Composite fiber structures with antiproliferative agents exhibit advantageous drug delivery and cell growth inhibition in vitro

PHARMACEUTICS, PREFORMULATION ANDDRUG DELIVERY

Composite Fiber Structures With Antiproliferative AgentsExhibit Advantageous Drug Delivery and Cell GrowthInhibition In Vitro

AMIR KRAITZER,1 YOEL KLOOG,2 RONI HAKLAI,2 MEITAL ZILBERMAN1

1Faculty of Engineering, Department of Biomedical Engineering, Tel-Aviv University, Tel-Aviv 69978, Israel

2Faculty of Life Sciences, Department of Neurobiology, Tel-Aviv University, Tel-Aviv 69978, Israel

Received 1 September 2009; revised 15 March 2010; accepted 22 April 2010

Published online 8 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22238

Corresponde6405842; Fax: þJournal of Pharm

� 2010 Wiley-Liss

ABSTRACT: Composite core/shell fiber structures loaded with the antiproliferative drugspaclitaxel or farnesylthiosalicylate (FTS) were developed and studied. The latter is a specificnontoxic Ras inhibitor with a mild hydrophobic nature, which can also be used for local cancertreatment and stent applications. The fibers were composed of a dense polyglyconate core and aporous drug-loaded poly(D,L-lactic-glycolic acid) shell, prepared using freeze drying of invertedemulsions. Our study focused on the release profile of the antiproliferative drugs from the fibers,the shell morphology and its degradation and erosion. The postfabrication antiproliferativeeffect of the drugs was tested in a cell culture. The process parameters were found to affectthe drug-release profile via two routes: (1) direct, through water uptake and swelling of thestructure leading to FTS release, or through degradation of the host polymer leading topaclitaxel release at a later stage; (2) indirect effect of the microstructure on the release profile.The fabrication process did not reduce the pharmacological activity of either paclitaxel or FTS.FTS-eluting composite fibers proved to effectively induce growth inhibition or cell death by agradient effect and dose-dependent manner. The combined effect of the targeted mechanismof FTS as a Ras inhibitor together with the localized and controlled release characteristics ofthe fiber is an advantageous antiproliferative quality. It is therefore suggested that our drug-eluting fibers may be used in biomedical applications that require short release (restenosis) orprolonged release (cancer therapy). � 2010 Wiley-Liss, Inc. and the American Pharmacists Association

J Pharm Sci 100:133–149, 2011

Keywords: paclitaxel; farnesylthiosalicylat
e (FTS); drug-eluting stents; drug-eluting fibers;local cancer treatment
INTRODUCTION

Antiproliferative drugs inhibit cell proliferation andare therefore effective in the treatment of cancer aswell as neointimal hyperplasia, which is known asthe main cause of restenosis. Paclitaxel is the mostpopular antiproliferative agent. Its chemical struc-ture is presented in Scheme 1a. Paclitaxel wasoriginally isolated from a trace compound found inthe bark of the Pacific Yew (Taxus brevifolia).1 Its

nce to: Meital Zilberman (Telephone: þþ972-3-þ972-3-6407939; E-mail: [email protected])

aceutical Sciences, Vol. 100, 133–149 (2011)

, Inc. and the American Pharmacists Association

JOURNAL O

antitumor activity was detected in 1967 by the USNational Cancer Institute (NCI) and was later foundto be a promising novel antineoplastic drug. It wasapproved by the FDA for ovarian cancer in 1992, foradvanced breast cancer in 1994, and for early stagebreast cancer in October 1999. Paclitaxel eventuallybecame a standard medication in oncology.1–3 It actsto inhibit mitosis in dividing cells by binding tomicrotubules and causes the formation of extremelystable and nonfunctional microtubules. Microtubuledisassembly is essential for the transition from theG2 to the M phase in the mitotic cycle, whereasstabilization arrests mitosis and cell proliferation.1

Slow release of perivascularly applied paclitaxeltotally inhibits intimal hyperplasia and prevents

F PHARMACEUTICAL SCIENCES, VOL. 100, NO. 1, JANUARY 2011 133

Scheme 1. The chemical structure of: (a) paclitaxel, (b) FTS.

