fluorescence excitation shifts from 334to 339 nm were used to determine CMC of the polymericnanocarrier

26 PNPC Physical Properties Dynamic light scattering(DLS) and atomic force microscopy (AFM) were carriedout to evaluate alterations in the zeta-potential particle sizeparticle size distribution shape and polydispersity index(PdI) of the polymeric nanoparticle [14ndash16] Nanoparticlezeta-potential and size distribution of PNPC (001M PBSpH 74) were analyzed by DLS (Zetasizer NanoZS MalvernInstruments UK) using an argon laser beam at 633 nm and90∘ scattering angle To determine PNPC shape a drop ofPNPC solution (005mgmL) was placed on freshly cleavedmica and was allowed to dry at room temperature Thesample was then mounted on a microscope scanner andimaged in semicontact mode with AFM (JPK InstrumentsCo Germany)

27 PNPC Encapsulation Efficiency and Drug Loading Inthis section measurements were performed in triplicateDifferent concentrations of curcumin (2 to 25mg) in acetonesolutionweremixedwith 100mgnanocarrier in 3mLofwaterand rotated with rotary evaporator till acetone evaporationPNPC was filtered using 022120583m syringe filter to remove

BioMed Research International 3

nonencapsulated curcumin and then lyophilized To disruptmicellar or vesicle structures 10mg lyophilized PNPC wasdissolved in 1mL methanol and vortexed with ultrasonicwaves for 10min to ensure encapsulated curcumin releaseCurcumin concentration was then determined with spec-trophotometer at 425 nm in comparison with methanol cali-bration curve for curcumin Drug loading (DL) and encapsu-lation efficiency (EE) of curcuminnanocarrier micelles werefinally quantified utilizing the following equation [15 16]

Drug loading content ()

=weight of curcumin in micelles

weight of micellestimes 100

Encapsulation efficiency ()

=weight of micelled curcuminweight of feeding curcumin

times 100

(1)

28 PNPC Preparation Curcumin (6mg) was dissolved inacetone and added into nanocarrier solution (100mg3mLwater) and then mixed and rotated with rotary evaporatorfor acetone evaporation PNPC was filtered using 022120583msyringe filter to remove nonencapsulated curcumin Thecomplex was then lyophilized and used for next experiments[13]

29 Stability Assays PNPC aqueous solution was kept at4∘C for 24 h and ten months at dark room temperaturePrecipitation indicates PNPC instability while clear solutionconfirms its stability [17] Dissolved lyophilized samples inwater were made by manual shaking without additionalheating or sonication PNPC size and distribution werecompared with freshly prepared PNPC by DLS [18] At theend of the first week curcumin loaded content and sizedistribution of PNPC were compared with freshly preparedPNPC by spectroscopy and DLS respectively

210 MTT Assay HuH-7 4T1 and normal fibroblast celllines were grown in Dulbeccorsquos modified Eaglersquos medium(DMEM GIBCO USA) containing 10 fetal bovine serum(FBS GIBCO USA) at 37∘C in humid atmosphere Cellviability measured by MTT (3-[45-dimethylthiazol-2-yl] 25-diphenyltetrazolium bromide) assay [19] Curcumin stocksolution (100 120583M) in DMEM was prepared from 10mMcurcumin in methanol Methanol percentage in the finalsolution was lower than 04 vv Identical cell numbers (1 times106 cells) in 200120583L DMEM containing 10 FBS were seededin triplicate on 96-well plates and incubated overnight Cellswere subsequently treated with various concentrations (010 20 30 and 40 120583M) of PNPC PNP alone curcuminand doxorubicin (as positive control) for 24 h and 48 hAfterwards 20120583L of MTT (5mgmL) was added to each welland incubated for an additional 4 h followedby adding 200 120583Lof dimethyl sulfoxide Relative cell viability was then deter-mined using a 96-well plate reader (TECAN Switzerland) at540 nm

211 Dosing Procedure One hundred sixteen BALBc micewere used to study the acute and chronic toxicity of PNPC[20] In acute toxicity protocol 2000mgkg of PNPC wasgiven as starting dose Then doses of 2000 1000 500 250125 625 3125 and 1563mgkg body weight of PNPC andPNP were intraperitoneally injected Animals were eutha-nized after 24 h In case of studying 24 h adverse reactionsthe dose was adjusted based on the average toxicity and thelast tolerated dose for the new group of mice Organ damagehistopathological findings abnormal hematologicalbloodchemical indices reduced organ weight ratio and bodyweight changes were amongst the toxicity signs

Based on the acute toxicity results chronic toxicity wascarried out using doses of 125 625 3125 and 1563mgkgof PNPC and PNP for 7 consecutive days The dose with noadverse reactions during 24 h was assigned as the survivaldose Survived animals were weighed on a daily basis andeuthanized one week later Hematology blood chemistryand histopathological tests were carried out Vital organsincluding heart liver spleen lung brain and kidney wereexcised and weighed

212 Hematology Blood Chemistry and HistopathologicalTests Animals were decapitated under general anesthesia toevaluate hematology clinical chemistry and histopathologyparameters Blood samples were taken for clinical chemistrytests Total leukocyte count (WBC) erythrocyte count (RBC)platelets (Plt) hemoglobin (Hgb) hematocrit (Hct) meancell volume (MCV) mean corpuscular hemoglobin (MCH)and mean corpuscular hemoglobin concentration (MCHC)neutrophils lymphocytes eosinophils and monocytes weremeasured using an animal blood counter (Celltac NihonKohden Tokyo Japan) Plasma urea nitrogen (BUN) creati-nine (Cr) sodium (Na) potassium (K) chloride (Cl) bicar-bonate (HCO3minus) calcium (Ca) magnesium (Mg) lactate(Lac) osmolarity (Osm) and glucose (Glu) were determinedusing CCX System (CCX WB Nova Biomedical USA)Plasma alkaline phosphatase (ALP) albumin (ALB) totalbilirubin (TBil) direct bilirubin (DBil) gamma-glutamyltranspeptidase (GGT) alanine transaminase (ALT) andaspartate transaminase (AST)were alsomeasured (Autoanal-yser Model Biotecnica BT 3500 Rome Italy) In additionliver kidney brain lung spleen and heart tissue sampleswere fixed and preserved in 4 buffered formaldehyde forat least 24 h Tissue blocks were prepared and evaluated forhistopathological changes

213 Tumor Transplantation Spontaneous mouse mammarytumor (SMMT) was aseptically separated from the breast-cancer-bearing BALBc mice cut into pieces of less than03 cm3 and subcutaneously transplanted into the animalsrsquoleft flank under ketamine and xylazine (10mgkg ip) anes-thesia [21]

214 PNPC Protective Effects on an Animal Model of BreastCancer Sixteen mice were divided into two equal groupsof nontreated (control) and treated for the study of PNPCprotective effects on a typical animal model of breast cancer


PNPC was given for 24 days from 3 days before to 21 daysafter tumor transplantation Animal survival rate and tumortake rate were measured at the end of the study

215 PNPC Therapeutic Effects on an Animal Model of BreastCancer Twenty-four mice were equally divided into neg-ative control positive control and PNPC groups to studynanocurcumin therapeutic effects on mice breast cancerPNPC was given for 24 days after tumor transplantationfrom day 14 up to day 38 In the positive control groupdoxorubicin (5mgkg) and cyclophosphamide (2mgmouse)were intraperitoneally coadministered in three separate dosesfor three consecutive weeks Normal saline was also givenin the negative control group Tumor volume was measuredweekly by a digital vernier caliper (Mitutoyo Japan) andreported according to the following formula [21]

119881 =1

6 (120587119871119882119863) (2)

where 119871 = length119882 = width and119863 = depth

216 Histopathological Assay The breast tumoral and adja-cent nontumoral mucosal tissues were fixed in 10 formalde-hyde passaged and embedded in paraffin Paraffin blockswere then sectioned by 3ndash5 120583m thickness for hematoxylinand eosin (HampE) staining [22] Slides were studied usingOLYMPUS-BX51microscope Digital photos were takenwithOLYMPUS-DP12 camera and graded by the Scarff-Bloom-Richardson Scale [23]

