Cytotoxicity and apoptosis enhancement in brain tumor cells upon coadministration of paclitaxel and...

PHARMACEUTICAL NANOTECHNOLOGY

Cytotoxicity and Apoptosis Enhancement inBrain Tumor Cells Upon Coadministration ofPaclitaxel and Ceramide in Nanoemulsion Formulations

ANKITA DESAI, TUSHAR VYAS, MANSOOR AMIJI

Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, 110 Mugar Life Sciences Building,Boston, Massachusetts 02115

Received 20 June 2007; revised 20 July 2007; accepted 2 August 2007

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21182

Corresponde3137; Fax: 617-

Journal of Pharm

� 2007 Wiley-Liss

ABSTRACT: The objective of this study was to examine augmentation of therapeuticactivity in human glioblastoma cells with combination of paclitaxel (PTX) and theapoptotic signaling molecule, C6-ceramide (CER), when administered in novel oil-in-water nanoemulsions. The nanoemulsions were formulated with pine-nut oil, which hashigh concentrations of essential polyunsaturated fatty acid (PUFA). Drug-containingnanoemulsions were characterized for particle size, surface charge, and the particlemorphology was examined with transmission electron microscopy (TEM). Epi-fluores-cent microscopy was used to analyze nanoemulsion-encapsulated rhodamine-labeledPTX and NBD-labeled CER uptake and distribution in U-118 human glioblastoma cells.Cell viability was assessed with the MTS (formazan) assay, while apoptotic activity ofPTX and CER was evaluated with caspase-3/7 activation and flow cytometry. Nano-emulsion formulations with the oil droplet size of approximately 200 nm in diameterwere prepared with PTX, CER, and combination of the two agents. When administeredto U-118 cells, significant enhancement in cytotoxicity was observed with combination ofPTX and CER as compared to administration of individual agents. The increase incytotoxicity correlated with enhancement in apoptotic activity in cells treated withcombination of PTX and CER. The results of these studies show that oil-in-waternanoemulsions can be designed with combination therapy for enhancement of cytotoxiceffect in brain tumor cells. In addition, PTX and CER can be used together to augmenttherapeutic activity, especially in aggressive tumor models such as glioblastoma. � 2007

Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2745–2756, 2008

Keywords: nanoemulsions; paclitaxel;
C6-ceramide; U-118 human glioblastomacells; combination therapy
INTRODUCTION

Gliomas are the most common type primarytumors of the brain, with an incidence of about

nce to: Mansoor Amiji (Telephone: 671-373-373-8886; E-mail: [email protected])

aceutical Sciences, Vol. 97, 2745–2756 (2008)

, Inc. and the American Pharmacists Association

JOURNAL O

25000 new cases per year in the United States.1,2

At least half of all gliomas exhibit very aggressiveand malignant behavior. Glioblastoma multi-forme (GBM), in particular, is clinically andpathologically malignant. Patients with GBMhave a very poor prognosis; with a mediansurvival of one year or less even with aggressivetherapy and fewer than 5% will survive five years.The available treatment modalities for GBM

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2746 DESAI, VYAS, AND AMIJI

include chemotherapy, conformal radiotherapy,stereotactic radiosurgery, interventional mag-netic resonance surgery, image-guided surgery,and interstitial brachytherapy.1,3–5 For systemicdelivery of chemotherapeutic agents to the brain,the major limitation is efficient permeabilityacross the blood-brain barrier.6,7 The tight junc-tions between densely packed endothelial cellsresults in a very high trans-endothelial electricresistance of 1500–2000 V cm2 in the braincapillaries as compared to 3–33 V cm2 inperipheral tissues.8 In addition to poor transport,brain capillary endothelial cells also express theactive drug efflux transporter pumps, such as P-glycoprotein (P-gp), and metabolizing enzymes,including cytochrome P-450.9

Paclitaxel is a potent anti-tumor chemother-apeutic, originally derived from the bark of thePacific yew tree (Taxus brevifolia),10 that is widelyused in the treatment of solid tumors, particularlyof the breast and ovaries.11 PTX exerts itscytotoxicity by inducing tubulin polymerizationresulting in unstable microtubules, which inter-feres with mitotic spindle function and ultimatelyarrests cells in the G2 mitosis phase of celldivision.11 Tumor cells exposed to PTX treatment,as a result of spindle stabilization, undergoprogrammed cell death 12. PTX has been shownto have high therapeutic activity in GBM, if thedrug can be administered efficiently, such as withconvection-enhanced delivery.13,14 Recent studiesshowed that coadministration of the P-gp inhi-bitor (valspodar) was found to enhance the brainuptake of PTX, a P-gp substrate, and improveanti-tumor therapeutic effect.15