134 KRAITZER ET AL.

luminal narrowing following balloon angioplasty.However, paclitaxel’s narrow toxic-therapeutic win-dow may cause side effects during therapy.4

Farnesylthiosalicylate (FTS, Salirasib) is a newrather specific nontoxic drug, which was recentlydeveloped at the Tel-Aviv University.5 Its chemicalstructure is presented in Scheme 1b. FTS acts as aRas antagonist6,7 and has a mild hydrophobic nature.In its active form (GTP-bound), Ras promotesenhanced cell proliferation, tumor cell resistance todrug-induced cell death, enhanced migration, andinvasion. Ras is therefore considered an importanttarget for cancer therapy as well as for therapy ofother proliferation diseases, including restenosis. Theapparent selectivity of FTS towards active (GTP-bound) Ras and the absence of toxic or adverse sideeffects was proven in animal models6 and in humans(Concordia Pharmaceuticals, Inc., Ft. Lauderdale,FL). FTS was found to be a potent inhibitor of intimalthickening in the rat carotid artery injury model,which serves as a model for restenosis, while it doesnot interfere with endothelial proliferation.6 Theincorporation of the new drug FTS in a stent coatingmay overcome the incomplete healing and lack ofendothelial coverage associated with current drug-eluting stents (DES).

DES significantly reduce the incidence of in-stentrestenosis (ISR), which was once considered a majoradverse outcome of percutaneous coronary stentimplantations. Localized release of antiproliferativedrugs interferes with the pathological proliferation ofvascular smooth muscle cells (VSMC), which is themain cause of ISR.8 Current drug-eluting biodegrad-able or biostable stent coatings that contain anti-proliferative drugs exhibit side effects due to delayedor incomplete healing. There is strong evidencelinking these side effects to the drug loading andthe release profiles of these coatings.9 Furthermore,these coating cannot carry sufficient amounts of drugbecause of the trade-off between the mechanicalproperties and drug loading. Completely biodegrad-able stents may eliminate late complications of stentimplantation by degrading into nontoxic substancesafter maintaining luminal integrity during the first9–12 months period when there is high risk forrestenosis.10 Biodegradable stents may also eliminatecurrent DES endothelial related limitations and

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 1, JANUARY 2011

suggest a larger drug reservoir. Although bothdrug-eluting and bioresorbable stents have long beenstudied, current biodegradable, or stable DES are farfrom optimal in terms of controlled release of drugswithin the therapeutic range and safe healing of thetissue.

Glioblastoma multiforme is the most common andmost aggressive primary brain tumor, with a mediansurvival time from diagnosis of up to 1 year.11 Theongoing problem in glioblastoma multiforme therapyis drug accessibility.11,12 The blood brain barrierprevents many chemotherapeutic agents from reach-ing these tumors in adequate concentrations. Oneapproach could be targeted drug delivery to aparticular site near the tumor.2 Achieving anadequate drug level near the tumor cell is of primaryimportance because an inadequate tumor cell drug-burden will lead to low cell killing and to a potentialfor early development of drug resistance.2 Drugdelivery systems explored so far for localized pacli-taxel delivery in cancer treatment include micro-spheres,13,14 surgical pastes,15 and implants.16 One ofthe first intracerebral delivery systems studied forglioblastoma treatment involved surface erodingbioresorbable polyanhydride polymers. Drug-loadedpolymer discs of poly(bis-(p-carboxy phenoxy) pro-pane-sebacic acid; PCPP-SA, 20:80) containing 20%(w/w) drug loading of 1,3-bis-(2-chloroethyl)-1-nitro-sourea] (BCNU), also known as carmustine, wereimplanted intracranially in cynomolgus monkeys.16

Carmustine delivered from PCPP-SA (20:80) wafershas been shown to have promising initial activity andlimited toxicity,17 which led to FDA approval of theGliadel1 wafer. However, relatively minor achieve-ments were obtained with this approach, due to theresistance of many brain tumors to carmustine,18 aswell as the low stability of the drug and its tendencyto ionize at physiological pH.16 None of theseapproaches, however, has shown significant improve-ment in patient outcomes. The major limitations ofthese implants are in attaining the required amountof drug for a given amount of time in a properdistribution of the antiproliferative drug.

Paclitaxel-loaded PLGA micro and nanofibers(diameters from around 30 nm to 10 mm) wererecently fabricated by electrospinning18 to treat C6glioma. Cell viability test results suggested that the

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ANTIPROLIFERATIVE AGENTS EXHIBIT DRUG DELIVERY AND CELL GROWTH INHIBITION 135

paclitaxel-loaded PLGA nanofibers were effectivefor 72 h incubation. Ranganath et al.19 developedpaclitaxel incorporated poly(D,L-lactide-co-glycolide;PDLGA) implants in the form of microfiber discsand sheets. Paclitaxel was released from the PDLGAcopolymer implants (85/15 PDLGA and 50/50PDLGA) for 80 days. An animal study confirmedbrain tumor growth inhibition after 24 days.