217 Immunohistochemistry Examinations Immunohisto-chemistry was carried out on 3ndash5 120583m tissue sections takenfrom the formalin-fixed paraffin blocks using avidin-biotinimmunoperoxidase method [24] For tumor cellsrsquo apoptoticand angiogenic studies sections were stained with polyclonalmouse anti-ratrabbit Bax Bcl-2 and CD31 antibodies(DAKO Corporation USA) according to the manufacturerrsquosinstructions Briefly the paraffin sections were deparaffinizedwith xylene and rehydrated through a series of descendinggraded ethanol solutions Slides kept into TBS-EDTAbuffer and put into a microwave oven for 15min at 90∘CEndogenous peroxidase activity was blocked by 03 H

2O2

buffer incubation for 15min Biotinylated secondary antibodyand avidin-biotin complex with horseradish peroxidase wereapplied followed by chromogen 331015840-diaminobenzidineaddition (Sigma Chemical)

To study tumor cell proliferative activity sections weretreated with 3 (vv) H

2O2at room temperature blocked

with 10 (vv) goat serum or rabbit serum (Nichirei TokyoJapan) and incubated with a rabbit monoclonal antibodyagainst Ki-67 (Thermo Fisher Scientific Inc San JoseCA) Sections were then incubated with biotinylated goatanti-rabbit IgG (Nichirei) and a solution of streptavidin-conjugated horseradish peroxidase (Nichirei) according tothe manufacturerrsquos recommendations

Criteria used to evaluate Bax Bcl-2 CD31 and Ki67markers were based on the estimated proportion of positivecells and estimated average staining intensity of positive cells

in cytoplasm (for Bax) membrane (for CD31) nucleus (forKi67) and membrane cytoplasm or nucleus (for Bcl-2)Semiquantitative score was adopted as follows [22 24]

no staining 0faintbarely staining up to 13 of cells 1moderate staining in 13 to 12 of cells 2strong staining in more than 12 of cells 3

218 Statistical Analysis Analysis of variance (ANOVA) andTukeyrsquos post hoc tests were used for comparison betweengroups Two-tailed Studentrsquos 119905-tests were used when compar-ing two groups Differences in tumor incidence (percentageof animals with breast cancer) were analyzed by Fisherrsquos exactprobability test Values were represented as mean plusmn SEM 119875 lt005 was considered to be statistically significant Statisticalanalysis was done using SPSS statistical software version 140

3 Results

31 The FT-IR Spectrum of the Polymeric Carrier The FT-IRspectrum of the polymeric carrier showed stretching band ofCndashH aliphatic at 2889 2947 and 2960 cmminus1 C=O stretchingvibration of ester bands could be seen at 1736 cmminus1 CndashH bending vibration of CH

2and CndashH bending vibration

of CH3can be seen in 1467 and 1343 cmminus1 respectively

CndashO stretching vibration was at 1112 cmminus1 as broad band(Supplemental Figure 2)

32 The 1H NMR Spectrum of the Polymeric Carrier Supple-mental Figure 3 shows 1H NMR spectrum of the polymericcarrier from 0 to 65 ppm in DMSO-d

5 Saturated protons in

fatty ester were at 08 12 15 2 and 23 ppm CH2protons

between unsaturated bonds in linoleate and linolenate were at26 ppm Residual DMSO-d

5

1H NMR signal was at 25 ppm(Supplemental Figure 3(a)) DMSO-d

5water was in 33 ppm

as broad band CH3protons of mPEG were in 32 ppm CH

2

protons of mPEG ethylene oxide units were multipeaks in35 ppm (Supplemental Figure 3(b)) CH

2protons of mPEG

ethylene oxide connected to fatty acid chloride were at 41 and42 ppm CndashH oleate linoleate and linolenate were in 53 62and 64 ppm respectively (Supplemental Figure 3(c))

33 Critical Micelle Concentration of Polymeric NanocarrierConcentration at crossover point in Figure 1 shows CMCof nanocarrier at 339334 nm intensity ratio and nanocarrierconcentration logarithm As noted CMC value is very lownear 003 gL With increasing nanocarrier concentrationflorescence intensity has significantly risen (Figure 1)

34 PNPC Physical Properties The size morphology andpolydispersity of the nanoparticles were evaluated usingdynamic light-scattering technique (DLS) andAFMmethods(Figure 2) The results show that two forms of particleswere produced in the process of synthesis micelles andpolymersomes with the average size of 1833 plusmn 532 nm and9944 plusmn 65 nm respectively The freshly prepared PNP with


mPEG-OAmPEG (control)

25

20

15

10

05

00

000

minus025

minus050

minus075

minus150

minus175

minus200

minus225

minus100

minus250

minus125

minus275

minus300

CMC

logC (concentration (gL))

I (339334)

I

(a)

0

200

400

600

320 330 340 350

Excitation wavelength (nm)

mPEG-control00050125

05

1

Flor

esce

nce i

nten

sity

(au

)

(b)

Figure 1 Determination of CMC of (a) pyrene excitation spectra shift and (b) four datasets from ten are displayed to simply show the belowand above CMC concentration when micelles developed C the concentration of polymeric nanoparticle (PNP)

05

101520

01 1 10 100 1000 10000

freshly prepared

after one week

average result of all samples

Volu

me (

)

Record 108 mPEG-OA nanoparticles



Size distribution by volume

Size (dmiddotnm)

(a)

0100 02 03 04 05 06 07 08 09 1000

01

02

03

04

05

06

07

08

09

1000

01

02

03

04

05

06

07

08

09

10

142 nm

35 nm

(120583m)

(b)

Figure 2 Morphology and particle size distribution of PNPC (a) Red line shows average results of freshly prepared PNPC samples (0ndash25curcumin encapsulation in PNP) It shows three particle sizes 183 plusmn 53 655 plusmn 30 and 283 plusmn 112 nm Green line shows average results ofPNPC samples after one week at 25∘C (0ndash25 curcumin encapsulation in PNP) It shows one particle size of 994 plusmn 426 nm Blue line showsaverage result of all samples with two sizes 183 plusmn 53 (they seem to bemicelles) and 994 plusmn 65 nm (they seem to be polymersomes) (b) Atomicforcemicroscopy (AFM) results AFM image of redissolved PNPC after freeze-drying (005mgmL) also showed two particle forms and sizesSmaller particles (lt40 nm) seem to be micelles and larger particles (gt40 nm) seem to be polymersomes

different curcumin content (0ndash25) weremonodisperse (PdI= 0332 plusmn 013) with three particle forms 535 micelles(1833 plusmn 53 nm) 388 polymersomes (655 plusmn 30 nm) and75 polymersomes (2836 plusmn 112 nm) However after oneweek of incubation at 25∘C the particle forms changed tobe more monodisperse (PdI = 0182 plusmn 0072) with 100polymersomes (9944 plusmn 4256 nm) The results of AFManalysis show that the shape of PNPC was in accordancewith DLS analysis (Figure 2(b)) However compared to DLS

analysis the AFM results show the larger size of PNPCwhich can be attributed to the expansion of spherical micellesor vesicles (polymersomes) in mica surface after drying itssolution Moreover the AFM results show that in PNPCgraph the z-dimensional (height) bar is smaller than X- andY-dimensional bars which support this claim (SupplementalFigure 4)The PNPC is indeed stable in the presence of oleatein this nanoformulation The negative zeta-potential wasfound to be minus293 plusmn 52mV at concentration of 005mgmL


Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)

Curcumin (mg100mg nanocarrier)

(a)

Encapsulation efficiency120

100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)

Figure 3 Encapsulation efficiency and drug loading of curcumin in PNPC (a) Encapsulation efficiency (EE) and (b) drug loading (DL) ofcurcumin

05

10152025

01 1 10 100 1000 10000


Record 125 nanocurcumin freshRecord 126 nanocurcumin after one week

Volu

me (

)

Size (dmiddotnm)

(a)

Stability after one week in PBS

Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)

Figure 4 Stability test of curcuminnanoparticle after one week at room temperature by DLS test

(slightly higher concentration thanCMCpoint 003mgmL)This low zeta-potential is at optimum range for stability ofPNPC and explains the reason of developing more uniformsize distribution (PdI = 0182 plusmn 0072) after one week at roomtemperature [25 26]