The short chain sphingolipids, such as cera-mide, which are second messengers in the cell,have been found to play a crucial role as mediatorsof apoptotic signaling.16–18 Either enhancement ofde novo production or exogenously administeredceramide has been shown to mediate apoptoticsignaling cascade via inhibition of pro-survivalpathways, mitochondrial dysfunction, and stimu-lation of caspase activity, which subsequentlyresults in DNA fragmentation and cell death.19,20

Moreover, chemotherapeutic agents like PTX canact as multiple cellular stressors, which can leadto de novo ceramide accumulation within the cells,and ultimately synergistically act to enhanceapoptosis and anti-cancer activity with exogen-ously administered ceramide.21–23 In a recentstudy, we have shown that short chain C6-ceramide (CER) coadministration with PTX cansignificantly augment therapeutic efficacy in

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SKOV-3 human ovarian adenocarcinoma cellsand tumor models.24,25 More importantly, CERcoadministration significantly enhanced the apop-totic activity and therapeutic effectiveness inmultidrug resistant tumor model.24 Therefore,in this study, we hypothesized that exogenouscoadministration of CER along with PTX, andtheir cellular internalization will be beneficialfrom the perspective of enhancing the rate andextent of apoptosis and improved therapeuticeffectiveness in the treatment of GBM. The majorshortcoming with PTX and CER chemotherapy isrestricted aqueous solubility, and hence limitedcellular permeation, which subsequently leads toinadequate cellular internalization. In addition,CER is prone to enzymatic degradation in thesystemic circulation and also does not penetratewell through the cell membranes.26,27 Thus, thereis a need for improved systemic delivery, whichcan solubilize and encapsulate PTX and CER,facilitate transport across the blood-brain barrier,provide cellular internalization, and protect thedrugs against enzymatic degradation for max-imum therapeutic effect in GBM.

Nanoemulsions are heterogeneous liquid dis-persions of oil and water, where the internal phaseis an oil droplet in the nanometer length scale,typically in the range of less than 200 nm.28–30

Nanoemulsions are versatile delivery systembased on selection of different edible oils, theratio of oil and water phase, the choice of surface-active agents (surfactants) used for stabilization,and surface immobilization of target-specificligands for disease-specific localization.30 In aprevious study,31 we have observed that when thenanoemulsions were made with oils rich inessential polyunsaturated fatty acids (PUFAs),PTX was efficiently solubilized in the oil dropletand there was a significant enhancement in thedrug absorption across the gastro-intestinal tractfollowing oral administration. In addition, thenanoemulsions made with pine-nut oil, which isrich in pinolenic acid and linoleic acid,32 andstabilized with Lipoid-801 were found to enhancethe delivery of PTX across the blood-brain barrierin mice (unpublished results). Pinolenic andlinoleic acids are examples of omega-6 fatty acidsthat are necessary for biological functions, but aremainly imported in the body through individual’sdiet. Selective brain uptake of essential PUFAhas been established by several studies. Forinstance, Edmond33 has shown that linoleic acidwith 18-carbon monocarboxylic acids with two cis-double bonds was imported in the brain, while

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PACLITAXEL AND CERAMIDE COADMINISTRATION FOR BRAIN TUMOR 2747

oleic acid containing one cis-double bond was not.These results suggest exquisite selectivity in thetransport of essential PUFA across the blood-brain barrier. In addition, nonessential fattyacids, including palmitic and stearic acids, werenot found in the brain. The beneficial role of PUFAin cancer has been a subject of intense study.34–36

In most experimental and clinical studies, dietarysupplements of omega-3 and omega-6 fatty acidcontaining oils have shown to reduce the tumorburden significantly.34,35

Although there have been a few reportedstudies that have examined augmentation ofanti-tumor effects of PTX with cell-permeableCER in different tumor cells,23,37 none to ourknowledge have examined intracellular deliveryusing nanoemulsions. In this study, we describethe development, characterization, cell kill effi-ciency, and apoptotic activity of PTX and CERwhen administered alone or in combination innanoemulsion formulations to U-118 humanglioblastoma cells.