We have recently developed and studied paclitaxel-eluting bioresorbable core/shell fiber structures forstent applications and local cancer treatment.20 Inour composite fibers, the dense core enables obtainingthe desired mechanical properties. The drug islocated in a porous shell so as not to affect themechanical properties. The shell is highly porous inorder to enable release of the relatively hydrophobicantiproliferative drugs in a desired manner. Thesefibers can be used for both applications, that is, forlocal cancer treatment implanted during tumorresection, or using stereotaxy and as basic elementsof DES. The drug-eluting coating (shell) can also beapplied on metal stents. Our structures are composedof a dense polyglyconate core and a porous drug-loaded PDLGA shell, prepared using freeze drying ofinverted emulsions. Paclitaxel’s release from theseporous structures is relatively slow due to itsextremely hydrophobic nature. We examined theeffect of the emulsion’s formulation on the paclitaxel’srelease profile and found that the host copolymer’scomposition exhibited the most profound effect.20 Inthis study, our composite fibers were loaded with theantiproliferative drugs paclitaxel or FTS. The effectsof the copolymer composition, its degradation profile,and the shell’s microstructure (dense vs. porous) onthe release profile of both drugs were studied. Thepostfabrication antiproliferative effect of the drugswas tested in cell culture and the growth inhibitioneffect of FTS-eluting fibers was tested in vitro.

MATERIALS AND METHODS

Materials

MaxonTM polyglyconate monofilament (3–0) sutures,with a diameter of 0.20–0.25 mm, Syneture (Covidien,Mansfield, MA) were used as core fibers. This polymercontains a 67.5:22.5 glycolide to trimethylene carbo-nate ratio.

Bioresorbable porous structures (the shell coating)were made of the following polymers:

� 7

DOI 1

5/25 poly(D,L-lactic-co-glycolic acid), inherentviscosity (i.v.)¼ 0.65 dL/g (in CHCl3 at 308C,approximately 97100 g/mol), Lactel AbsorbablePolymer. DURECT Corporation, (Pelham, AL)This polymer is termed 75/25 PDLGA.

0.1002/jps JO

� 5

URNA

0/50 poly(D,L-lactic-co-glycolic acid), inherentviscosity (i.v.)¼ 0.56 dL/g (in CHCl3 at 308C,approximately 31300 g/mol), Absorbable Poly-mer Technologies, Inc. This polymer is termed50/50 PDLGA.

The incorporated agents/drugs were either FTS orpaclitaxel:

� F
arnesylthiosalicylate (FTS, Salirasib) was a giftfrom Concordia Pharmaceuticals.
� P
aclitaxel (GenexolTM) was purchased from SamYang Corp. (Seoul, Korea).
Reagents used:

� 1
,1,1,3,3,3-Hexafluoro-2-propanol (H1008) waspurchased from Spectrum Chemical Mfg. Corp(Gardena, CA, USA).
� C
hloroform, CHCl3, HPLC grade, and methylenechloride, CH2Cl2, HPLC grade were purchasedfrom Frutarom Ltd. (Haifa, Israel).
� A
cetonitrile (methyl cyanide), CH3CN, HPLCgrade was purchased from Mallinckrodt Baker,Inc. (Phillipsburg, NJ, U.S.A).
� D
imethyl sulphoxide (DMSO), AR grade, waspurchased from Merck & Co., Inc. (WhitehouseStation, NJ, USA).
� E
thanol (ethyl alcohol) absolute, AR grade, waspurchased from Merck.
Cell Lines

Three cell lines were used for the in vitro cell cultures:

� H
uman glioblastoma (U87) kindly donated byEric Holland (Memorial Sloan-Kettering CancerCenter, New York, NY).
� E
J cells (Rat-1 fibroblast cells) were transfectedusing a method that is described elsewhere.21
� A
549 cell lines were obtained from AmericanType Culture Collection (ATCC) (Manassas,VA, USA).
Preparation of Core/Shell Fiber Structures

Fiber Surface Treatment

The sutures were surface-treated in order to enhancethe adhesion between the core fiber and the coating.The polyglyconate fibers were slightly stretched onspecial holders and dipped in 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluorisopropanol) for 40 s. The fiberswere then washed with ethanol and dried at roomtemperature.

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136 KRAITZER ET AL.

Emulsion Formation

A known amount of 50/50 PDLGA or 75/25 PDLGAwas dissolved in chloroform to form an organicsolution and FTS or paclitaxel was added to thesolution. Double-distilled water was then poured intothe organic phase (in a scintillation vial) andhomogenization of the emulsion was performed usinga homogenizer (Polytron PT3100 Kinematica, 12 mmrotor) operating at 16500 rpm (medium rate) for2 min. As a reference sample for the FTS formulationwe chose an emulsion formulation containing 12.5%(w/v) polymer in chloroform, 2% (w/w) FTS (relative tothe polymer load), and an organic to aqueous (O:A)phase ratio of 4:1 (v/v). As a reference sample for thepaclitaxel formulation we chose an emulsion formu-lation containing 17.5% (w/v) polymer, 1.43% (w/w)paclitaxel, O:A phase ratio of 2:1 (v/v).