35 PNPC Encapsulation Efficiency and Drug Loading Theaverage drug encapsulating efficiency and drug loading ofPNPC were 64 and 597 plusmn 21 (ww) respectively Thepercentage of drug loading at different ratios of curcumin to100mg of nanocarrier was plotted in Figure 3 Curcumin didnot precipitate at 6 loading after 10 months of incubation(Supplemental Figure 5)

36 PNPC Stability PNPC was stable more than 300 daysat 4∘C although loading curcumin higher than 6 into

nanoparticles leads to some precipitation of curcumin (Sup-plemental Figure 7) Lyophilized PNPC samples in waterdissolved by manual shaking without additional heatingor sonication Then size and distribution of PNP werecompared with freshly prepared PNPC by DLS (Fig-ure 2(a)) After one week at 25∘C as mentioned PNPCsamples inclined to develop more uniform polymersomesnanoparticles (PdI = 0182 plusmn 0072) Furthermore spec-troscopy analysis showed that one-third of curcumin incurcuminnanocarriers micelles (pH = 72) was conserved atroom temperature after one week (Figure 4)

37 PNPC Effects on HuH-7 and 4T1 Cell Lines PNPCsignificantly suppressed the proliferation of HuH-7 and 4T1cancerous cells in a dose- and time-dependent manner incomparison with curcumin and PNP groups (119875 lt 005)


0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

Concentration (120583M)

Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120

0 20 40Concentration (120583M)

PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)

Figure 5 Cytotoxic effects of PNPC on mouse mammary (4T1) and human hepatocellular (HuH-7) carcinoma cells Cells were treated withdifferent concentrations of PNPC for 24 h (a) and 48 h (b) on 4T1 cell line and 24 h (c) and 48 h (d) on HuH-7 cell line Data reported aremean plusmn SD lowast119875 lt 005 compared to curcumin PNPC = the polymeric nanoparticle curcumin PNP = polymeric nanoparticles

PNPC IC50for 4T1 cells was 29 120583Mwithin 24 h (Figure 5(a))

which relatively reduced to 24 120583M in 48 h (Figure 5(b))In addition PNPC IC

50for HuH-7 cells was 25 120583M within

24 h (Figure 5(c)) which relatively reduced to 19120583M in48 h (Figure 5(d)) We showed concentration about 48 and40 120583M for IC

50of free curcumin for 24 and 48 h respectively

Therefore we showed that IC50of the free curcumin is signifi-

cantly higher than PNPC and PNPC significantly suppressedcell growth compared to free curcumin (119875 lt 005) Inaddition no significant toxicitywas observed for voidmPEG-OA nanocarrier (PNP) even at concentrations above the40 120583M PNPC was not toxic to normal human fibroblasticcells (HFSF-PI3) [13] and this data is in accordance withother researcher results reviewed by Ravindran et al [27]PNPC showed nonsignificant changes in comparison withthe positive control group PNPC and doxorubicin had noeffect on normal human fibroblasts cells (data not shown)

38 PNPC Toxicity The main toxicity signs to PNPC andPNP in various doses are summarized in Tables 1 and 2and Supplemental Tables 1ndash5 In acute toxicity groups dosesof 2000 1000 500 and 250mgkg of PNPC are associatedwith death or severe poisoning symptoms AdditionallyPNPC and PNP caused hematological hepatocellular andrenal toxicity at the acute dose of 125mgkg (Table 1 andSupplemental Table 1) Higher doses provoked severe adversereactions and death especially at 250mgkg PNPC

In the chronic toxicity groups doses up to 625mgkgPNPC brought about no death but at 125mgkg PNPC 2 outof 6 mice died Weight loss diarrhea imbalance and asciteswere seen at 125mgkg of PNPC after oneweek of consecutiveinjections Survived animals (3125mgkg and lower) had noclinical differences compared with the control group PNPCshowed remarkable safety rates up to 3125mgkg (Tables 1and 2 and Supplemental Tables 1 and 2)


Table 1 Acute toxicity effects of PNPC on hematological and blood chemical indices in mice

ParametersGroups

Control PNPC3125mgkg

PNPC625mgkg

PNPC125mgkg

Animal weight (g) 243 plusmn 22 25 plusmn 31 247 plusmn 25 232 plusmn 22RBC (Millinmm3) 608 plusmn 02 68 plusmn 08 72 plusmn 05 82 plusmn 04lowast

HCT () 384 plusmn 17 377 plusmn 12 385 plusmn 25 425 plusmn 2lowast

Hgb (gdL) 10 plusmn 12 113 plusmn 18 117 plusmn 13 134 plusmn 16lowast

MCV (FL) 51 plusmn 07 501 plusmn 08 493 plusmn 15 491 plusmn 12MCH (pg) 165 plusmn 02 164 plusmn 03 155 plusmn 04 153 plusmn 03MCHC (molL) 323 plusmn 07 324 plusmn 05 322 plusmn 04 325 plusmn 03Plt (1000mm3) 567 plusmn 50 546 plusmn 91 570 plusmn 45 540 plusmn 38WBC (1000mm3) 67 plusmn 08 87 plusmn 2 79 plusmn 15 81 plusmn 22Neutrophils () 355 plusmn 2 366 plusmn 12 38 plusmn 5 382 plusmn 6Monocytes () 21 plusmn 3 12 plusmn 09 31 plusmn 1 21 plusmn 08Lymphocytes () 535 plusmn 64 52 plusmn 9 572 plusmn 5 57 plusmn 4Na (mML) 1503 plusmn 29 1465 plusmn 33 1542 plusmn 21 1571 plusmn 15lowast

K (mML) 52 plusmn 05 58 plusmn 13 61 plusmn 17 82 plusmn 22lowast

Cl (mML) 111 plusmn 4 117 plusmn 3 114 plusmn 2 117 plusmn 3lowast

HCO3minus (mML) 226 plusmn 32 173 plusmn 16 175 plusmn 22 163 plusmn 27lowast

Osm (mOsmkg) 307 plusmn 47 307 plusmn 52 3122 plusmn 7 320 plusmn 8lowast

Ca (mML) 072 plusmn 014 063 plusmn 01 065 plusmn 02 066 plusmn 03Mg (mML) 029 plusmn 004 030 plusmn 003 025 plusmn 004 031 plusmn 003Glu (mgdL) 194 plusmn 33 204 plusmn 48 182 plusmn 30 176 plusmn 25Lac (mML) 54 plusmn 12 52 plusmn 13 52 plusmn 2 57 plusmn 18Urea (mgdL) 212 plusmn 26 213 plusmn 36 26 plusmn 5 43 plusmn 7lowast

Cr (mgdL) 048 plusmn 008 051 plusmn 007 055 plusmn 007 092 plusmn 012lowast

AST (UL) 455 plusmn 45 538 plusmn 85 777 plusmn 80 1280 plusmn 152lowast

ALT (UL) 68 plusmn 88 706 plusmn 87 86 plusmn 21 258 plusmn 86lowast

ALP (UL) 763 plusmn 50 794 plusmn 67 825 plusmn 36 1187 plusmn 92lowast

GGT (UL) 55 plusmn 05 mdash mdash 62 plusmn 06ALB (mgdL) 3 plusmn 02 mdash mdash 28 plusmn 04TBIL (mgdL) 05 plusmn 003 mdash mdash 047 plusmn 005DBIL (mgdL) 035 plusmn 005 mdash mdash 032 plusmn 004Values are means plusmn SEM lowast119875 lt 005 compared to control PNPC = the polymeric nanoparticle curcumin RBC = red blood cell HCT = hematocrit MCV= mean corpuscular volume MCH = mean corpuscular hemoglobin MCHC = mean corpuscular hemoglobin concentration WBC = white blood cells Plt= platelets Na = sodium K = potassium Cl = chloride HCO3minus = bicarbonate Osm = osmolarity Ca = calcium Mg = magnesium Cr = creatinine Lac =lactate Glu = glucose AST = aspartate transaminase ALT = alanine transaminase ALP = alkaline phosphatase GGT = gamma-glutamyl transpeptidase ALB= albumin TBIL = total bilirubin and DBIL = direct bilirubin