MATERIALS AND METHODS

Materials

Extra-virgin pine-nut oil, containing high con-centrations of pinolenic acid (25% by weight) andlinoleic acid (40% by weight), was purchased fromSiberian Tiger Naturals, Inc. (Cabot, VT). Eggphosphotidylcholine (Lipoid1 E80) and Lipoid1 PE18:0/18:0/poly(ethylene glycol) 2000 (PEG-2kD-modified phospholipid) were obtained from LipoidGMBH (Ludwigshfaen, Germany). Marketed for-mulation of paclitaxel for injection (Onxol1),containing 6 mg/mLPTX in Cremophore1 EL/ethanol (50:50), was purchased from Ivax Phar-maceuticals (Miami, FL). Synthetic C6-ceramide(N-hexanoyl-D-erythro-sphingosine) was pur-chased from Avanti Polar Lipids (Alabaster,AL). Rhodamine-conjugated PTX was purchasedfrom Natural Pharmaceuticals, Inc. (Beverley,MA) and N-a-6-aminocaproyl-D-erythro-sphingo-sine (C6-NBD-ceramide), propidium iodide (PI),and YO-PRO1-1 were all purchased from Mole-cular Probes (Eugene, OR). CellTiter 961 AQueous

cell proliferation assay based on tetrazolium dye[i.e., 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho-xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] (MTS)and Apo-ONE1 Homogeneous Caspase-3/7 Assaywere purchased from Promega Corporation

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(Madison, WI). Human glioblastoma (U-118) cellswere obtained from American Type CultureCollection (Rockville, MD). Dulbecco’s modifica-tion of Eagle’s medium (DMEM) and trypsinEDTA were purchased from Mediatech, Inc.(Herdon, VA). All other chemicals and solventswere of analytical reagent grade and were usedwithout further purification. Deionized distilledwater (Barnsted/Thermolyne, Dubuque, IA) wasused exclusively in preparation of all aqueoussolutions.

Preparation of Drug-Containing Nanoemulsions

Oil-in-water nanoemulsions were formulated usingan ultrasound technique as previously described.31

Briefly, the aqueous phase was prepared usingdeionized water (2 mL) and egg phosphotidylcho-line (Lipoid1 E80, 40 mg). Subsequently, Lipoid1

PE (20 mg) was added and stirred for anadditional 15 min at room temperature to obtaina homogenous dispersion. The mixture was thenstirred at 5000 rpm for 10 min using a FisherPowerGen1 125 homogenizer. The oil phase,consisting of pine-nut oil (0.5 mL), was takenseparately in a glass vial. Following this, theaqueous phase and the oil phase were heated to70–758C for 5–7 min and mixed rapidly using amagnetic stirrer. The crude emulsion mixture wasthen sonicated at 21% amplitude and 50% dutycycle using Vibra-Cell1 VC 505 ultrasoundinstrument (Sonics and Materials, Inc., Newtown,CT) for 10 min to reduce the oil droplet size.The formed nanoemulsions were stored in arefrigerator at approximately 48C until furtheruse.

For incorporation of PTX and CER into thenanoemulsions, stock solutions of both activepharmaceutical ingredients were made in dehy-drated ethanol. PTX stock solution was made fromthe paclitaxel for injection (Onxol1) formulationand CER powder was appropriately dissolved inethanol and the ethanolic solution was added tothe oil phase of the nanoemulsion. Ethanol inthe oil phase was allowed to completely evaporateunder high vacuum prior to the addition of theaqueous phase. The final concentrations of PTXand CER as single agents in the nanoemulsionswere 0.4% (w/v). For combination therapy, the twodrug formulations were mixed to achieve appro-priate dose of each before addition to the cells inculture.

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Characterization of the Nanoemulsion Formulations

Particle Size Analysis

For each batch of control and drug-loaded nano-emulsions, they were characterized for particlesize and size distribution by dynamic light scat-tering method using Brookhaven Instrument’s(Holtsville, NY) ZetaPALS1 90Plus instrument.Nanoemulsion sample (20 mL) was diluted to2.5 mL in deionized distilled water and theparticle size was determined at 908 scatteringangle and 258C temperature. At the time ofparticle size determination, average count ratewas maintained between 50–500 kcps to achievereproducibility. The mean effective hydrodynamicdiameter of the oil droplets on log-normal sizedistribution mode was considered for comparativeevaluation.

