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Small Molecule Therapeutics

Novel Curcumin-Loaded Magnetic Nanoparticles forPancreatic Cancer Treatment

Murali M. Yallapu1, Mara C. Ebeling1, Sheema Khan1, Vasudha Sundram1, Neeraj Chauhan1,Brij K. Gupta1, Susan E. Puumala2, Meena Jaggi1,3, and Subhash C. Chauhan1,3

AbstractCurcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed

potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of

CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and

poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded

magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic

efficacy of this novelMNP-CUR formulation inpancreatic cancer.Humanpancreatic cancer cells (HPAF-II and

Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a

result, theMNP-CUR formulation effectively inhibited growth ofHPAF-II and Panc-1 cells in cell proliferation

and colony formation assays. TheMNP-CUR formulation suppressed pancreatic tumor growth in anHPAF-II

xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory

effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA),

B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell

surface–associated Mucin 1 (MUC1), collagen I, and enhanced membrane b-catenin expression. MNP-CUR

formulationdidnot showany signof hemotoxicity andwas stable after incubationwithhumanserumproteins.

In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as

compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation

was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In

conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of

pancreatic cancer. Mol Cancer Ther; 12(8); 1471–80. �2013 AACR.

IntroductionPancreatic cancer ranks fourth in causing cancer-related

deaths in the United States (1). Recent statistics estimatedthat 44,030 individuals were diagnosed with pancreaticcancer and 37,660 patients died in 2011 from this highlydevastating disease (1). In spite of significant progress inthe area of cancer therapeutics, pancreatic cancer stillremains by and large untreatable with an extremely poor5-year survival rate (1). Poor vasculature and the hyperfi-brotic and desmoplastic tumormicroenvironment of pan-

creatic cancer are primarily responsible for reduceduptake of chemotherapeutic drugs and low therapeuticoutcome (2, 3). Gemcitabine is a standard chemothera-peutic drug used for the treatment of pancreatic cancer;however, survival is increased by only 5 to 12 months(4, 5). In addition, this therapeutic approach may lead tosevere toxicity and may also result in chemo-resistance.Therefore, developing a safer and more efficient thera-peutic modality to improve management of pancreaticcancer is highly desirable. Because of their potent anti-cancer activity, a few natural dietary ingredients areconsidered as complementary and alternative medicineby the National Cancer Institute for cancer prevention/treatment options. Treatment that can modulate tumormicroenvironment using a natural dietary agentwould beconsidered a novel therapeutic strategy.

Curcumin (CUR; chemical structure, Fig. 1A) is awidelyknown natural bioactive polyphenolic component of tur-meric. CUR is also a "Generally Recognized As Safe"compound by the Food and Drug Administration (FDA;ref. 6). CURexhibits both anticancer and cancer preventionactivities by suppressing key elements of initiation, pro-motion, andmetastasis of awide range of cancer(s) (7). Themultitargeting role of CUR enables it to conduct a widespectrum of actions against cancer(s) (8). Epidemiological

Authors' Affiliations: 1Cancer Biology Research Center; 2Methodologyand Data Analysis Center, Sanford Research/University of South Dakota;and 3OB/GYN, Basic Biomedical Science Division, Sanford School ofMedicine, University of South Dakota, Sioux Falls, South Dakota

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for M.M. Yallapu, S. Khan, N. Chauhan, M. Jaggi, S.C.Chauhan: Department of Pharmaceutical Sciences, University of Tennes-see Health Science Center, Memphis, Tennessee.

Corresponding Author: Subhash C. Chauhan, Department of Pharma-ceutical Sciences, University of Tennessee Health Science Center, 19 SManassas Avenue, Memphis, TN 38163. Phone: 901-448-2175; Fax: 901-448-1051; Email: [email protected]

doi: 10.1158/1535-7163.MCT-12-1227

�2013 American Association for Cancer Research.

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Therapeutics

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Published OnlineFirst May 23, 2013; DOI: 10.1158/1535-7163.MCT-12-1227

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studies revealed that populations with CUR consumptionhave lower incidence and risk of various types of cancersincludingpancreatic cancer (9–11).BecauseCURpossessesexceptional therapeutic properties, more than 20 clinicaltrials are underway to assess CUR’s efficacy in cancertreatment(s) (as of January 2013). In vitro studies have

concluded that CUR exhibits anticancer effects at micro-molar concentrations. However, achieving these concen-trations at the tumor site in humans is highly challengingand has not yet been accomplished due to its highermetabolic activity and low bioavailability (10, 12). CURnanoformulations can be a useful delivery module (12).CUR nanoformulations have showed an enhanced deliv-ery of CUR in biologically active form to various cancercells (13). Although many nanotechnology investigationshave primarily focused on either improving stability orbioavailability of CUR, limited studies have been pursuedrelated to therapeutic effects on pancreatic cancer (14–18).To date, no study has reported on magnetic nanoparticle(MNP)-mediatedCURdelivery for treatment of pancreaticcancer. Herein we report, for the first time, the effects ofa novel CUR-loaded MNP (MNP-CUR) formulation(Patent# PCTUS2011/063723) on humanpancreatic cancercells in vitro and in vivo and elucidate its potential trans-lational capabilities. In addition, we have used advancedandappropriate quantitativemethods to study the efficacyof this novel formulation in both in vitro and xenograftmouse models.