Hematological markers were measured by a completeblood count analysis No inflammatory responses were seenin acute and chronic toxicity groups since total leukocytecounts remained within normal range (Tables 1 and 2 andSupplemental Table 1) In addition significant increases inRBC Hct and Hgb were seen in acute 125mgkg PNPCand chronic 625mgkg PNPC groups compared to controlanimals (119875 lt 005) (Table 1 and Supplemental Table 1)Na K and Cl were also statistically escalated while HCO3minusdrastically decreased in acute 125mgkg PNPC (Table 1)and chronic 625mgkg PNPC-treated mice compared withcontrol groups (119875 lt 005) (Table 2)

Plasma BUN and Cr levels were measured for kidneyfunction assessment while ALP TBil DBil GGT ALB ALTand AST were done for liver function evaluation BUN andCr were significantly higher in animals of acute 125mgkgPNPC (Tables 1 and 2) and chronic 625mgkg PNPC groupscompared to control animals (119875 lt 005) (Table 2 andSupplemental Table 2) Furthermore liver function testsshowed drastic increases in AST ALT andALP levels in acute125mgkg PNPC group (Table 1 and Supplemental Table 1)and chronic 625mgkg PNPC group (Table 2 and Supple-mental Table 1) compared to control mice (119875 lt 005) On theother hand albumin level significantly lowered at 625mgkg


Table 2 Chronic toxicity effects of PNPC on hematological and blood chemical indices and the organs weight percentage in mice

ParametersGroups


PNPC3125mgkg

PNPC625mgkg

Animal weight (g) 243 plusmn 22 24 plusmn 32 233 plusmn 34 183 plusmn 27lowast

RBC (Millinmm3) 608 plusmn 008 63 plusmn 007 69 plusmn 03 77 plusmn 04lowast

HCT () 384 plusmn 17 366 plusmn 21 374 plusmn 12 403 plusmn 32Hgb (gdL) 10 plusmn 12 12 plusmn 11 98 plusmn 09 123 plusmn 1MCV (FL) 51 plusmn 07 528 plusmn 06 504 plusmn 2 495 plusmn 15MCH (pg) 165 plusmn 02 173 plusmn 03 157 plusmn 09 156 plusmn 05MCHC (molL) 323 plusmn 07 328 plusmn 04 32 plusmn 07 322 plusmn 06Plt (1000mm3) 567 plusmn 50 519 plusmn 75 539 plusmn 53 594 plusmn 41WBC (1000mm3) 67 plusmn 08 69 plusmn 1 65 plusmn 18 77 plusmn 21Neutrophils () 325 plusmn 2 41 plusmn 8 385 plusmn 11 42 plusmn 9Monocytes () 21 plusmn 3 44 plusmn 21 18 plusmn 1 38 plusmn 22Lymphocytes () 665 plusmn 64 53 plusmn 11 608 plusmn 58 51 plusmn 13Na (mML) 1503 plusmn 29 1497 plusmn 32 1512 plusmn 38 1574 plusmn 15lowast

K (mML) 52 plusmn 05 57 plusmn 16 49 plusmn 04 43 plusmn 08Cl (mML) 111 plusmn 4 1122 plusmn 21 1127 plusmn 18 1155 plusmn 23HCO3minus (mML) 226 plusmn 32 167 plusmn 49 199 plusmn 32 189 plusmn 27Osm (mOsmkg) 307 plusmn 47 3013 plusmn 12 3069 plusmn 82 3158 plusmn 7Ca (mML) 072 plusmn 014 061 plusmn 014 074 plusmn 007 097 plusmn 06Mg (mML) 029 plusmn 004 030 plusmn 002 032 plusmn 004 035 plusmn 003Glu (mgdL) 194 plusmn 33 174 plusmn 31 172 plusmn 40 212 plusmn 30Lac (mML) 54 plusmn 12 48 plusmn 1 51 plusmn 11 43 plusmn 15Urea (mgdL) 212 plusmn 26 194 plusmn 32 20 plusmn 26 245 plusmn 38Cr (mgdL) 048 plusmn 008 042 plusmn 005 053 plusmn 003 098 plusmn 008lowast




GGT (UL) 55 plusmn 05 mdash 59 plusmn 08 64 plusmn 1ALB (mgdL) 3 plusmn 02 mdash 34 plusmn 06 23 plusmn 03lowast

TBIL (mgdL) 05 plusmn 003 mdash 047 plusmn 013 056 plusmn 01DBIL (mgdL) 035 plusmn 005 mdash 038 plusmn 008 034 plusmn 005 body weight

Heart 052 plusmn 006 047 plusmn 004Liver 63 plusmn 035 42 plusmn 026lowast

Spleen 043 plusmn 005 058 plusmn 007Lung 073 plusmn 009 071 plusmn 007Kidney 153 plusmn 015 146 plusmn 022Brain 112 plusmn 011 119 plusmn 015

Values are means plusmn SEM lowast119875 lt 005 compared to control PNPC = the polymeric nanoparticle curcumin RBC = red blood cell HCT = hematocrit MCV= mean corpuscular volume MCH = mean corpuscular hemoglobin MCHC = mean corpuscular hemoglobin concentration WBC = white blood cells Plt= platelets Na = sodium K = potassium Cl = chloride HCO3minus = bicarbonate Osm = osmolarity Ca = calcium Mg = magnesium Cr = creatinine Lac =lactate Glu = glucose AST = aspartate transaminase ALT = alanine transaminase ALP = alkaline phosphatase GGT = gamma-glutamyl transpeptidase ALB= albumin TBIL = total bilirubin and DBIL = direct bilirubin

in chronic PNPC group compared to control animals (Table 2and Supplemental Table 2) Liver and kidney weights weredecreased but spleen weight increased in 125mgkg PNPCand PNP groups compared to controls groups (Table 2) It

seems that the liver and the kidney were the target organs forthe polymeric carrier toxicity in high doses

In this study we also injected 3125mgkg PNPC for 7consecutive days and animals were euthanized at the end of


(A) (a)

(B)

(C)

(b)

(c)

Figure 6 Light microscopic analysis of organs impressed from PNPC (125mgkg) Mild Kupffer cells hyperplasia and sinusoidal distentionin favor of congestion were seen in the liver tissue [(A) (20x) (a) (40x)] Moderate congestion sinusoidal dilatation with preserved whitelymphoid pulp was seen in the spleen tissue [(B) (20x) (b) (20x)] The glomeruli were unremarkable but mild peritubular congestion andproteinaceous cast formations were noted in some kidney tubules [(C) (20x) (c) (40x)]

2 4 and 12 weeks No adverse reactions observed in hemato-logical blood chemical and histological examinations in thisprotocol (Supplemental Table 3)

We also compromised the toxicity of PNPC and PNPeach other In the acute toxicity no significant differencewas observed in hematological and blood chemical exami-nations in various doses of PNPC and PNP (SupplementalTable 4) But in the chronic groups high BUN and ASTlevels and low albumin serum level were seen at 625mgkgPNP group compared to PNPC animals (SupplementalTable 5)

39 Histopathology Examinations In order to obtain anaccurate diagnosis of PNPC and PNP toxicity onmicroscopiclevels major organs were histopathologically evaluatedCompared with control animals treated with 125mgkgPNPC and PNP developed slight abdominal ascites kidneyliver and spleen congestion after one-week consecutive injec-tions AST ALT ALP and GGT drastic increases in thesegroups may be assumed as liver dysfunction These changeswere also congruent with elevated renal biomarkers In theliver mild Kupffer cells hyperplasia and sinusoidal distentionin favor of congestion were seen (Figure 6(A)) In addition


we observed moderate congestion sinusoidal dilatation withpreserved white lymphoid pulp in the spleen tissue (Fig-ure 6(B)) In the kidney the glomeruli were unremarkablebut mild peritubular congestion and proteinaceous castformations were noted in some tubules (Figure 6(C)) In allof the histopathological examinations we observed minimalhepatic and renal toxicity No histological abnormality wasfound in other major organs such as the brain the heart andthe lung Additionally no significant pathologic changes werefound in themajor organs of animals treatedwith 3125mgkgPNPC and lower doses compared with the control group so3125mgkg dose was ruled out as the toxic dose