Surface Charge Measurements

The surface charge (zeta potential) measurementswere carried out by the diluting control and drug-loaded nanoemulsion formulations using deio-nized distilled water in disposable cuvets. Therefractive index was kept at 1.33 and the viscosityat 1 cps to mimic the conditions in water. Smallvolume of the nanoemulsion (20 mL) was dilutedto 2.5 mL with deionized distilled water, andapproximately 1.5 mL of the diluted nanoemul-sion, placed in a cuvet, and was connected to anelectrode at 4 V and 2 Hz field frequency forsurface charge determination using ZetaPALS1

instrument. The electrophoretic mobility of thenanodroplets was measured and converted to zetapotential using a built-in software, which uses theSmoluchowski equation to calculate the values.38

Transmission Electron Microscopy (TEM)

To observe the morphology of the oil droplets inthe nanoemulsions, each batch was also char-acterized by TEM using a negative stainingtechnique. Approximately, 50 mL of the controland drug-loaded nanoemulsion formulations wereadded to the 200 mesh Formwar-coated coppergrids (Electron Microscopy Science, Hatfield, PA).The samples were then negatively stained with50 mL of 10% (w/v) phosphotungstic acid andallowing the staining to proceed for 10 minat room temperature. Excess liquid was drain-ed off with a Whatman filter paper, which wasplaced on the edge of the copper grid. Copper gridcontaining the nanoemulsion sample as a dry


film was placed on a TEM sample holder andobserved with a JEOL 100-X transmission elec-tron microscope (Peabody, MA) equipped with20 mm aperture and at an accelerating voltageof 80 kV.

Uptake and Distribution of the Nanoemulsionsin U-118 Glioblastoma Cells

Preparation Rhodamine-PTX andNBD-CER Nanoemulsions

PTX and CER were replaced by rhodamine-labeled paclitaxel (rhodamine-PTX) and NBD-C6-ceramide (NBD-CER), respectively, and addedto the oil phase. The fluorescently labelednanoemulsions were formulated using the sameprocedure as described above.

Cell Culture Conditions

Human glioblastoma cells (U-118) purchased fromAmerican Type Culture Collection and werecultured in modified Dulbecco’s modified Eagle’smedium (DMEM) at 378C in 5.0% CO2 atmo-sphere. When the cells reached approximately80% confluency in culture flasks, they weredetached with trypsin-EDTA and reseeded in6-well microplates with a clean glass coverslip at adensity of 10000 cells per well.

Evaluation of Cellular Uptake and Distribution

Nanoemulsion containing rhodamine-PTX andNBD-CER was diluted with DMEM and an aliquotwas added to the U-118 cells. At specific timeintervals ranging from 30 min to 6 h, the mediumwas removed and the cells were washed with freshDMEM. After the final wash, the coverslips weremounted on clean glass slides with Fluromount-G1

mounting medium (Southern Biotechnology Asso-ciates, Birmingham, AL). Differential interfer-ence contrast (DIC) and epi-fluorescent imageswere acquired at 40� magnifications using NikonTE-2000 U (Melville, NY) scanning fluorescenceconfocal microscope and the digital images wereprocessed using the Adobe Photoshop1 software.

Cell Viability Studies

The cytotoxicity studies were performed with bothaqueous solution and the nanoemulsion formula-tions containing different concentration of PTX,CER and combination of PTX and CER. For the

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cytotoxicity study, the MTS cell viability reagentwas prepared by dissolving 3-(4,5-dimethylthia-zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS reagent) andphenazine ethosulfate in Dulbecco’s sterile phos-phate-buffered saline (pH 6.0) and was stored at�208C for up to 7 days. U-118 cells were allowed toadhere on the surface of 96-well microplates at adensity of 5000 cells per well. After approximately18 h to allow the cells to adhere, PTX, CER, andcombination of PXT and CER was added to thecells in culture either in aqueous solution or innanoemulsion formulations prepared by dilutingappropriately with DMEM. Following 1 h ofincubation with the drug, the cells were washedwith DMEM to wash away any drug that did notenter the cells. The PTX, CER, and combination ofPTX and CER treated cells were kept in DMEMfor 6 days at 378C. The cells were incubated for6 days with the control and nanoemulsionsformulations in order to insure that maximumcell-kill effectiveness is observed, especially forPTX, which is a cell cycle specific drug. Cellstreated with DMEM alone (without any drugs)were used as negative control and those treatedwith 250 mg/mL of polyethyleneimine (Mol. wt.10 kDa), a cytotoxic cationic polymer, were used aspositive control.