Core/Shell Fiber Structure Formation

The treated core polyglyconate fibers were dip-coated(while placed on holders) in fresh emulsions andthen frozen immediately in a liquid nitrogen bath.The holdersþ samples were then placed in a pre-cooled (�1058C) freeze dryer (Virtis 101 equippedwith a nitrogen trap) capable of working with organicsolvents (freezing temperature of the condenser wasapproximately �1058C) and freeze dried in order topreserve the temporal state of the emulsion in a solidform. The freeze dryer chamber pressure was reducedto 100 mTorr while the temperature remained con-stant (�1058C) in order to sublimate the water andsolvents. Room temperature was then slowly restoredin order to evaporate residual solvent vapors. Thesamples were then stored in desiccators until use. Atthe end of the process, the shell’s microstructurereflects the emulsion’s stability.

In Vitro Drug Release Studies

The composite core/shell fiber structures wereimmersed in phosphate-buffered saline (PBS) at378C and pH 7.4 for 35 days (FTS) or 37 weeks(paclitaxel), in triplicates, in order to determine therelease kinetics from the drug-loaded compositestructures. Each test vial contained two fibers;each fiber was 5 cm long. The release studies wereconducted in closed glass tubes containing 3 mL PBSmedium, using a horizontal bath shaker operated at aconstant rate of 130 rpm. The medium was removed(completely) periodically, at certain sampling times,extracted from the aqueous medium as described inDrug Extraction Procedure Section and measuredusing HPLC. Fresh medium was then introduced. Atthe end of the experiment the fibers were immersed inmethylene chloride and the residual amount of drugwas measured.


The drug content of the medium samples wasdetermined using Jasco High Performance LiquidChromatography (HPLC) (Great Dunmow, Essex,UK) equipped with a UV 2075 plus detector and aquaternary gradient pump (PU 2089 plus). A reversephase column (ACE 5 C18, inner diameter¼ 4.6 mm,length¼ 250 mm) was used for FTS measurements,equipped with a column guard and kept at roomtemperature (258C). The mobile phase consisted of amixture of acetonitrile and phosphate buffer (30 mM,pH¼ 4.5) at a ratio of 70/30 (v/v), respectively, at aflow rate of 1 mL/min without gradient. The paclitaxelcontent of the medium samples was determined usinga reverse phase column (Zorbax1, Santa Clara, CA;ODS 5 mm, inner diameter d¼ 4.6 mm, length¼150 mm), and kept at 258C. The mobile phaseconsisted of a mixture of acetonitrile and double-distilled water (55/45, v/v) at a flow rate of 1 mL/min.One hundred microliter samples were injected withan autosampler (AS 2057 Plus). UV detection wascarried out at 227 nm for paclitaxel and 322 nm forFTS. The area of each eluted peak was integratedusing the EZstart software version 3.1.7.

Drug Extraction Procedure

FTS or paclitaxel extraction from the medium wasperformed as follows: the 3 mL PBS/drug medium wascompletely removed at each time point and placed ina scintillation vial. Three milliliter acetonitrile and1 mL methylene chloride were added and methylenechloride evaporation was performed under a nitrogenstream (99.999% grade). Medium (50/50, v/v, acet-onitrile/PBS) was added until reaching 4 mL in eachtest tube. The drug concentration was then estimatedusing HPLC. An extraction factor was used forcorrection. Known weights of drug were dissolvedin 3 mL acetonitrile and 3 mL PBS and 1 mLmethylene chloride was added. The known concen-trations were subjected to the same extractionprocedure as the unknown concentrations in orderto determine the efficiency of the extraction proce-dure. The recovery efficiency of the method for FTSwas 88.4% and the value of the measured drug wascorrected accordingly. The recovery efficiency of themethod for paclitaxel was 75% and the value of themeasured drug was corrected accordingly.

Residual Drug Recovery from the Composite Fibers

On the final day of the in vitro trial, residual drugfrom the composite fibers was measured as follows:the fibers were placed in 1 mL methylene chloridefor 10 min and the coating shell was dissolved. Sixmilliliters of a 50/50 acetonitrile/water solution werethen added and the polyglyconate core was removed.Methylene chloride evaporation was performed undera nitrogen (99.999%) stream. Medium (50/50, v/v,acetonitrile/water) was added until reaching 4 mL in

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each test tube and the drug concentration was esti-mated by HPLC using the same method as describedabove. A calibration curve was calculated usingknown amounts of drug under the same conditions.

The cumulative release profiles were determinedrelative to the initial amount of drug in the compositefibers (quantity released during the incubationperiodþ the residue remaining in the fibers). Allexperiments were performed in triplicates. Resultsare presented as means� standard deviations.