Materials and MethodsChemicals, reagents, and antibodies

All chemicals and reagents were acquired from Sigma-Aldrich Corporation and cell culture plastic ware andconsumableswerepurchased fromBDBiosciences, unlessotherwise mentioned. The preparation and characteriza-tion of theMNP-CUR formulation was conducted follow-ing our optimized/patented composition protocol (19).Monoclonal antibodies against B-cell lymphoma-extralarge (Bcl-xL; Abcam), induced myeloid leukemia celldifferentiation protein (Mcl-1; Abcam), proliferatingcell nuclear antigen (PCNA; Cell Signaling Technology),collagen I (Abcam), b-actin (Sigma), cell surface–associ-ated Mucin 1 (MUC1; Cell Signaling Technology), andb-catenin (gift of Dr. K. Johnson, University of NebraskaMedical Center, Omaha, Nebraska) were used for immu-nohistochemistry and Western blot analyses.

Cancer cell lines and animalsHPAF-II and Panc-1 human pancreatic cancer cell lines

were purchased from American Type Cell Culture (Man-assas, VA), cultured asmonolayer in 75 cm2 culture flasksin Dulbecco’s Modified Eagle Medium (DMEM)-F12/DMEM medium (HyClone Laboratories, Inc.) and sup-plemented with 10% heat-inactivated FBS (Atlanta Biolo-gicals), 1%penicillin, and 1% streptomycin (Gibco BRL) at37�C in a humidified atmosphere (5% CO2 and 95% airatmosphere). To maintain authenticity of the cell lines,frozen stocks were prepared from a parent stock andevery 6 months a new frozen stock cell line was used forthe experiments. All animal experiments were carried outusing 6- to 8-week-old male athymic nude/nude mice(Harlan Laboratories) and all procedures were approvedby the Sanford Research/University of South DakotaInstitutional Animal Care and Use Committee.

Figure 1. MNP-CUR formulation efficiently internalizes and exhibitsanticancer activity in pancreatic cancer cells. A, schematicrepresentations of CUR's chemical structure and MNP-CURnanoformulation and a TEM image ofMNP-CUR formulation (10.5� 0.54nm, calculated from 20 randomly chosen nanoparticles using ImageJSoftware). B, MNP-CUR nanoformulation cellular internalization inpancreatic cancer cells. Cancer cells (2 � 105) were treated with a 25 to200 mg MNP-CUR formulation for 6 hours and then processed forPrussian blue staining. Prussian blue stains in cancer cells are identifiedwith black arrows. C, the anticancer effects of CUR and MNP-CUR onHPAF-II and Panc-1 cancer in cell proliferation assays. Cancer cells weretreatedwith 5 to 20 mmol/LCUR/MNP-CUR for 2 days,washedwith PBS,trypsinized, and cell number was counted using a hemocytometer.Number of cells were counted in at least 6 treatments for eachconcentration and presented as mean � SEM. D, effects of CUR andMNP-CUR on the growth of pancreatic cancer cells in colony formationassays.Cancer cellswere treatedwith2 to 8mmol/LCUR/MNP-CUR for 7days, followed with a medium without CUR/MNP-CUR for 7 days. Thecolonies were rinsed with PBS, fixed with methanol, and stained withhematoxylin. Representative colony images are presented as inserts ingraph. Note: Zero (0) on X-axis for cell proliferation and colony formationrepresents controls for CUR andMNP-CUR, respectively. The number ofcolonies was countedmanually. Data represent mean�SEMof triplicateprocedures. �, P < 0.05.

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Cellular uptakeCellular uptake of MNP-CUR formulation in HPAF-II

and Panc-1 cancer cells was evaluated by Prussian bluestain and 1,10-phenanthroline photometric method.For the Prussian blue staining experiment, 2 � 105 cellswere seeded in 6-well plates in 2 mL medium and cellswere allowed to attach to the plates overnight. Thesecells were treated with 25–to 200 mg MNP-CUR formu-lation for 6 hours. After treatment, cells were washed(with PBS), fixed (with methanol), and incubated subse-quently with a mixture of 2% potassium ferrocyanide,2% hydrochloric acid for 30 minutes, and nuclear fastred-aluminum sulfate solution (0.1%) for 5–to 10 minutesat 25�C. The representative images of internalized MNP-CUR in cancer cells were captured at 200� magnificationusing an Olympus BX 41 Microscope (Olympus Corpora-tion). For iron content estimation, pancreatic cancer cells(2� 105) were treatedwith 100 mgMNP-CUR formulation,washed, and trypsinized using 0.25% Trypsin-EDTA, andcell pellets were washed twice with 1� PBS, collected,and dissolved in hydrochloric acid to determine ironcontent using 1,10-phenanthroline photometric method asdescribed in our recent publications (19, 20).

Cell proliferationCells (5 � 104) were seeded in 6-well plates in 2 mL

media and allowed to adhere overnight. Cells were thenincubated with 5, 10, and 20 mmol/L of free CUR or CURequivalent MNP-CUR formulation for 2 days at 37�C. Inthis experiment, equivalent amounts of DMSO or MNPs(no CUR) in PBS served as controls for CUR and MNP-CUR, respectively. The media was removed after 2 days,cells were washed with PBS, trypsinized, and collected inculture medium and cell number was counted using ahemocytometer (21). Each concentration treatment wasdone at least 6 times.

Clonogenic assayCells (500) were seeded in 6-well plates in 2 mL growth

media and incubated for 2–to 3 days to allow grow intocolonies. Cells were treated with 2, 4, and 8 mmol/L freeCUR or MNP-CUR for 7 days and then media withoutdrugs for 7 days at 37�C. Subsequently, cells in all plateswere gently rinsed with PBS, fixed with methanol, andstainedwith hematoxylin. A population of at least 50 cellswas considered a colony and the number of colonies wascounted and quantified according to our standard previ-ously reported protocol (22).