Following a 6-day incubation period, the mediafrom each well was aspirated and replaced withdiluted MTS reagent in supplemented DMEM(1:10, v/v). After addition of the MTS reagent, the96-well microplates were again incubated for 4 hat 378C. The absorbance of formazan producedby viable cells was measured using a BioTek-HTUV–Vis/fluorescence microplate reader at 490 nm.The percent cell viability was calculated based onthe absorbance of the drug-treated cells over theabsorbance of control (DMEM alone) cells andmultiplied by one hundred. In all cases, controlexperiments were conducted simultaneously withaqueous solution or nanoemulsions that did nothave any drugs.

Cellular Apoptosis Analysis

In order to examine the mechanism of enhancedcell kill with the addition of CER, the percent ofcontrol and treated cells undergoing apoptosisand necrosis was measured by detection of theactivities of caspases 3 and -7 a using homogenousCaspase-3/7 assay and flow cytometric assayusing both YO-PRO1-1 and PI dyes. Like the

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cytotoxicity studies, cellular apoptotic necrosisanalysis was performed using both the aqueoussolution and the nanoemulsion formulations.Concentration of PTX, CER and combination ofPTX and CER were chosen from the results ofcytotoxicity study and only those concentrationswere included in apoptotic analysis which gavehighest cell death in cell viability studies.

Caspase-3/7 Assay

U-118 cells were allowed to adhere on the surfaceof 96-well microplates at a density of 5000 cells perwell. After approximately 18 h to allow the cells toadhere, PTX, CER, and combination of PTX andCER was added to the cells culture either inaqueous solution (prepared with DMEM medium)or in nanoemulsion formulations. Following 1 h ofincubation with the drug, the cells were washedwith DMEM to wash away any drug that did notenter the cells. The treated cells were incubatedfor 48 h. At the end of incubation period, caspasesubstrate and Apo-ONE1 Caspase-3/7 buffer werethawed and mixed to make the Apo-ONE1

Caspase-3/7 reagent. This prepared Apo-ONE1

Caspase-3/7 reagent was added to each well bymaintaining 1:1 volume ratio of reagent volumeand treated cell culture volume (100 mL reagentþ100 mL DMEM medium). The blank control wasused as a measure of background fluorescenceassociated with the culture system and Apo-ONE1 Caspase-3/7 reagent and was subtractedfrom the experimental values.

The contents of wells were mixed using a plateshaker at 300–500 rpm from 30 s up to read time.The 96-well plate was incubated at room tem-perature for 3 h and the florescence of each wellwas measured at an excitation wavelength of490 nm and emission wavelength of 520 nm usingBioTek-HT UV–Vis/fluorescence microplate reader.Caspase-3/7 activity was reported as percent acti-vation relative to untreated control.

Flow Cytometric Assay

For these studies, the U-118 cells were grown insupplemented DMEM in T25 cell-culture flaskwith at a seeding density of 1� 106 cells per flask.Similar to cell viability studies described above,the cells were treated with aqueous solutions andnanoemulsion formulations containing PTX at adose of 100 nM, CER at a dose of 10 mM, andcombination of PTX and CER. Following 48 h afterdrug treatment, the cells were harvested with



trypsin EDTA and were washed in ice-coldphosphate-buffered saline. After centrifugation,the cell density was adjusted to 1� 106 cells/mL.Cells that did not receive any drug treatmentserved as a negative control. YO-PRO1-1 stocksolution (1 mL was added to each 1 mL of cellsuspension. In addition, PI stock solution wasadded in the same concentration as YO-PRO1-1 toindicate necrotic cells. After the incubation periodfor 30 min on ice, stained cells were analyzed byflow cytometry using the BD Biosceinces FACS-caliber1 (San Jose, CA) instrument equipped withan argon 488 laser. The FL1 channel (530/30emission) was used to detect cells containing YO-PRO1-1 and the FL2 channel (585/42 emission)was used to detect cells with PI. The flowcytometry results were analyzed using CellQuestPro1 software.