310 PNPC Protective Effects on Animal Breast Cancer In theprotective study PNPC was injected for 24 consecutive daysfrom 3 days before to 21 days after tumor transplantationAt the end of the study the tumor take rate was at 375in comparison with control group (875 119875 lt 005)Additionally PNPC-treated mice had a greater survival rateof at least 20 weeks after tumor transplantation compared tothe control animals who died by the end of the eighth week

311 PNPC Therapeutic Effects on Animal Breast CancerPNPCwas given for 24 days after tumor transplantation fromday 14 up to day 38 to evaluate the therapeutic effects of PNPConmice breast cancer At the end of the third week the tumordisappeared in 3 and the tumor volume decreased in 3 andincreased in other two mice in PNPC group Animals gainedweight in PNPC group compared to the negative controlTheaverage tumor volume and weight were significantly less thanthe negative control in the second and thirdweeks after PNPCtreatment (119875 lt 005) The mean final tumor volume reachedapproximately 390mm3 and 1420mm3 in PNPC-treated andcontrol animals respectively (Figure 7) The mean tumorvolume and weight in the positive control group showedinsignificant changes with PNPC group

312 The Tumor Type and Characterization Spontaneousmouse mammary tumor was invasive ductal carcinomamalignant cells with hyperchromatic and polymorphismnuclei and a low to moderate rate of mitosis Tumor cellinfiltration was observed in the surrounding tissues and nestsof carcinoma cells with grade IIIII based on Scarff-Bloom-Richardson Scale (Supplemental Figure 6)

313 Metastases At the end of the study all animals under-went routine surgery There were no signs of metastasis inmajor organs of both PNPC-treated and control groups

314 Immunohistochemistry Examinations Immunohisto-chemistry examinations in PNPC-treated animals showedincreased proapoptotic Bax protein expression in breasttumor in comparison with control (Figure 8 and Supple-mental Figure 7A) Antiapoptotic Bcl-2 protein expressionconceivably lowered after PNPC therapy (Figure 8 andSupplemental Figure 7B) In addition treatment with PNPCcaused a significant reduction in Bcl-2 activity and Bcl-2Baxratio compared to the control group (119875 lt 005)

0

05

1

15

2

14 21 28 35Days

Negative controlPositive control

PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)

Figure 7 The polymeric nanoparticle curcumin effects on tumorsize (cm3) in mouse mammary tumor The positive control ldquodox-orubicin and cyclophosphamide were used as positive controlrdquo Datareported are mean plusmn SD lowast119875 lt 005 compared to negative controlPNPC = polymeric nanoparticle curcumin

0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast

Figure 8Thepolymeric nanoparticle curcumin effects on immuno-histochemical markers in mouse mammary tumor Data reportedare mean plusmn SD lowast119875 lt 005 compared to control PNPC = polymericnanoparticle curcumin

Angiogenesis also dramatically decreased in breasttumors of PNPC-treated animals CD31 activity reduced inPNPC-treated mice tumors compared to control animals(Figure 8 and Supplemental Figure 7C) Moreover most ofthe tumor cells represented high Ki67 marker in controlgroup The mean proliferative cell number in PNPC-treatedmice was lower than the control group (119875 lt 005) (Figure 8and Supplemental Figure 7D)

4 Discussion

Our study showed that the polymeric nanocarrier is anegative amphipathic and biodegradable polymer suitablefor drug delivery They are new types of biocompatiblepolymeric chains taken from plant fatty acids suitable forcurcumin bioavailability Figure 1 shows that with increasing


nanocarrier concentration to more than CMC the flores-cence intensity has significantly risen This is due to the self-aggregation of micelle blocks and development of hydropho-bic compartment inside themicelles and subsequent increaseof pyrene florescence Additionally CMC low value indicatesmicelles thermodynamic stability which is enhanced afterextreme in vivo dilution and may improve circulation timecompared to surfactant micelles [14] Another advantage ofthis nanocarrier is its small size which provokes passivetargeting of the tumor tissues by increasing permeability andretention Moreover the stability of encapsulated curcuminin the polymeric nanoparticle is quite appropriate comparingwith free curcuminwhich degrades to about 90 after 30min[28] Therefore this nanocarrier conserves curcumin fromdecomposition and degradation and can be considered as anappropriate drug carrier for in vitro and in vivo experiments

The major goal of our study was to develop polymericnanoparticle curcumin as an applicable strategy in can-cer treatment Toxicity is a crucial factor for nanoparticlesafety in nanomedicine arena Understanding of variousdeterminants of nanoparticle toxicity helps establishing suit-able strategies for selecting appropriate compositions todevelop biocompatible and efficacious polymeric carriersin nanomedicine requests In our study the polymericnanoparticle curcumin significantly suppressed proliferationof human and mouse carcinoma cells in vitro Based onhematological blood chemical and histopathological exam-inations minimal hepatic and renal toxicity was seen withhigh PNPC doses In addition in vivo results showed thattumor incidence weight and size were significantly declinedin PNPC-treated animals PNPC also induced proapoptoticBax protein expression and reduced antiapoptotic Bcl-2protein levels relative to the control group Moreover pro-liferative and angiogenic markers were lowered in PNPC-treated animals These findings point to the features of thepolymeric carrier as a promising drug delivery system forcancer therapy

In the present study we showed that monotherapy withPNPC at high doses (250mgkg) and polymeric nanocarrieralone caused the animal acute death Daily administration ofPNPC and PNP at higher doses (125mgkg) for one weekhas also been life-threatening Macroscopic changes suchas ascites weight loss and imbalance together with otheruntoward sequels like splenomegaly were also seen in someanimals Histological examinations revealed macrophageinfiltration and splenic hyperplasia Sodium and K+ levelswere significantly increased at high PNPC doses compared tothe control animalsThis indicates that fluid balance is some-how affected by PNPC administration and is in agreementwith ascites and diarrhea observed in some animals at thesame dose groups RBC Hct and Hgb levels were drasticallyincreased at 625mgkg and higher doses compared to thecontrols Increased Cr level may be due to Cr release fromdamaged muscular cells [21] We are not sure about thereversibility of PNPC and PNP induced blood chemistryand hematological changes since it is beyond the scope ofthe present study Significant decrease in liver weight andalbumin level together with increased ALT AST ALP andGGT in PNPC- or PNP-treated animals points to the hepatic

effects of high doses and may by induced by polymericnanocarrier metabolism Nevertheless striking liver changeson the above mentioned parameters were seen in animalstaking very high PNPC and PNP dosesThese outcomes mayhave been primary or secondary effects from nanocarrier insome major organs such as liver kidney and spleen Ourresults support this hypothesis that mPEG may trigger liverdysfunctions together with other organ toxicities [29 30]

Our important finding is that the polymeric nanocarrierdramatically increased tumor-suppressing effects of cur-cumin both in cell culture and in a typical animal model ofbreast cancer Our results also showed that tumor incidenceweight and size were significantly declined in PNPC-treatedgroup Fairly complete tumor regression after 24 days is adramatic finding with PNPC treatment observed in someanimals We also observed a decrease in tumor incidenceas well as tumor size in both protective and PNPC-treatedmice compared to the control group Tumor incidenceand size are indicators of proliferation and angiogenesis[31] Changes in tumor growth characteristics observed inanimals treated with PNPC suggest antiproliferative andantiangiogenic effects of PNPC Lower tumor incidence andsmaller tumor size may have been attributed to the directeffects of PNPC owing to its potential antiproliferative andantiangiogenic roles or its indirect strong oxidative response[32] It should be noted that other pathways involved inthe protective effects of PNPC are being pursued in ourlaboratory

Apoptosis role in breast carcinogenesis has been exten-sively studied It has been shown accordingly that resis-tance to apoptosis in premalignant breast epithelial cells candevelop breast tumors [33] In order to gain insight intothe mechanisms involved in apoptosis induction mediatedby PNPC in breast cancer we studied its effects on theexpression of Bcl-2 and Bax proteins under in vivo situationsMany genetic alterations of breast cancer are actually derivedfrom an imbalance between pro- and antiapoptotic membersof the Bcl-2 family [34] It has generally been establishedthat oncoprotein Bcl-2 duels with its counteracting twina protein known as Bax Overexpression of Bax promotescell death conversely Bcl-2 functions as a suppressor ofapoptosis A decrease in Bcl-2Bax ratio has been consideredas a reliable indicator of the overall propensity of a cell toundergo apoptosis [34] In the present study PNPCdecreasedBcl-2Bax ratio by suppressing Bcl-2 expression and BaxstimulationThis may be indicative of the Bcl-2 family role inapoptosis inductionmediated by PNPC inmice breast cancer[35 36] These findings reveal a new therapeutic potentialfor PNPC in different tissues malignancies via apoptosisinduction