In vivo antitumor activity and survival studyApproximately 6- to 8-week-old male athymic nude

(nu/nu)micewere inoculated subcutaneously at their leftflank with 5� 106 HPAF-II human pancreatic cancer cellsdispersed in a 200 mL solution of PBS and Matrigel (1:1ratio; BD Biosciences; ref. 21). These mice were used toevaluate both antitumor activity and survival analysis.Onday 13 (postinjection with cancer cells), the animals wererandomly distributed into 4 groups (8 mice per group).

Two treatment groups were administered intratumoralinjections of 20 mgCURdissolved in 100 mL of 0.1%Tween20 or 20 mg equivalent CUR containing MNP-CUR dis-persed in 100 mL PBS. Similarly, control groups weretreated with either Tween 20 or empty MNPs (no CUR).The tumor size was measured on day 7, 13, 20, 24, 28, 32,35, and 40 using a digital Vernier caliper. The tumor sizedata were presented up to day 28 because of statisticalanalysis consideration. The tumor volumewas calculatedusing the ellipsoid volume equation: tumor volume(mm3)¼ p/6� L�W�H, where L is length,W is width,andH is height (21). The animalswere sacrificed at the endof the treatment or when tumor volume reached 1,000mm3. Survival of mice was followed until day 40 and aplot was generated using Origin 6.1 software. Tumortissues were collected for immunohistochemical andimmunofluorescence analyses as well as the analysis ofMNP-CUR accumulation.

Immunohistochemical and immunofluorescenceanalyses

Immunohistochemical and immunofluorescence anal-yses were conducted to investigate pathways that areinvolved in MNP-CUR–mediated suppression of pancre-atic tumor growth. For immunohistochemistry analysis,10% formalin fixed tumor tissues were processed asdescribed earlier (21, 23) and tissue slides were incubatedwith primary antibodies against Bcl-xL (1:400), Mcl-1(1:300), PCNA (1:2000), b-catenin (1:200), and MUC1(1:250). Protein expression was detected using MACH 4Universal HRP polymer detection kit (Biocare Medical)and 3,30-diaminobenzidine (DAB substrate kit; BiocareMedical) as described earlier (21). Finally, slides werewashed with water, counterstained with hematoxylin,dehydrated and mounted with Ecomount (Biocare Med-ical), immunostainingwas assessedusing anOlympusBX41 Microscope (Olympus Corporation). Similarly forimmunofluorescence analysis, rehydrated tissue slideswere blocked using 4% normal donkey serum (JacksonImmunoResearch Laboratories) followed by incubationwith primary rat anti-mouse collagen I (1:200; Chondrex,Inc.) and mouse anti-human collagen I (1:200; Abcam).Afterwashingwith PBS, tissue slideswere incubatedwithsecondary antibodies Alexa-Fluor 488 and Alexa-Fluor568 (1:200; Life Technologies). 40,6-Diamidino-2-pheny-lindole was used to visualize nuclei. A laser scanningconfocal microscope (Nikon TIRF) was used with a 20�Apochromat objective andoptical Z sectionswere taken at�0.8 mm.Magnification, pinhole settings, laser, and detec-tor gains, which were set below saturation were identicalacross samples (21). Western blot experiments and anal-yses were followed as described earlier (19).

Accumulation of MNP-CUR in pancreatic tumorsParaffin-embedded tumor tissues were processed

according to our protocol (21) and tissue sections weresubsequently immersed in 10% potassium ferrocyanideand 10%hydrochloric acid solution for 20minutes at 25�C

MNP-Curcumin Formulation for Pancreatic Cancer

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and in 0.1%nuclear fast red-aluminumsulfate solution for5 minutes at 25�C. Following dehydration, tissues wereviewed and representative images captured at 400�mag-nification using an Olympus BX 41 Microscope.

Bioavailability studies of MNP-CUR formulationTo determine bioavailability of CUR in blood serum in

nu/numouse, 50 mg CUR in Tween 20 or CUR equivalentMNP-CUR formulation in PBS solutionwas administeredintraperitoneally (i.p.) using a 30G needle. At predeter-mined time intervals (5, 10, 15, 30, 60, 120, 360, 720, and1440 min), blood was drawn from tails and serum wasimmediately collected by centrifugation at 2,000 rpm for5 minutes (Centrifuge 5415D; Eppendorf AG). Serumsamples were stored at �80�C until further use. Serumsamples (3–5 mL) were lyophilized using a LabconcoFreeze Dry System (�48�C, 133 � 10�3 m Bar; Labconco)and CUR was extracted from serum by incubating 2 daysin acetonitrile. CUR concentrations were determinedusing an UltiMate high-performance liquid chromatog-raphy (DionexCorporation) equippedwithUltiMate 3000injector, RS variable wavelength detector, and anAcclaimpolar advantage column of 3 mm 120 A (4.6 � 150 mm).The mobile phase consisted of 1% citric acid:acetonitrile(50:50, v/v). Chromatograms were collected at 430 nm.Linear calibration curve for CURunder similar conditionswas obtained in the range of 1 to 10 ng/mL.