In Vitro Degradation of the Porous PDLGA Structure

Porous 50/50 PDLGA or 75/25 PDLGA film structureswere fabricated using the emulsion formulation of thereference samples but without drug. The invertedemulsion was prepared as described earlier, pouredinto an aluminum plate, quenched in liquid nitrogen,and freeze dried. Each sample (three repetitions),approximately 1 cm2, was incubated in 40 mL PBScontaining 0.05% (v/v) sodium azide (as preservative)at 378C under static conditions for 5 weeks (50/50PDLGA) or 17 weeks (75/25 PDLGA). PBS was addedwhen the pH was out of range (between 7 and 8) orwhen the PBS volume dropped below 40 mL. Thesamples were taken out at weekly intervals, driedusing a vacuum oven (358C for 2 h), and stored ina dessicator. These samples were then used formolecular weight measurements (2.7), weight lossmeasurements (2.8), and morphological characteriza-tion (2.9).

Weight–Average Molecular Weight Measurements

The weight–average molecular weight of the sampleswas determined by gel permeation chromatography(GPC). The dried sample was dissolved in methylenechloride before elution to achieve a minimal concen-tration of about 0.13% (w/v). The GPC (Waters 21515isocratic pump, operating temperature 408C using acolumn oven) was equipped with a refractive indexdetector (Waters Corporation, Milford, MA, USA;Waters operating temperature 408C) and calibratedwith poly-L-lactic acid MW kit standards (Poly-sciences Inc., Warrington, PA). Data were analyzedusing the Breeze version 3.3 software. The sampleswere dissolved in methylene chloride, filtered, andeluted through 4 Styragel columns (model WAT044234HR1 THF, WAT044237 HR2, WAT044225 HR4,WAT054460 HR5, 300� 7.8 mm2, 5 mm particle dia-meter) equipped with a guard column at a flow rate of1 mL/min. The elution medium was Baker analyzedHPLC grade methylene chloride.

Weight Loss Profile of the Porous PDLGA Structure

Porous 50/50 and 75/25 PDLGA film structures werefabricated as described above in the paragraph aboutthe in vitro degradation study. Samples (three

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repetitions) were taken out at weekly intervals,filtered using a 70 mm porcelain Buchner funnelequipped with a Whattman 2 mm filtration paper anddried in a vacuum oven (358C for 2 h).

Mass loss was measured using a Mettler–Toledomicrobalance. The normalized mass loss was calcu-lated by comparing the mass at a given time point (wt)with the initial mass (w0) as shown in Eq. (1). Theresults are presented as means� standard deviations(n¼ 3).

Normalized weight ¼ wt

w0� 100% ð1Þ

Water Uptake of the Porous PDLGA Structure

Porous 50/50 and 75/25 PDLGA film structures werefabricated as described above in the paragraph aboutthe in vitro degradation study. Samples (triplicates)were taken out periodically and immediately sub-jected to measurement of wet weight, after surfacewater was removed with a clean-wipe tissue. Wateruptake, that is, adsorption and absorption of eachsample during the swelling period, was determinedaccording to equation 2 (w is the wet weight at eachtime point and w0 is the dry weight measured beforethe incubation):

Water uptake ¼ w�w0

w0� 100% (2)

Encapsulation Efficiency

The encapsulation efficiency (EE) is determined bycalculating the overall measured cumulative releasedrug (Wcd) divided by the theoretical drug incorpo-rated in a given coating weight according to thetheoretical drug load percentage. The theoreticaldrug load is the concentration of drug incorporatedwithin the emulsion (C0) multiplied by the weight ofthe coating. The coating weight is the initial weightof the coated fiber (Wcf) minus the weight of thefiber denuded from the coating at the end of theexperiment.

EE ¼ Wcd

C0ðWcf �WcÞ� 100% (3)

Morphological Characterization

The morphology of the composite core/shell fiberstructures (cryogenically fractured surfaces) wasobserved using a Jeol JSM-6300 scanning electronmicroscope (SEM) with an accelerating voltage of5 kV. The mean pore diameter of the observedmorphologies was analyzed using the Sigma ScanPro software.

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138 KRAITZER ET AL.