In addition tumor growth noticeably depends on angio-genesis where simultaneous enhances in the tumor vascula-ture supply nutrients and oxygen to the growing neoplasticcells is required [37] Growth and progression of breasttumor as well as growth of most of other cancers areangiogenesis-dependent processes High angiogenic activityin the primary tumor seems to be well correlated withuntoward sequels in patients suffering from breast cancerBreast cancer is considered to be an angiogenic carcinoma


due to high expression of proangiogenic factors [38] Wefound in vivo anticarcinogenic properties of nanocurcuminas effective as in vitro antiproliferative and antiangiogeniceffects of curcumin These compounds presumably changethe balance of pro-and antiangiogenic factors in tumor tissuesand fix the effective delivery system of therapeutic drugs totumor cells in a larger scale Our results provide support fora potential therapeutic role of PNPC in breast cancer viatheir antiangiogenic and antiproliferative properties In factit seems that the polymeric nanocarrier increases tumor cellaccess to curcumin and in turn it causes tumor cells death

In some previous reports polymeric nanocarriers wereused for curcumin tissue delivery Bisht et al (2007) syn-thesized a polymeric nanoparticle of curcumin (50ndash100 nmrange) in an encapsulated formulation utilizing micellaraggregates of cross-linked and indiscriminate acrylic copoly-mers of N-isopropylacrylamide (NIPAAM) with N-vinyl-2-pyrrolidone (VP) [15] An ideal drug delivery platform mustbe biodegradable biocompatible and free from incidentaladverse effects Bishtrsquos nanocarriers are nondegradable andharmful to health due to NIPAAM VP and poly(ethyleneglycol) monoacrylate monomers use But in our nanocarrierform fatty acids with biodegradable and biocompatibleproperties were adopted to produce the least health harmfuleffects unless in very high doses In addition Anand etal (2010) used PLGA-poly-ethylene glycol nanoparticles todeliver encapsulated curcumin with 975 efficiency butlow drug loading [39] Our data reveals for the first timethat nanopolymeric compounds not only boost curcuminsolubility and uptake in cell lines but also increase its toxicityon cancer cells This issue with the biodegradable abilityshows that polymeric carriers are unique host cell drug inanimal models as per the following aspects

(a) They are inexpensive neutral nontoxic biodegrad-able and easy to use

(b) Pharmaceutical agents can create a weak structuralcombination helping their easy cellular separation

(c) Although nanocarriers are biodegradable they arestable and resistant not only in dry environments butalso in fluids with low temperature

5 Conclusion

In summary we showed that PNPC is effective in sup-pressing tumor growth both in vitro and in vivo Tumorgrowth in PNPC-treated mice was significantly suppressedandor almost completely stopped at the end of the treat-ment Our results also suggested PNPC appropriate dose(3125mgkgdaily for 3 weeks) Lower doses effectiveness orfewer PNPC shots are currently being investigated in ourlaboratory The present study provides persuasive evidencefor polymeric nanocarrier superior biocompatibility in phar-macological arena which in turn can reduce anticancer drugside effects with excellent tumor-suppressing response

Conflict of Interests

The authors reported no conflict of interests The authorsalone are responsible for the content of the paper

Acknowledgment

This study was supported by a grant of Tehran University ofMedical Sciences

References

[1] M Bayet-Robert and D Morvan ldquoMetabolomics revealsmetabolic targets and biphasic responses in breast cancer cellstreated by curcumin alone and in association with docetaxelrdquoPLoS ONE vol 8 no 3 Article ID e57971 2013

[2] PAnandA BKunnumakkara RANewman andB BAggar-wal ldquoBioavailability of curcumin problems and promisesrdquoMolecular Pharmaceutics vol 4 no 6 pp 807ndash818 2007

[3] M Kakarala D E Brenner H Korkaya et al ldquoTargeting breaststem cells with the cancer preventive compounds curcumin andpiperinerdquo Breast Cancer Research and Treatment vol 122 no 3pp 777ndash785 2010

[4] S S Dhule P Penfornis T Frazier et al ldquoCurcumin-loaded120574-cyclodextrin liposomal nanoparticles as delivery vehiclesfor osteosarcomardquoNanomedicine Nanotechnology Biology andMedicine vol 8 no 4 pp 440ndash451 2012

[5] N Ghalandarlaki A M Alizadeh and S Ashkani-EsfahanildquoNanotechnology-applied curcumin for different diseases ther-apyrdquo BioMed Research International vol 2014 Article ID394264 23 pages 2014

[6] T G Shutava S S Balkundi P Vangala et al ldquoLayer-by-layer-coated gelatin nanoparticles as a vehicle for delivery of naturalpolyphenolsrdquo ACS Nano vol 3 no 7 pp 1877ndash1885 2009

[7] B Le Droumaguet J Nicolas D Brambilla et al ldquoVersatileand efficient targeting using a single nanoparticulate platformapplication to cancer and alzheimerrsquos diseaserdquo ACS Nano vol6 no 7 pp 5866ndash5879 2012

[8] F D Ledley ldquoNonviral gene therapy the promise of genes aspharmaceutical productsrdquo Human Gene Therapy vol 6 no 9pp 1129ndash1144 1995

[9] N Suwannateep S Wanichwecharungruang S F Haag etal ldquoEncapsulated curcumin results in prolonged curcuminactivity in vitro and radical scavenging activity ex vivo on skinafter UVB-irradiationrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 82 no 3 pp 485ndash490 2012

[10] E Babaei M Sadeghizadeh Z M Hassan M A H Feizi FNajafi and S M Hashemi ldquoDendrosomal curcumin signifi-cantly suppresses cancer cell proliferation in vitro and in vivordquoInternational Immunopharmacology vol 12 no 1 pp 226ndash2342012

[11] M Khaniki S Azizian A M Alizadeh H Hemmati N Ema-mipour andMAMohagheghi ldquoThe antiproliferative and anti-cancerogenic effects of nano-curcumin in rat colon cancerrdquoTehran University Medical Journal vol 71 no 5 pp 277ndash2842013

[12] M N Sarbolouki A M Alizadeh M Khaniki S Azizianand M A Mohaghgheg ldquoProtective effect of dendrosomal cur-cumin combination on colon cancer in ratrdquo Tehran UniversityMedical Journal vol 69 no 11 pp 678ndash685 2012


[13] M TMirgani B IsacchiM Sadeghizadeh et al ldquoDendrosomalcurcumin nanoformulation downregulates pluripotency genesvia miR-145 activation in U87MG glioblastoma cellsrdquo Interna-tional Journal of Nanomedicine vol 9 no 1 pp 403ndash417 2014

[14] A SahuU BoraNKasoju andPGoswami ldquoSynthesis of novelbiodegradable and self-assembling methoxy poly(ethyleneglycol)-palmitate nanocarrier for curcumin delivery to cancercellsrdquo Acta Biomaterialia vol 4 no 6 pp 1752ndash1761 2008

[15] S Bisht G Feldmann S Soni R Ravi C Karikar and A Mait-ra ldquoPolymeric nanoparticle-encapsulated curcumin (ldquonanocur-cuminrdquo) a novel strategy for human cancer therapyrdquo Journal ofNanobiotechnology vol 5 article 3 2007

[16] Z Ma A Haddadi O Molavi A Lavasanifar R Lai andJ Samuel ldquoMicelles of poly(ethylene oxide)-b-poly(120576-capro-lactone) as vehicles for the solubilization stabilization and con-trolled delivery of curcuminrdquo Journal of Biomedical MaterialsResearch Part A vol 86 no 2 pp 300ndash310 2008

[17] M Gou KMen H Shi et al ldquoCurcumin-loaded biodegradablepolymeric micelles for colon cancer therapy in vitro and invivordquo Nanoscale vol 3 no 4 pp 1558ndash1567 2011