Biodistribution studiesTo examine MNP-CUR biodistribution in different

organs in mice, tumor-bearing mice were injected intra-tumorally or i.p. with 20 mg CUR equivalent MNP-CURformulations in 100 mL PBS solution using a 30G needle.After 24 hours, animalswere euthanized and tumor, liver,spleen, and brain tissues were collected and stored at�80�C. Tissue homogenates were prepared by grindingat 8,000 rpm in PBS using a Power Gen 125 homogenizer(125W, 115V, 50/60 Hz; Fisher Scientific) and lyophilizedfor quantitative analysis of iron of MNP-CUR. Knownquantities of lyophilized tissue samples were dissolved inconcentrated hydrochloric acid and iron content ofMNPswas determined following 1,10-phenanthroline photo-metric method (24). The iron levels of control group mice(no MNP treatment) organs were subtracted to obtainabsolute iron levels in tissues after treatment withMNP-CUR formulation. All organs were saved for Prus-sian blue staining analysis.

Protein adsorptionFor this study, 1 mg of MNP-CUR formulation was

incubated in 100 mg of fibrinogen, immunoglobulin G(IgG), transferrin, or human serum albumin (HSA) solu-tions at 37�C. Within 2 hours of incubation, the plasmaproteins formed a corona layer on each MNP-CUR nano-particle. The adsorbed proteins on the MNP-CUR formu-lation were recovered by centrifugation at 12,000 rpm for15 minutes, redispersed, and run for SDS-PAGE gelto quantify. Protocol for running gel, staining of protein

with SimplyBlue SafeStain solution (Coomassie G-250stain; Life Technologies), anddensitometrymethodswerefollowed as previously reported (25). The variation innanoformulation structure was also examined usingtransmission electron microscope (TEM) before and afterHSA incubation (25).

HemocompatibilityTo examine hemocompatibility of the MNP-CUR for-

mulation, 12.25 mL human red blood cells (RBC) in 100 mLRPMI-1640 (healthy male Donor No. 53554, RegistrationNo. 2577632; Biological Specialty Corp.) was incubatedwith 100 mmol/L MNP-CUR at 37�C in Eppendorf tubes.After 2 hours of incubation, the RBCs were collected bycentrifugation and observed for ultrastructural morphol-ogies using TEM. The no-treatment group and dendrimerformulations were used as negative and positive controls,respectively.

Statistical analysesDescriptive data are presented as the mean � SEM.

ANOVA models and t tests were used to evaluate differ-ences in in vitro properties. Linear mixed regression anal-yseswere conducted to determine the tumor volumes as afunction of time. Transformations of continuous variableswere conducted to meet model assumptions. Time todeath (or euthanization) was used for Kaplan–Meieranalysis and equality of survival was determined usinglog-rank analysis. All analysis was conducted using SAS9.3 (SAS Institute, Inc.). P values of <0.05 were consideredsignificant. All graphs were plotted using Origin 6.1software.

ResultsOptimal size of MNP-CUR formulation for cancertherapeutics

In this study we have investigated utilization of "triplecrown MNPs" as a delivery vehicle for CUR (MNP-CUR;ref. 19). The optimized MNP-CUR was formulated with810:300 mg Fe3þ:Fe2þ salts, 200 mg cyclodextrin, 250 mgPluronicF-127 (19)with 10% (w/w)CUR loading (Fig. 1A).An average individual MNP-CUR particle grain size of10.5 � 0.54 nm, observed under JEOL 1210 TEM (JEOLLtd.; Fig. 1A), showed theultra small particle size nature ofthis formulation. However, this formulation showed 109nmparticle sizewith�0.99mV jpotential indynamic lightscattering analysis (19). These nanoparticle characteristicsare suitable for optimal cancer therapeutics purpose.

MNP-CUR effectively internalizes in pancreaticcancer cells

Qualitative analysis of Prussian blue staining in HPAF-II and Panc-1 cells showed a dose-dependant uptake ofMNP-CUR formulation (Fig. 1B). The uptake pattern wasdifferent in both cancer cells and this observation isconsistent with uptake of other magnetic drug nanofor-mulations in cancer cells (26, 27). However, the amount ofnanoparticle uptake in both cell lines was very close (i.e.,

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54.06% and 53.86%) when incubatedwith 100 mg ofMNP-CUR formulation (Supplementary Fig. S1A).

MNP-CUR inhibits proliferation and clonogenicpotential of pancreatic cancer cellsIn cell proliferation experiments, CUR and MNP-CUR

exhibited a dose-dependant inhibition of cell proliferation(Fig. 1C). At all 3 tested concentrations, MNP-CUR had avery similar cytotoxicity oncancer cells as compared to freeCUR treatment. This indicates the MNP-CUR formulationpreserved the inherent biological activity of CUR. In addi-tion, the effect of MNP-CUR was tested in the colonyformation assay using HPAF-II and Panc-1 pancreaticcancer cells. CUR and MNP-CUR formulation effectivelyinhibited the clonogenic potential of HPAF-II and Panc-1cancer cells compared to control-treated cells (Fig. 1D). Incell proliferation and colony formation assay, only �53%internalized MNP-CUR and �40% released CUR fromMNP-CUR(19) caused cytotoxicity equivalent to freeCUR.