Data Analysis

All of the results are reported as mean� standarddeviation and the differences between the controland test groups were tested using student’s t-test.Results were considered statistically significantbetween the control and test treatment at the levelof p< 0.05.

RESULTS AND DISCUSSION

Nanoemulsions are heterogeneous liquid disper-sions made with either oil-in-water or water-in-oil, wherein the droplet size of the internal phaseis reduced to nanometer scale (�200 nm) byapplying high shear stress using ultrasoundor microfluidizing instruments.30 Amphiphillicmolecules or surfactants are incorporated to allowfor stabilization of the nanoemulsions due toadsorption of these agents at the oil–water inter-face and lowering of the interfacial tension. Since

Table 1. Properties of the Control and Drug-Containing N

Abbreviation Nanoemulsion Formulation

NE-STD Blank nanoemulsionNE-PTX Paclitaxel containing nanoemulsionNE-CER C6-ceramide containing nanoemulsionNE-PTXþCER Paclitaxel and C6-ceramide containing

nanoemulsion

aMean�SD (n¼ 5).


the nanoemulsions can be made by using differenttypes of edible oils, different ratios of oil and waterphases, and wide selection of surfactants andcharge-inducing agents, they offer tremendousversatility in medical and pharmaceutical appli-cations as a multifunctional nanocarrier systemfor delivery of drugs and imaging agents.29 Whenhydrophobic drug molecules are incorporated intothe nanoemulsions, the pharmacokinetic anddistributive pattern of the agents upon systemicadministration will be dictated by the propertiesof the nanoemulsion rather than the physico-chemical properties of the drug molecules. Basedon this fact, variety of different anticancertherapies can be incorporated into the nanoemul-sion systems for target-specific systemic deliveryto tumor site. In addition, based on the judiciousselection of the oils used in the preparation of thenanoemulsions, there is an opportunity to engi-neer the formulation such that it can facilitatetransport across formidable biological barriersincluding the blood-brain barrier.

In this study, we have developed oil-in-waternanoemulsions using pine-nut oil, which has ahigh concentration of pinolenic and linoleic acids,an omega-6 PUFA. The nanoemulsion formula-tion was used to encapsulate PTX and CER forcombination therapy against GBM. As shown inTable 1, the average particle diameter of thecontrol nanoemulsion (without any drugs) was200 nm. When PTX, CER, and combination of PTXand CER were included in the nanoemulsions, theaverage hydrodynamic particle diameter remain-ed at approximately 200 nm. The surface chargeon the nanoemulsion droplets is representative ofthe ionization of the components forming theinterfacial layer. In this formulation, the inter-facial layer is formed by adsorption of eggphosphatidylcholine (Lipoid1-E80) and PEG-modified phospholipids (Lipoid1-PE). The zetapotential values of PTX, CER, and combination ofPTX and CER in the nanoemulsions systems were

anoemulsion Formulations

Hydrodynamic Diameterof the Oil Droplets (nm)

Surface Charge of theOil Droplets (mV)

199.5� 1.6a �38.2� 4.1209.0� 1.5 �46.6� 1.3194.5� 1.6 �30.9� 3.9212.5� 1.6 �31.3� 2.5

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Figure 1. (A) Schematic illustration of paclitaxel (PTX)- and C6-ceramide (CER)-containing oil-in-water nanoemulsions and (B) the transmission electron micrograph(TEM) of the blank nanoemulsion formulation. In the TEM image, the scale barrepresents a distance of 100 nm.


similar to the control nanoemulsion suggestingthat the drugs were incorporated in the oildroplets and did not adsorb at the interfaciallayer to affect the charge. In addition, TEManalysis of the blank nanoemulsions, shown inFigure 1, was exactly the same in terms of particlemorphology as the ones that contained PTX andCER. There were no identifiable solid crystals in

Figure 2. Epi-fluorescent microscopy imagshowing uptake of paclitaxel- and ceramide-cointerference contrast image of the U-118 cellsnanoemulsion uptake imaged with red emiss(CER) nanoemulsion uptake imaged with grof B and C images showing colocalization onanoemulsions.