Cell Culture Procedures

U87 and EJ cells were grown in Dulbecco’s modifiedEagle’s medium (DMEM) containing 10% Fetal calfserum (FCS), 5% L-glutamine, and 5% penicillin/streptomycin (P/S) at 378C in a humidified atmo-sphere of 95% air and 5% CO2. A549 cell lines weregrown in medium containing 10% FCS, 5% (P/S), asrecommended by ATCC.22

Control and Postfabrication Drug Effect onCell Cultures

In order to estimate the effects of postfabrication drugand control drug on cell growth, cells were plated at adensity of 8000 cells (for FTS treatment) or 10000 cells(for paclitaxel treatment) per well in 24-well plates, towhich either 0.1% DMSO (vehicle control), postfab-rication drugs (FTS or paclitaxel) or control drugswere added after 24 h. Solutions of the postfabricationdrug and control drug were prepared before eachexperiment in growth medium which contained 0.1%DMSO. Postfabrication drugs (FTS or paclitaxel)were premeasured using HPLC (see In Vitro DrugRelease Studies). Control drug was measured grav-imetrically and both solutions were diluted to theircorresponding concentrations. The treatment wellscontained either a single 50 nM dose of paclitaxel,or 25, 50, 75, and 100 mM FTS. The cells were thengrown for 1 more day (paclitaxel) and 3 days (FTS)after treatment, detached and counted using ahemocytometer. The data are presented as meanviability percentage� standard deviation.

Drug Extraction of Postfabrication FTS and Paclitaxel

Designated drug-containing PDLGA films werefabricated using the freeze drying of an invertedemulsion method. These films, 0.3–0.7 g in weight,were then dissolved in 3 mL chloroform. In orderto extract their drug content, 10 mL absolute ethanolwere added to the PDLGA/chloroform solutionallowing the solidification of the polymer under a99.999% N2 stream until the chloroform evaporatedcompletely. The stock solution was then divided intoEppendorf tubes; each dose stock contained eitherFTS in 200 mL or paclitaxel in 150 mL absoluteethanol. The concentration of the postfabricationdrug stock was then measured by HPLC using themethod described in In Vitro Drug Release StudiesSection subsequent to ethanol evaporation. Themeasured FTS stock concentration was 258.5 mg/mLand the measured paclitaxel stock concentration was0.95 mg/mL. The Eppendorf tubes were kept at �48Cuntil the day of the trial. On the day of the trial theEppendorf tubes were completely evaporated for 3 hunder a 99.999% N2 stream, yielding a drug powder,which could be used for further dilutions. The reasonfor adding ethanol during the process was to


eliminate the relatively high polymer content andtherefore obtain the drug only. This method ensuresthat the drug will affect the cell viability rather thancausing polymer artifacts.

Three-point calibration graphs were obtained, withR2¼ 0.9947 for paclitaxel and R2¼ 0.9994 for FTS.Known weights were measured gravimetrically anddiluted with ethanol to obtain concentrations of 179,358, and 715 FTS mg/mL and 0.2, 0.42, 1.0 mg/mLpaclitaxel. The test tubes were evaporated and theircontent was diluted with 1 mL acetonitrile (FTS) and1 mL (50/50 ddw/acetonitrile; paclitaxel). The treat-ment of the drugs was similar to the method for thepostfabrication of drugs described above.

Determination of Ras and Ras-GTP in thePostfabrication and Control FTS Preparations

In order to examine the effect of postfabrication FTSand the control-FTS on Ras protein levels in thecultured cells, EJ cells were plated at a density of1� 106 cells/10 cm dish and grown for 24 h. The cellswere then treated with postfabrication FTS (50 mM),control FTS (50 mM), or control (0.1% DMSO). Thecells were then lysed for 24 h after the drug treatmentin 0.5 mL Ras-binding domain (RBD) lysis buffer.23

Debris was removed by centrifugation at 14000 rpmfor 10 min. Total Ras was determined in a 50 mgsample by immunoblotting with pan anti-Ras Ab (firstAb; Calbiochem, La, Jolla, CA) and goat anti-mouseHRP (second Ab) followed by enhanced chemilumi-nescence (ECL).24 Ras activity was determined byRBD-GST pull-down assays. Briefly, lysates con-taining 500 mg protein were incubated with GST-RBD sepharose beads. Since the RBD of Raf bindsonly Ras-GTP, using pull-down assays followed byWestern immunoblotting with pan anti-Ras Ab24

represents Ras activity. Protein bands werevisualized.

Effect of FTS-Loaded Fiber on Cell Cultures

A quantitative and qualitative comparison was madebetween the number of cells incubated in the presenceof 50/50 PDLGA-FTS shell-coated fibers (fast releasefiber), fibers without incorporated drug (control fiber),and 75/25 PDLGA shell-coated fibers (slow releasefibers). Between one and three fibers were used,depending on the trial setting; each fiber wasapproximately 1 cm long. The calculated drug amountin these fibers is estimated at 3.5 mg FTS per 1 cmfiber, which corresponds to 0.01 mmol or 10 mM whenreaching equilibrium in a well containing 1 mLvehicle. The fibers did not undergo sterilization,since they were kept in a sterile environment untiltreatment immediately after their fabrication, whichinvolves chloroform. Cells (either EJ, A549, or U87)were plated in 24 wells at a density of 8000 cells

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per well and grown for 24 h. The fibers were thenapplied and the cells were grown for an additional5–8 days and were then photomicrographed orcounted using a hemocytometer, depending on thetrial setting.