MNP-CUR decreases tumor growth and improvessurvival of tumor-bearing miceIn vivo therapeutic efficacy of free CUR and the MNP-

CUR formulation was examined in the HPAF-II tumorxenograft model following intratumoral administration.After cancer cells postinjection, on day 28, MNP-CURtreatment significantly inhibited tumor growth by71.2% compared to emptyMNP-treated controls,whereasthe treatment with CUR inhibited tumor growth by 35.9%(Fig. 2A). Tumor size steadily increased in Tween 20 orMNP groups. A significant difference in tumor volumewas observed for MNP versus MNP-CUR (P ¼ 0.02), butnot for Tween 20 versus CUR (P ¼ 0.30). Higher tumorgrowth inhibition observed with MNP-CUR formulationmay be attributed to long-term sustained release of CURfrom MNP-CUR formulation within tumors. It was evi-dent from the uptake experiment using flow cytometerthat the MNP-CUR formulation can provide higher con-centrations of CUR within cells compared to free CUR(Supplementary Fig. S1B). The flow cytometer measure-ments are based on the inherent fluorescence signals fromtheCUR in solutionorCUR innanoparticles.Toprove thatMNP-CUR remains in tumors after intratumoral injectionand protects CUR’s biological activity, we examined theaccumulation of MNP-CUR formulation on the histologicsections of tumor tissues by Prussian blue staining. Dis-tinct Prussian blue stains were found throughout theregion of the tumor(s) [Fig. 2B(a–e)]. Most of the tissuesshowed a similar pattern of Prussian blue staining but afew tumor slides revealed only peripheral stains [Fig. 2B(f)]. The observed patterns of nanoparticle existence intumors are in accordance with a previous study (25). TheKaplan–Meier curves of mice treated with Tween 20 orempty MNPs exhibited a lower survival rate, but thiswas not statistically significant (Fig. 2C). Median sur-vival was 40 days for MNP and 24 days for Tween 20.Both the CUR and the MNP-CUR groups had survivalrates above 0.5 by the end of the study period.

MNP-CUR formulation effectively modulates keyoncogenic molecular targets

A markedly decreased (�70%) staining of Bcl-xL andMcl-1 was observed in both CUR- andMNP-CUR–treatedanimals compared to vehicle control groups (Fig. 3A).Interestingly, MNP-CUR–treated animals showed rela-tively less PCNA staining (�75%) compared to free CUR(�55%). In addition, we investigated the expression of adual-function protein, b-catenin (a modulator of cell–celladhesion and wnt signaling), in these tumor tissues.The deregulated expression or function of this proteinresults indecreasedcell–cell adhesiondue to lossof surfaceb-catenin and in enhanced cell proliferation due to thenuclear function of b-catenin as a cotranscription factor(16, 28). A distinct increase in membrane b-catenin was

Figure 2. MNP-CUR formulation efficiently inhibits tumor growth andincreases survival of HPAF-II tumor xenograft–bearing mice. A, single-time intratumoral dose of MNP-CUR (20 mg in PBS) more efficientlyinhibited growth of HPAF-II tumor xenografts as comparedwith freeCUR(20 mg in Tween 20) and controls (blank MNPs and Tween 20).Representative photographs of tumor-bearing mice are shown as inset.B, photomicrographs of MNP-CUR–treated tumors that were processedfor Prussian blue staining. C, Kaplan–Meier survival plot of mice treatedwith Tween 20, MNPs, CUR, and MNP-CUR. Data represents at least6 mice/group (mean � SEM). �, P < 0.05.






detected in MNP-CUR–treated tumors (�80%) comparedto free CUR–treated tumors (�60%) and control animals(Fig. 3A). MUC1 is a transmembrane O-glycosylated pro-tein that is overexpressed in pancreatic cancer (29) andmodulates various signaling pathways (including b-cate-nin), resulting in the overexpression of tumorigenic fac-tors and formation of tumors. A striking downregulation(�75–80%) of MUC1 protein expression was observedupon CUR or MNP-CUR treatment (Fig. 3A). Theseresults were also observed in vitro in HPAF-II cancer cellline models (Fig. 3B). To elucidate the role of CUR andMNP-CUR on tumor tissue fibrosis (desmoplasia), tumor

tissues were double stained with anti-human collagen-1(green) and anti-mouse collagen-1 (red). There was amarked reduction in both the human and mouse collagenlevels in the tumors of mice treated with MNP-CUR (Fig.4A), which implicated a decrease in host–tumor interac-tions. In vitro collagen production also significantlydropped to 24% to 25% upon treatment with MNP-CUR(Fig. 4B). CUR treatment did not efficiently regulate col-lagenproduction, that is, collagenproductionwas�71% to84%. All together, these data indicate an enhanced efficacyofMNP-CURtreatment at themolecular level compared tocontrol and free CUR.

MNP-CUR increases bioavailability and tumortargeting

Up to a 2.5-fold increase in bioavailability of CURin blood plasma was observed with the MNP-CUR for-mulation as compared to free CUR (Fig. 5A). A consider-able amount of CUR in MNP-CUR (1792.19 � 644 ng)was observed in 1 mL serum at 6 hours whereas only

Figure 3. Effects of CUR and MNP-CUR treatment on the expression ofcell proliferation, cell survival, and cell–cell adhesion molecules. A, theexpression of PCNA, Bcl-xL, Mcl-1, MUC1, and b-catenin in HPAF-IIpancreatic tumor xenografts after treatmentwith an intratumoral injectionof CUR/MNP-CUR (20 mg/mice). Representative images were obtainedusing an Olympus BX 41 Microscope. MNP-CUR treatment suppressedexpression of prosurvival proteins (PCNA, Bcl-xL, Mcl-1, andMUC1) andenhanced membrane b-catenin localization compared with controltreatment tumors. (original magnification 400�). B, cancer cells weretreated with CUR or MNP-CUR (10–30 mmol/L) for 36 hours and proteinlysates were collected and the expression of PCNA, Bcl-xL, Mcl-1, andMUC1 proteins was analyzed by immunoblotting. b-Actin was used asthe loading control. DMSO and blank MNPs served as controls for CURand MNP-CUR, respectively. The levels of each protein change arequantified using AlphaEase Fc software and relative data are normalizedwith respect to b-actin levels and included under each blot, consideringcontrol as 1.0.