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any of the TEM images to suggest that PTX andCER were precipitating in the aqueous phase ofthe nanoemulsions. TEM image in Figure 1 alsoconfirms that the oil droplets were spherical anduniformly distributed. It is important to note thatthe oil droplet particle size in the TEM image wasmuch smaller than the hydrodynamic diametermeasured by the light scattering instrument.

es of human U-118 glioblastoma cellsntaining nanoemulsions. (A) Differential, (B) rhodamine-labeled paclitaxel (PTX)ion filter, (C) NBD-labeled C6-ceramideeen emission filter, and (D) an overlayf PTX and CER in the cells with the


Figure 3. The percentage viable cells as a function ofpaclitaxel (PTX), ceramide (CER), and combinationPTX and CER when administered in aqueous solutionor in nanoemulsion formulations to U-118 human glio-blastoma cells. (A) Percent viable cells following treat-ment with varying PTX concentrations, (B) percentviable cells following treatment with varying CER con-centrations, and (C) percent viable cells following treat-ment with combination of PTX and CER at differentconcentrations. The cytotoxicity was measured by theMTS (formazan) cell viability assay after 6 days ofincubation at 378C. ‘‘Control’’ represents treatmentwith either aqueous solution or nanoemulsion thatdid not have any therapeutic agent. The results repre-sent mean�SD (n¼ 4).



In order to examine where PTX and CERcontaining nanoemulsions were internalized inU-118 glioblastoma cells, rhodamine-labeled PTXand NBD-labeled CER were incorporated and epi-fluorescence confocal microscopy studies were per-formed. As shown in Figure 2, the nanoemulsionsdo efficiently deliver the payload in the gliomacells. Rhodamine-PTX and NBD-CER fluores-cence emission could be overlayed (Fig. 2D) toshow that both of these drugs were indeedsimultaneously delivered to the cell and remainedencapsulated in the oil droplets of the nanoemul-sions during this period of time.

The cell-kill efficiency of PTX, CER, andcombination of PTX and CER in aqueous solutionand in the nanoemulsion formulations wasexamined in U-118 glioblastoma cells using theMTS assay. Cell viability with 250 mg/mL ofpolyethyleneimine (Mol. wt. 10 kDa), a cytotoxiccationic polymer, when used as a positive control,was always less than 20%. Based on our studies ofCER coadministration in SKOV-3 human ovariancancer cells,25 the final concentrations of PTXselected for these studies were 10 and 100 nM andthe final concentrations of CER of 1–10 mM.Combination of PTX and CER was examined at10 nm PTXþ 1 mM CER, 100 nm PTXþ 1 mMCER, 10 nM PTX and 10 mM CER, and 100 nMPTX and 10 mM CER. The results, shown inFigure 3, express the percent viable cells remain-ing as a function of treatment following 6 days ofdrug exposure at 378C. When PTX was adminis-

Figure 4. Evaluation of caspase-3/7 activity as afunction of treatment with paclitaxel (PTX), ceramide(CER), combination PTX and CER when administeredin aqueous solution or in nanoemulsion formulations toU-118 human glioblastoma cells. The caspase-3/7 activ-ity was measured after 48 h of incubation with thedifferent treatment options using the Apo-ONE1 Cas-pase-3/7 kit. ‘‘Control’’ represents treatment with eitheraqueous solution or nanoemulsion that did not have anytherapeutic agent. The results represent mean�SD(n¼ 4).

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Figure 5. Flow cytometric analysis of apoptotic activity in U-118 human glioblastomacells treated with paclitaxel (PTX), ceramide (CER), combination PTX and CER whenadministered in aqueous solution or in nanoemulsion formulations. The scatter plots aswell as the percent of cells undergoing apoptosis are shown. Flow cytometry analysis wasperformed using YO-PRO1 and propidium iodide fluorescent dyes following 48 h oftreatment with the drugs. ‘‘Control’’ represents treatment with either aqueous solutionor nanoemulsion that did not have any therapeutic agent. The results representmean�SD (n¼ 4).


tered at 10 and 100 nM concentrations, highercytotoxicity was observed with the nanoemulsionas compared to the aqueous solution formulation.For instance, at 10 nM PTX dose, there were onaverage 63% viable cells with the nanoemulsion ascompared to over 82% viable cells with the samedose administered in aqueous solution. In addi-tion, CER in solution was also not effective ininducing cytotoxicity in U-118 cells. This may bedue to the fact that CER is very hydrophobic andsusceptible to enzymatic degradation. When CERwas administered at 10 mM dose in the nanoemul-sion formulation, the cell viability decreased to

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69%. Combination of PTX and CER administeredin the nanoemulsion was also significantly moreeffective than the aqueous solution. At 10 nM and10 mM doses of PTXþCER combination, the cellviability decreased to 85% with the aqueoussolution and over 53% with the nanoemulsionformulation. Additionally, when the PTX dose wasincreased to 100 nM and CER remained at 10 mM,the cell viability decrease was even more profoundat 50% for aqueous solution and 33% for thenanoemulsion formulation.