[18] L Song Y Shen J Hou L Lei S Guo and C Qian ldquoPolymericmicelles for parenteral delivery of curcumin preparation char-acterization and in vitro evaluationrdquo Colloids and Surfaces APhysicochemical and Engineering Aspects vol 390 no 1ndash3 pp25ndash32 2011

[19] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[20] R A Sharma A J Gescher and W P Steward ldquoCurcumin thestory so farrdquoEuropean Journal of Cancer vol 41 no 13 pp 1955ndash1968 2005

[21] M Mohsenikia A M Alizadeh S Khodayari et al ldquoTheprotective and therapeutic effects of alpha-solanine on micebreast cancerrdquo European Journal of Pharmacology vol 718 no1-3 pp 1ndash9 2013

[22] A M Alizadeh M Faghihi V Khori et al ldquoOxytocin pro-tects cardiomyocytes from apoptosis induced by ischemia-reperfusion in rat heart role of mitochondrial ATP-dependentpotassium channel and permeability transition porerdquo Peptidesvol 36 no 1 pp 71ndash77 2012

[23] J-L Achard I van Praagh and V Feillel ldquoScarff-Bloom-Richardson (SBR) grading a pleiotropic marker of chemosen-sitivity in invasive ductal breast carcinomas treated by neoadju-vant chemotherapyrdquo International Journal of Oncology vol 20no 4 pp 791ndash796 2002

[24] A M Alizadeh M Khaniki S Azizian M A MohaghgheghiM Sadeghizadeh and F Najafi ldquoChemoprevention ofazoxymethane-initiated colon cancer in rat by using a novelpolymeric nanocarrier-curcuminrdquo European Journal ofPharmacology vol 689 no 1ndash3 pp 226ndash232 2012

[25] A Vila A Sanchez M Tobıo P Calvo and M J AlonsoldquoDesign of biodegradable particles for protein deliveryrdquo Journalof Controlled Release vol 78 no 1ndash3 pp 15ndash24 2002

[26] R H Muller C Jacobs and O Kayser ldquoNanosuspensions asparticulate drug formulations in therapy rationale for develop-ment and what we can expect for the futurerdquo Advanced DrugDelivery Reviews vol 47 no 1 pp 3ndash19 2001

[27] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[28] Y-JWangM-H Pan A-L Cheng et al ldquoStability of curcuminin buffer solutions and characterization of its degradationproductsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 15 no 12 pp 1867ndash1876 1997

[29] J Heyes K Hall V Tailor R Lenz and I MacLachlanldquoSynthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug deliveryrdquo Journal ofControlled Release vol 112 no 2 pp 280ndash290 2006

[30] X Wei C Gong M Gou et al ldquoBiodegradable poly(120576-capro-lactone)-poly(ethylene glycol) copolymers as drug deliverysystemrdquo International Journal of Pharmaceutics vol 381 no 1pp 1ndash18 2009

[31] K Maeda Y-S Chung S Takatsuka et al ldquoTumour angiogen-esis and tumour cell proliferation as prognostic indicators ingastric carcinomardquo British Journal of Cancer vol 72 no 2 pp319ndash323 1995

[32] J-M Zingg S T Hasan and M Meydani ldquoMolecular mecha-nisms of hypolipidemic effects of curcuminrdquo BioFactors vol 39no 1 pp 101ndash121 2013

[33] E C M Mommers P J van Diest A M Leonhart C J LM Meijer and J P A Baak ldquoBalance of cell proliferation andapoptosis in breast carcinogenesisrdquo Breast Cancer Research andTreatment vol 58 no 2 pp 163ndash169 1999

[34] J B Baell and D C S Huang ldquoProspects for targeting theBcl-2 family of proteins to develop novel cytotoxic drugsrdquoBiochemical Pharmacology vol 64 no 5-6 pp 851ndash863 2002

[35] D Karunagaran R Rashmi and T R Santhosh Kumar ldquoInduc-tion of apoptosis by curcumin and its implications for cancertherapyrdquo Current Cancer Drug Targets vol 5 no 2 pp 117ndash1292005

[36] R J Anto A Mukhopadhyay K Denning and B B AggarwalldquoCurcumin (diferuloylmethane) induces apoptosis throughactivation of caspase-8 BID cleavage and cytochrome c releaseits suppression by ectopic expression of Bcl-2 and Bcl-xlrdquoCarcinogenesis vol 23 no 1 pp 143ndash150 2002

[37] J Folkman ldquoWhat is the evidence that tumors are angiogenesisdependentrdquo Journal of the National Cancer Institute vol 82 no1 pp 4ndash6 1990

[38] S B Fox D G Generali and A L Harris ldquoBreast tumourangiogenesisrdquo Breast Cancer Research vol 9 no 6 article 2162007

[39] P Anand H B Nair B Sung et al ldquoDesign of curcumin-loaded PLGAnanoparticles formulationwith enhanced cellularuptake and increased bioactivity in vitro and superior bioavail-ability in vivordquo Biochemical Pharmacology vol 79 no 3 pp330ndash338 2010

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Autoimmune Diseases


Anesthesiology Research and Practice

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


nanoparticles has promising results for the poor water solu-ble hydrophobic agents like curcumin Feasible preparationand use size fitness environmental sustainability and shellviability are all important factors for a suitable carrier [8]According to some studies encapsulated form of curcuminimproves its medical properties and solubility [9] Previousstudies showed that dendrosome a diblock nanostructuremade by oleic acid (OA) and polyethylene glycol (PEG 400Dalton) with anticancer and proapoptosis effects is a suitableoption for curcumin encapsulation [10ndash12] In the presentresearch we used modified 2000 Dalton molecular weightmonomethoxy-PEG (mPEG) for curcumin encapsulationRecently our research group evaluated morphology andphysical behavior of mPEG

2000-OA [13] Based on CMC

measurement this novel nanocarrier can be considered as anappropriate drug delivery system for curcumin delivery incancer cells A unique feature of mPEG

2000-OA is the ease

with which the structure of their monomers can be variedin order to provide suitable inert drug porters for targetcells In this study we exploited mPEG

2000-OA for curcumin

encapsulation as polymeric nanoparticle curcumin (PNPC)and investigated its efficiency drug loading and stability withmore focus on clinical observations hematologicalbloodchemistry tests and histological examinations In additionPNPCrsquos protective and therapeutic effects were also assessedin an animal model of breast cancer Our results shed newlight on PNPC potential biocompatibility in in vitro and invivo biological systems

2 Materials and Methods

21 Materials Curcumin (gt99) doxorubicin hydrochlo-ride cyclophosphamide ketamine and xylazine were pur-chased from Sigma Aldrich Co (St Louis MO USA) Mate-rials for the polymeric nanocarrier (OM2000) preparationincluding oleoyl chloride and methoxy polyethylene glycol2000 (mPEG 2000) were got from Sigma-Aldrich Co (StLouis MO USA) Chloroform and triethyl amine werealso purchased from Merck-Serono Company (Germany)The human hepatocellular (HuH-7) mouse mammary (4T1)carcinoma cell lines and normal human fibroblast cells wereprocured from Pasteur Institute of Iran (Pasteur InstituteTehran Iran) Polyclonal mouse anti-ratrabbit Bax Bcl-2and CD31 antibodies (DAKO Corporation USA) and rabbitmonoclonal antibody against Ki-67 (Thermo Fisher ScientificInc San Jose CA) were purchased

22 The Study Design Studies were conducted in five seriesof experiments including (i) PNPC synthesis (ii) PNPCstability and drug loading (iii) PNPC effects on cell viabilityof mammary and hepatocellular carcinoma cells (iv) PNPCacute and chronic toxicity and (v) PNPC protective andtherapeutic effects on animal model of breast cancer

23 Animals All animal studies have been conductedaccording to relevant national and international guidelines ofthe Weatherall report and Institutional Animal Care and Use

Committee (IACUC) of Tehran University of Medical Sci-ences Inbred female BALBcmice (6ndash8 weeks old purchasedfrom Iran Pasteur Institute) were maintained under 12-hourdark and light cycle with free access to food and water