Figure 4. Effect of CUR and MNP-CUR on collagen I. A, HPAF-II tumorxenografts were double stained with anti-human collagen I (green) andanti-mouse collagen I (red). MNP-CUR treatment showed a markeddecrease in both human and mouse collagen I levels in confocalmicroscopy analysis. B, HPAF-II cancer cells were treated with CUR orMNP-CUR (10–30 mmol/L) for 36 hours and protein lysates werecollected and the expression of collagen I (mature and precursor) wasanalyzed by immunoblotting. b-Actin was used as the loading control.DMSO and blank MNPs served as controls for CUR and MNP-CUR,respectively.

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766.54 � 256 ng of CUR in Tween 20 was present. Thisincreased serum bioavailability implies greater stabilityand prolonged sustained blood circulation of MNP-CURunder physiological conditions (30, 31), showing thatMNPspossess strong properties as effective drug carriers.To investigatekeydifferences inbiodistributionpatterns

of the MNP-CUR formulation injected intratumorally andi.p., the presence of MNPs was examined in tumor, liver,spleen, and brain during the 24 hours after administration

(Fig. 5B). Other organs, including pancreas, exhibited veryminimal or undetectable levels ofMNPs (data not shown).Intratumoral administration obviously resulted in highconcentrations of MNP-CUR in tumor (1.04 � 0.48 mgFe/g tissue), followedby liver (0.39� 0.17mgFe/g tissue),spleen (0.059 � 0.2 mg Fe/g tissue), and brain (0.14� 0.02mg Fe/g tissue). Interestingly, fairly high accumulation ofMNP-CUR in tumor (0.48� 0.29mg Fe/g tissue) aswell asin spleen (0.78 � 0.29 mg Fe/g tissue) was also achievedwith i.p. administration. This observation was furtherconfirmed by Prussian blue staining of tumor tissues. Thisdata also indicate thatMNP-CUR formulation is capable oftargeting pancreatic tumors fairlywell with the help of the"enhanced permeation and retention" (EPR) effect evenwith systemic (i.p.) administration (Fig. 5C).

MNP–CUR interaction with plasma proteins andRBCs

SDS-PAGE revealed the following order of quantity ofbound plasma proteins onMNP-CUR: Transferrin >HSA> fibrinogen > IgG (Supplementary Fig. S2). Relativeprotein binding quantification by densitometry showedthe protein affinity toward nanoparticles (Fig. 6A). Thisorder variation occurred due to differences in the

Figure 5. Serum bioavailability and biodistribution of MNP-CURformulation. A, comparative serum bioavailability of CUR andMNP-CURformulations in a tumor-bearingmousemodel. Enlarged 5, 10, 15, 30, 60,and 120minutes bioavailability data area shown as inset. CUR andMNP-CUR (50 mg per mice) were administered via intraperitoneal injection andblood samples from tail bleedwere collected at different timepoints. CURlevels in serum were determined using a Dionex HPLC instrumentequipped with an Acclaim Polar Advantage II C18 column (3 mm 120 Å4.6 � 150 mm) with the RS variable wavelength detector at 420 nm. B,comparative biodistribution in tumor, liver, spleen, and brain tissues afterintratumoral and intraperitoneal administration of MNP-CUR formulation.Mice were treated with MNP-CUR particles (20 mg per mice) and ironcontent of particles in tissue was estimated using 1,10-phenanthrolinecolorimetric method. C, visual evidence of nanoparticles in tumor tissuewas detected with Prussian blue staining method. Images were capturedusing an Olympus 1 � 71 microscope equipped with a DP71 camera(Olympus).

Figure 6. Hemocompatibility of MNP-CUR formulation. A, serum proteinsadsorption of MNP-CUR formulation. MNP-CUR formulation (1 mg) wasincubated with 100 mg plasma proteins (fibrinogen, IgG, transferrin, andHAS) for 2 hours, centrifuged, and bound proteins on NPs (palette)were estimated with the help of SDS-PAGE and Coomassie G-250staining (Supplementary Fig. S2). Relative adsorbed protein density wasquantified by densitometry using AlphaEase Fc software and dataare presented as the mean of 3 repeats for each protein (mean � SEM).�, P < 0.05 compared with fibrinogen and IgG. B, morphologicalobservation of red blood cells (RBCs) upon treatment with MNP-CURformulation. Control RBCs and RBCs treated with dendrimer formulationwere considered to be negative and positive controls for hemolysis,respectively. TEM images of RBCs reveal bound/entrapped MNP-CURnanoparticles (black circle in MNP-CUR) and secreted proteins andmembrane damage (black arrows in dendrimer-treated group).






formation of protein corona on nanoparticles that resultedfrom nanoparticle–protein complex interplay factors (32).A similar protein binding order had been noticed in CURassemblies (33). Human serum proteins have prevalentinteractions with nanoparticles but higher aggregation ofthose complexes lead to opsonization; however, this wasnot observed with the MNP-CUR formulation.