To further confirm the role of CER in inducingapoptosis in caspase-3/7 activation studies were



performed using the Apo-ONE1 Caspase-3/7 kit.The results of caspase activation, as shown inFigure 4, show that PTX and CER coadministra-tion was significantly more effective in enhancingthe caspase-induced apoptotic activity. For thesestudies, PTX and CER were administered indivi-dually at 100 nM and 10 mM doses, respectively,and the combination of PTX and CER had thesame doses. There was baseline caspase activationof approximately 30–40% in the absence of anytreatment. At 100 nM PTX administration,caspase-3/7 activation was similar at around66% when administered in aqueous solution orin nanoemulsion. On the other hand, when CERwas administered 10 mM dose, significantlygreater caspase activity was observed with thenanoemulsion (90%) as compared to the aqueoussolution (52%). These results collaborate with thecell viability data to confirm that CER cannot beeffective when administered in aqueous solutionformulation. When PTX and CER were adminis-tered in combination, 74% caspase-3/7 activitywas observed with the aqueous solution and 100%with the nanoemulsion formulation.

Further studies of apoptotic activity of U-118cells upon treatment with PTX and CER, either assingle agent or in combination, was carried outusing YO-PRO1 and PI dyes. Figure 5 shows cellsexposed to the PTX and CER treatment, either assingle agents or in combination. When PTX andCER were added in combination, there wassignificantly higher apoptotic activity in U-118glioblastoma cells as displayed by an increase inthe amount of apoptotic staining. The greenfluorescent dye YO-PRO-11 stains apoptotic cellsand the red fluorescent PI stains necrotic cells(and dead cells), based on their ability to permeatethe cell membrane. Given the recent observationsof apoptotic cell death mechanisms that portraya necrosis-like morphology, in which CER isthought to play an important role,39,40 the sumof green (apoptotic) and red (necrotic) fluorescencewas counted towards total apoptotic activity, butwith cytometry gating set to quantify only wholecells (thereby excluding dead cells and cellfragments). When the cells were exposed to blanknanoemulsion, there was a slight increase inapoptotic activity (13%). When PTX and CER wereadded at 100 nM and 10 mM dose, respectively, assingle agents, the percent of cell undergoingapoptosis values were 12% and 10% for aqueoussolution and 18% and 16% for the nanoemulsionformulations. Combination of PTX and CER led tosubstantial increase of 22% in cellular apoptosis


with aqueous solution and more than 32% withthe nanoemulsion formulation.

CONCLUSIONS

Nanoemulsions containing PTX, CER and combi-nation of PTX and CER successfully preparedusing an ultra-sonication technique. Particle sizeand zeta potential data demonstrated the forma-tion of physically stable nanoemulsions. Whenadministered in the nanoemulsion formulations,PTX and CER were delivered inside the U-118glioblastoma cells and resulted in significantenhancement in cytotoxicity. The enhanced cyto-toxicity was attributed to an increase in apoptoticactivity following treatment with combinationPTX and CER therapy. The results of thesestudies clearly demonstrate a significant thera-peutic benefit of coadministration of PTX and CERin nanoemulsions for the treatment of GBM.

ACKNOWLEDGMENTS

This study was supported by the National CancerInstitute of the National Institutes of Healththrough the Nanotechnology Platform Partner-ship grant (R01-CA119617). The authors aregrateful to Professor Vladimir Torchilin of North-eastern University (Boston, MA) and his labassociates for providing technical assistance withthe use of flow cytometry in these studies. Inaddition, Dr. Gary Laevsky of the Keck Micro-scope Facility and Mr. William Fowle of the Elec-tron Microscopy Facility, both at NortheasternUniversity (Boston, MA) are acknowledged fortheir assistance with the epi-fluorescence andtransmission electron microscopy, respectively.

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