24 Polymeric Nanocarrier Synthesis Polymeric nanoparticle(PNP OM2000) was synthesized by esterification of oleoylchloride (301 g 001mol) and methoxy polyethylene glycol2000 (20 g 001mol) in the presence of triethyl amine (12 g0012mol) and chloroform as solvent at 25∘C for 2 h (seeSupplemental Figure 1 in Supplementary Materials avail-able online at httpdxdoiorg1011552015824746) Triethylamine hydrochloride was filtered from organic phase Chlo-roform was then evaporated and OM2000 dried in a 40∘Cvacuum oven for 4 h with 96 purity Fourier transforminfrared (FT-IR) spectroscopic measurements in the potas-sium bromide (KBr) pellets were done using a Perkin-ElmerspectrometerThe 1HNMR spectrumswere carried out usingBruker 400MHZ in DMSO-d

5

25 Critical Micelle Concentration Determination Criticalmicelle concentration (CMC) of PNP was determined bydetecting shifts in the pyrene fluorescence absorbance spectra[14] In this regard 3mL of pyrene solution (6 times 10minus6M)in acetone was added to a glass test tube and evaporated toremove the solvent Then 5mL of PNP (0005 to 1mgmL)was conjugated with phosphate-buffered saline (PBS 001MpH 74) added to the glass test tubes to reach to 6 times 10minus7Mpyrene final concentration The solutions were vortexed andconditioned at 37∘C overnight Fluorescence excitation spec-tra of pyrene (300ndash350 nm) were measured at an emissionwavelength of 390 nm with slit widths of 25 and 50 nm(Perkin-Elmer Fluorimeter USA) for excitation and emis-sion respectively The fluorescence excitation shifts from 334to 339 nm were used to determine CMC of the polymericnanocarrier

26 PNPC Physical Properties Dynamic light scattering(DLS) and atomic force microscopy (AFM) were carriedout to evaluate alterations in the zeta-potential particle sizeparticle size distribution shape and polydispersity index(PdI) of the polymeric nanoparticle [14ndash16] Nanoparticlezeta-potential and size distribution of PNPC (001M PBSpH 74) were analyzed by DLS (Zetasizer NanoZS MalvernInstruments UK) using an argon laser beam at 633 nm and90∘ scattering angle To determine PNPC shape a drop ofPNPC solution (005mgmL) was placed on freshly cleavedmica and was allowed to dry at room temperature Thesample was then mounted on a microscope scanner andimaged in semicontact mode with AFM (JPK InstrumentsCo Germany)

27 PNPC Encapsulation Efficiency and Drug Loading Inthis section measurements were performed in triplicateDifferent concentrations of curcumin (2 to 25mg) in acetonesolutionweremixedwith 100mgnanocarrier in 3mLofwaterand rotated with rotary evaporator till acetone evaporationPNPC was filtered using 022120583m syringe filter to remove








times 100

(1)












119881 =1

6 (120587119871119882119863) (2)




2O2









3 Results









5




2


2protons of mPEG






25

20

15

10

05

00

000

minus025

minus050

minus075

minus150

minus175

minus200

minus225

minus100

minus250

minus125

minus275

minus300

CMC


I (339334)

I

(a)

0

200

400

600

320 330 340 350



05

1

Flor

esce

nce i

nten

sity

(au

)

(b)


05

101520

01 1 10 100 1000 10000

freshly prepared

after one week


Volu

me (

)





Size (dmiddotnm)

(a)

0100 02 03 04 05 06 07 08 09 1000

01

02

03

04

05

06

07

08

09

1000

01

02

03

04

05

06

07

08

09

10

142 nm

35 nm

(120583m)

(b)





Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)


(a)


100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)


05

10152025

01 1 10 100 1000 10000



Volu

me (

)

Size (dmiddotnm)

(a)


Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)








0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








times 100

(1)












119881 =1

6 (120587119871119882119863) (2)




2O2









3 Results









5




2


2protons of mPEG






25

20

15

10

05

00

000

minus025

minus050

minus075

minus150

minus175

minus200

minus225

minus100

minus250

minus125

minus275

minus300

CMC


I (339334)

I

(a)

0

200

400

600

320 330 340 350



05

1

Flor

esce

nce i

nten

sity

(au

)

(b)


05

101520

01 1 10 100 1000 10000

freshly prepared

after one week


Volu

me (

)





Size (dmiddotnm)

(a)

0100 02 03 04 05 06 07 08 09 1000

01

02

03

04

05

06

07

08

09

1000

01

02

03

04

05

06

07

08

09

10

142 nm

35 nm

(120583m)

(b)





Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)


(a)


100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)


05

10152025

01 1 10 100 1000 10000



Volu

me (

)

Size (dmiddotnm)

(a)


Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)








0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




119881 =1

6 (120587119871119882119863) (2)




2O2









3 Results









5




2


2protons of mPEG






25

20

15

10

05

00

000

minus025

minus050

minus075

minus150

minus175

minus200

minus225

minus100

minus250

minus125

minus275

minus300

CMC


I (339334)

I

(a)

0

200

400

600

320 330 340 350



05

1

Flor

esce

nce i

nten

sity

(au

)

(b)


05

101520

01 1 10 100 1000 10000

freshly prepared

after one week


Volu

me (

)





Size (dmiddotnm)

(a)

0100 02 03 04 05 06 07 08 09 1000

01

02

03

04

05

06

07

08

09

1000

01

02

03

04

05

06

07

08

09

10

142 nm

35 nm

(120583m)

(b)





Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)


(a)


100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)


05

10152025

01 1 10 100 1000 10000



Volu

me (

)

Size (dmiddotnm)

(a)


Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)








0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



25

20

15

10

05

00

000

minus025

minus050

minus075

minus150

minus175

minus200

minus225

minus100

minus250

minus125

minus275

minus300

CMC


I (339334)

I

(a)

0

200

400

600

320 330 340 350



05

1

Flor

esce

nce i

nten

sity

(au

)

(b)


05

101520

01 1 10 100 1000 10000

freshly prepared

after one week


Volu

me (

)





Size (dmiddotnm)

(a)

0100 02 03 04 05 06 07 08 09 1000

01

02

03

04

05

06

07

08

09

1000

01

02

03

04

05

06

07

08

09

10

142 nm

35 nm

(120583m)

(b)





Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)


(a)


100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)


05

10152025

01 1 10 100 1000 10000



Volu

me (

)

Size (dmiddotnm)

(a)


Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)








0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Curcumin loading10

10 12 14 16 18 20 22 24 26

9

8

8

7

6

6

5

4

4

3

2

2

10

0

Dru

g lo

adin

g (

)


(a)


100

80

60

40

20

400

8 12 16 20 24

Enca

psul

atio

n effi

cien

cy (

)


(b)


05

10152025

01 1 10 100 1000 10000



Volu

me (

)

Size (dmiddotnm)

(a)


Total curcumin (mg)

Cur

cum

in co

nten

t (m

g)

0 2 4 6 8 10 12 14 16 18 20 22 24 260

1

2

3

4

5

6

7

After one weekFresh

(b)








0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60

80

100

120

140

0 10 20 30 40 50

PNPCCurcumin

PNPDOX


Cel

l via

bilit

y(

)4T1 24h

(a)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCCurcumin

PNPDOX

4T1 48h


Cel

l via

bilit

y(

)

(b)

0

20

40

60

80

100

120


PNPC CurcuminDOXPNP

Cel

l via

bilit

y(

)

HuH-7 24h

(c)

0

20

40

60

80

100

120

0 10 20 30 40 50

PNPCPNP

CurcuminDOX


Cel

l via

bilit

y(

)HuH-7 48h

(d)













ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



ParametersGroups


PNPC625mgkg

PNPC125mgkg



















ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



ParametersGroups


PNPC3125mgkg

PNPC625mgkg

















(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(A) (a)

(B)

(C)

(b)

(c)












0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








0

05

1

15

2

14 21 28 35Days


PNCP

lowastlowastlowast

lowast

Mea

n tu

mor

size

(cm

3)


0

05

1

15

2

25

3

Bax Bcl-2 Ki67 CD31

Mea

n ex

pres

sion

Parameters

ControlPNPC

lowastlowastlowast

lowast



4 Discussion
















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















5 Conclusion




Acknowledgment


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Encapsulation of Curcumin in Diblock Copolymer Micelles for Cancer Therapy

Documents

Transcript of Encapsulation of Curcumin in Diblock Copolymer Micelles for Cancer Therapy