Furthermore, we compared the hemocompatibility ofMNP-CUR formulation with human RBCs (100 mmol/LCUR equivalent MNP-CUR formulation) to negative con-trol (no treatment) and positive control (dendrimer for-mulation) groups. RBCs treated with MNP-CUR retainedtheir morphology similar to negative control RBCs (Fig.6B). This behavior is achieved due to the pluronic layers(bound polyethylene glycol chains) in the formulation.However, some nonspecific binding of MNP-CUR withRBCswasalso observed (Fig. 6B, black circle),which is dueto entrapment ofnanoparticles in the cell pellet. In contrast,the dendrimer formulation caused secretion of hemoglo-bin and membrane proteins due to compromised RBCmembranes and hemolytic activity (Fig. 6B, black arrows).

DiscussionOur systematic approach for CUR encapsulation in

MNPs (MNP-CUR formulation) showed minimizeduptake in RAW 247.1 cells (macrophages) which can pre-vent rapid clearance while enhancing synchronized inter-nalization in cancer cells (19) including pancreatic cancercells (Fig. 1B). The MNP formulation is also able to encap-sulate other clinically relevant drugs such as doxorubicinand gemcitabine without significantly altering their struc-ture. Effective internalization ofMNPs in cancer cells is animportant characteristic for drug delivery. A layer of bio-adhesive material, Pluronic F127, on the MNPs greatlyimproved their cellular uptake and reducedparticle aggre-gation (34). Sustained release of CUR fromMNPs will notonly improve anticancer efficacy but also help preventrelapse and drug resistance (35). Herein, we report for thefirst time, the antitumor efficacy of an MNP-CUR formu-lation in pancreatic cancer treatment using in vitro (Fig. 1Cand D) and in vivo (Fig. 2A–C) models. Such equivalentbiological activities against various cancer cells wereobserved for drugs in solution and MNP drug formula-tions (26, 36). As a drug carrier, MNPs can effectivelyincrease the stability of drugs, protect them from degra-dation, promote targeting efficacy, and reduce side effects.In this study, tumorgrowth inhibitionwasobservedwith asingle dose administration of MNP-CUR formulation. Amore significant therapeutic effect can be expected from amultiple dose treatment schedule for a longer duration.MNP-CUR treatment not only suppressed tumor growth(Fig. 2A) but also increased the survival of animals in apancreatic cancer xenograft mouse model (Fig. 2C). Nosigns of behavioral abnormalities, undesirable side effects,or genotoxicity were observed during the course of treat-ment because MNPs are made with iron salts (Fe2þ andFe3þ).Humanpancreatic cancer is known tobe sensitive tomultiple chemotherapeutic agentsbutoftendevelopsdrug

resistance. Previous studies have shown injectable drug–gel formulations maintained high tumoral drug load con-centrations compared to free drug solutions (37). Ourstudy shows that in a pancreatic cancer xenograft mousemodel,humanpancreatic tumors can retain theMNP-CURformulation even after 2 to 3 weeks (Fig. 2B). Similarsustained release of CUR was found at the site of injectionof CUR microparticles in mice (38). This data suggest theutility of our unique delivery vehicle to effectively deliveranticancer drugs to pancreatic tumors. These formulationscan be used for intratumoral therapy through endoscopicultrasound procedures or percutaneously guided intratu-moral injections to shrink the tumors and prevent tumorrecurrence (39).

This study also suggests that the MNP-CUR formula-tion efficaciously decreased pancreatic tumor growth viaalterations in the expression profiles of cell survival asso-ciated proteins (Fig. 3A). Interestingly, the MNP-CUR–treated tumors exhibited higher levels of membraneb-catenin compared to control and free CUR treatments(Fig. 3A). b-Catenin is a well-known modulator of tumor-igenesis (40). The deregulated expression or function ofthis protein results in decreased cell–cell adhesion due toloss of surface b-catenin and enhanced cell proliferationdue to its increasednuclear transcriptional activity (22, 40).In fact, aberrant nuclear localization and expression ofb-catenin has been considered to be one of the primaryfactors in pancreatic tumorigenesis (41). In addition, re-searchers have confirmed that MUC1 overexpression,aberrant localization, and abnormal posttranslationalmodification (phosphorylation, hypo-glycosylation, andsulphation) are highly associated with pancreatic cancerprogression (29, 42). The intracellular, c-terminal portionof MUC1 plays a critical role in signal transduction byregulating various signaling pathways, including theb-catenin signal pathway. This in turn results in the tran-scription/overexpression of pro-growth factors. In addi-tion, MUC1 contains a large extracellular ectodomain thatprotrudes above the cell surface (200–2000 nm); hence,overexpression or aberrant baso-lateral localization ofMUC1mayalso reduce cell–cell adhesion, thereby enhanc-ing the tumorigenesis of pancreatic cancer (29, 43). CURandMNP-CUR treatments decrease the levels of MUC1 inthe tumor tissues and, therefore, possess a tumor-suppres-sing function by downregulating pro-survival proteins(Bcl-xL,Mcl-1, and PCNA; Fig. 3A andB).Moreover, uponMNP-CUR treatment, the enhanced membranous b-cate-ninmight enhance cell–cell adhesionwithin the tumor andprevent tumor metastasis. Therefore, a significant long-term implication of decreased MUC1 expression andincreased membranous b-catenin of the cells in the tumormay lower the risk of metastasis (a phenomenon that isprimarily responsible for cancer-related deaths).