Statistical Analysis

Results are presented as means� standard devia-tions. Statistical significance was determined usingthe unpaired Student’s t-test or ANOVA (Tukey–Kramer) methods using SPSS. The results areconsidered statistically significant at p< 0.05, unlessotherwise stated.

RESULTS AND DISCUSSION

The dense core of our composite fibers enablesobtaining the desired mechanical properties andthe drug is located in a porous shell so as not toaffect the mechanical properties. The shell is highlyporous so as to enable release of the relativelyhydrophobic antiproliferative drugs in a desiredmanner. In order to characterize our drug-elutingcore/shell fiber platform, we studied FTS andpaclitaxel release from the fibers in light of the shells’morphology and degradation weight loss profiles.In view of the drug release profiles obtained in vitro,we sought to test the core/shell system using in vitrocell cultures. We studied the activity of the drugspostfabrication and then the overall effect of thesystem as a tumor-targeted antiproliferative releasedevice using cancer cell lines. The trial settingmeasures the antiproliferative property of the fibersand is believed to predict in vivo models for localtreatment of cancer and restenosis.

The freeze-drying technique that was used for theshells’ preparation is unique in being able to preservethe temporal state of the emulsion in a solid form.We used this technique in order to produce invertedemulsions in which the continuous phase containedpolymer and drug dissolved in a solvent, with waterbeing the dispersed phase. In a previous study20 weinvestigated the effects of the inverted emulsion’sparameters, that is, polymer content, drug content,

Table 1. The Structural and Drug Load Characteristics of Fib

Sample Pore Diameter (mm) Porosity (%)

FTS-loaded samples50/50 PDLGA 2.9�1.1 84.2� 4.575/25 PDLGA 4.5�1.3 76.2� 2.3

Paclitaxel-loaded samples50/50 PDLGA 4.1�1.3 67.0� 6.075/25 PDLGA 6.4�2.3 69.0� 6.0

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organic to aqueous (O:A) phase ratio and copolymercomposition on the shell microstructure and on thepaclitaxel release profile from the fibers. Ourexperience showed that the effect of the copolymercomposition, that is, the relative quantities of lacticacid (LA) and glycolic acid (GA) in the copolymer, onthe drug release profile and on the shell microstruc-ture was the most pronounced of all parameterstested.20 In addition, for each drug (FTS or paclitaxel)we found the optimal formulation (see Preparation ofCore/Shell Fiber Structures Section), which enabledus to obtain a relatively stable emulsion as may beinferred by the shell’s bulk porous microstructure.Furthermore, in this study 50/50 and 75/25 PDLGAwere chosen as host polymers due to their relativelyfast degradation rate in order to be able to releasethe hydrophobic antiproliferative agents at anappropriate rate.

The diameter of the treated core fibers (i.e., withoutthe coating) was in the range of 200–250 mm and ashell thickness of 30–70 mm was obtained. The shell’sporous structure (resulting from the freeze dryingof the inverted emulsions) contained round-shapedpores in all the specimens that were based onrelatively stable emulsions, usually within the 2–7 mm range, with a porosity of 67–85%. The EE of thestudied samples was in the range of 47.5–64.6 for theFTS incorporated coatings and in the range of 47.3–54.2 for the paclitaxel incorporated coatings (Tab. 1).The structural characteristics of the shell and EEvalues of the examined specimens are summarized inTable 1. The 50/50 PDLGA is less hydrophobic thanthe 75/25 PDLGA due to its higher GA content. Theinterfacial tension (difference between the surfacetensions of the organic and the aqueous phases) of the50/50 PDLGA emulsion is therefore lower and theinverted emulsion is more stable. This results in alower shell pore size of the 50/50 PDLGA for bothpaclitaxel and FTS-loaded fibers (Tab. 1).

Drug Release and Degradation

The drug release profiles from a shell based on 50/50PDLGA and from a shell based on 75/25 PDLGA arepresented in Figure 1 for fibers loaded with paclitaxel(Fig. 1a) and FTS (Fig. 1b). The degradation profilesof the two copolymers are presented in Figure 2a

er Shells Loaded With Anti-Proliferative Agents

Encapsulation Efficiency (%) Drug Amount (mg/cm fiber)

56.7�7.9 0.336�0.8251.8�4.3 0.374�0.57

53�1.2 0.122�0.4548�0.7 0.113�0.12


Figure 1. The effect of the copolymer composition on thecumulative drug release profile from core/shell fiber struc-tures ( —50/50 PDLGA, —75/25 PDLGA): (a) paclitaxelrelease, (b) FTS release. Plots of dMt/dt versus sqrt (1/t) forthe first 5 weeks of release (in the small frames) indicatediffusion-controlled region.