Themajor extracellularmatrix component produced bymyofibroblasts, collagen I, not only functions as a scaffoldfor the tissue but also regulates the expression of genesassociated with cellular signaling, metabolism, gene tran-scription, and translation. Thus, production of collagen I

Yallapu et al.





affects fundamental cellular processes that are essentialfor tumor progression, such as cell survival, apoptosis,andcellular invasion (44, 45). Thisdata indicate thatMNP-CUR efficiently inhibited the collagen I levels of bothhuman and mouse origins within tumors (Fig. 4). Thissupports the finding that MNP-CUR induced a decreasein host–tumor interactions, thus may be involve in pre-venting pancreatic fibrosis.The prolonged circulation time and an EPR effect of

nanoformulations are general strategies used to targettumor(s). The MNP-CUR formulation showed a 2.5-foldincrease in CUR concentration in serum (Fig. 5A). Thepreferential tumor accumulation of MNP-CUR (Fig. 5BandC) is anticipateddue to leakyvasculature and lack of alymphatic system in tumors. It is possible to furtherimprove the accumulation of the MNP-CUR formulationby application of an external magnetic field gradient (46).In addition, in this study we observed an appropriatebinding of transferrin andHSAproteins to theMNP-CURnanoparticles to form a "hard corona" (Fig. 6A) whichdefines the long-lived equilibrium state of novel MNP-CUR nanoparticles (47). In general, enhanced adsorptionof plasma proteins onto the surface of nanoparticleswould lead to opsonization due to aggregation. However,we did not observe any aggregation property of MNP-CUR nanoparticles and hemotoxicity, indicating a long-term circulation of particles in the blood (Fig. 6A). Ourparticles gained this specific property due to the poly(propylene oxide) chain anchoring on the MNP-CURformulation that minimizes the shear forces when nano-particles are exposed to biological fluids. This observationwas further confirmed by hemocompatibility of our for-mulation by evaluating RBC’s ultrastructural morpholo-gy which suggested no signs of interaction of MNP-CURwith blood components and hemolysis (Fig. 6B).Active and specific targeting of drug delivery systems

has now become an interesting concept in cancer thera-peutic research. The advantageof thisMNP formulation isthat CUR or other anticancer drug(s) can be loaded intothe layers of cyclodextrin and F127 polymer. In addition,pancreatic cancer can be specifically targeted usinga novel anti-MUC13 monoclonal antibody. Most otherCUR nanoformulations either inefficiently load higheramounts of CUR or lack targeting chemistry. Our recentstudy has shown an overexpression of MUC13 in pan-creatic cancer (21). A MUC13-targeted nanoformulationmay lead to specific targeting of pancreatic cancer andpossibly enhance further tumor uptake, thereby improv-ing the efficacy of chemotherapeutic drugswhile preserv-ing imaging properties for simultaneous real-time mon-itoring of the disease condition.

ConclusionsOur data show that our MNP-CUR formulation can be

efficiently internalized in human pancreatic cancer cellsand induce potent anticancer effects. The in vivo tumorgrowth inhibition and improved survival rate show thatthe MNP-CUR formulation has superior anticancer activ-ity compared to free CUR. In addition, the MNP-CURformulation has exhibited an enhanced serum bioavail-ability of CUR along with appreciable tumor uptake andhemocompatibility. In conclusion, results presented inthis study suggest thatMNP-CUR is an excellent prospectfor effective pancreatic cancer treatment/managementbecause of enhanced bioavailability of CUR and its mul-tifocal therapeutic effects.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.M. Yallapu, S.C. ChauhanDevelopment of methodology: M.M. YallapuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.M. Yallapu, M.C. Ebeling, S. Khan, V. Sun-dram, N. Chauhan, M. JaggiAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M.M. Yallapu, M.C. Ebeling, V. Sundram,S.E. Puumala, M. Jaggi, S.C. ChauhanWriting, review, and/or revision of the manuscript: M.M. Yallapu, M.C.Ebeling, S. Khan, V. Sundram, S.E. Puumala, M. Jaggi, S.C. ChauhanStudy supervision: S.C. ChauhanOther: Performed mouse experiments with curcumin-loaded nanoparti-cles and sacrificed mouse and collected organs to study biodistributionof curcumin-loaded nanoparticles, B.K. Gupta.

AcknowledgmentsThe authors thank C. Christopherson (Center for Health Outcomes and

Prevention, Sanford Research/University of South Dakota) for criticallyreading themanuscript and for editorial assistance. The authors also thankA. Miller (Methodology Data Analyst, Sanford Research/University ofSouth Dakota) and N. Bauer (Cancer Biology Research Center, SanfordResearch/University of South Dakota) for statistical analysis and cell-counting experiments, respectively. The authors also acknowledge theImaging,Molecular Pathology, FlowCytometry and Tumor Biology Coresof Sanford Research which are supported by P20 GM103548-02 grantawarded to Dr. K. Miskimins.

Grant SupportThis study was supported by Governor’s Cancer 2010, Department of

Defense (PC073887), and the National Institutes of Health (RO1 CA142736and U01 CA162106) to S.C. Chauhan and M. Jaggi, and a pilot grantthrough P20 GM103548-02 to M.M. Yallapu.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 21, 2012; revised May 14, 2013; accepted May 16,2013; published OnlineFirst May 23, 2013.

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2013;12:1471-1480. Published OnlineFirst May 23, 2013.Mol Cancer Ther Murali M. Yallapu, Mara C. Ebeling, Sheema Khan, et al. Cancer TreatmentNovel Curcumin-Loaded Magnetic Nanoparticles for Pancreatic

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