140 KRAITZER ET AL.

and their weight loss profiles are presented inFigure 2b. Paclitaxel’s cumulative release exhibitedthe following three phases:

Figure 2. Degradation profiles (a), weight loss profile(b), and water uptake (c) of —PDLGA 50/50, —PDLGA
(a) T
75/25 porous structures.

JOURNA

he first phase of release (phase a) occurredduring weeks 1–8, in which the drug wasreleased in an exponential manner, that is,the rate of release decreased with time. Sucha release profile is typical of diffusion-con-trolled systems. It is known according to Higu-chi’s model that a purely diffusion-controlleddrug release region can be described as fol-lows25:

dMt

dt¼ A

2

ffiffiffiffiffiffiffiffiffiffiffiffiffi2PCO

t

r(4Þ

where Mt is the cumulative amount of drugreleased at time t, A is the surface area fordiffusion, P is the permeability of the drugwithin the polymer matrix and Co is the initialdrug concentration within the system. A plotof dMt/dt versus sqrt (1/t) for the first 5 weeks ofrelease shows a curve, which is very close tostraight line (small frame of Fig. 1a), whichindicates a diffusion-controlled region.

L OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 1, JANUARY 2011

A minor initial burst release was obtainedduring the first day of release. The paclitaxelrelease from the porous shell was relativelyslow for both types of host polymer, 50/50PDLGA and 75/25 PDLGA, mainly due to pacli-taxel’s extremely hydrophobic nature. More-over, the release rate decreased with time,since the drug had a progressively longer dis-tance to pass and a lower driving force fordiffusion.

(b) T
he second phase of release (phase b) occurredduring weeks 5–20, in which the drug wasreleased at a constant rate. The rate of pacli-taxel release from the 50/50 PDLGA hostpolymer was significantly higher than thatobtained from the 75/25 PDLGA. This differ-ence is attributed to a difference in the degra-
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DOI 10.


dation rate of these two copolymers, whichassists the drug’s diffusion. The 50/50 copoly-mer degrades faster than the 75/25 copolymerand therefore releases the drug at a faster rate.The degradation rate of the 50/50 PDLGAis indeed significantly higher than that of the75/25 PDLGA, as inferred by the slope of theirmolecular weight profile (Fig. 2a).

(c) T
he third phase of release (phase c) occurredduring weeks 21–38, when the porous shellstructure was already destroyed due to inten-sive degradation. In fact, at this stage most ofthe shell remains are no longer attached to thecore fiber and the core fiber also undergoeserosion.
Most of the encapsulated paclitaxel was releasedfrom the 50/50 PDLGA shell during phases a and b.However, in our system the highly hydrophobicpaclitaxel is probably attached to the surface of thehydrophobic 75/25 PDLGA even after intensivedegradation. There may be specific hydrophobicinteractions between the paclitaxel and the poly-mer.26 It is clear that the paclitaxel release profileduring phases a and b corresponds to the degradationprofile of the porous host PDLGA shell. Intensivedegradation of the host polymer is necessary in orderto obtain release of the highly hydrophobic paclitaxel.Overall, about 10 mg paclitaxel, corresponding to 90%of the loaded drug, was released from the 50/50PDLGA shell, whereas about 4 mg paclitaxel, corre-sponding to 30% of the loaded drug, was releasedfrom the 75/25 PDLGA shell. Other investigatorsworking on paclitaxel-eluting systems also reportedits relatively slow release rate from various polymericsystems.27,28

The FTS release profiles from our fiber platform aredifferent than the paclitaxel release profiles. Theyexhibited a burst effect accompanied by a releaserate, which decreased with time (Fig. 1b). The 50/50PDLGA fiber released 21% of the encapsulated drugduring the first day of release, whereas the 75/25PDLGA fiber released only 6%. This difference isattributed mainly to differences in the hydrophilic/hydrophobic balance of these two copolymers. The 50/50 PDLGA copolymer contains more GA groups andfewer LA groups along the polymer chain and istherefore less hydrophobic than the 75/25 PDLGAand probably exhibits higher water uptake during theinitial phase of release. Consequently, this enablesmore rapid water inflow which results in a higherburst release. Furthermore, the rate of release fromthe 50/50 PDLGA formulation is slightly higher thanthe rate obtained with the 75/25 PDLGA formulationand after 2 weeks of degradation the 50/50 formula-tion released 72% of the drug, whereas the 75/25formulation released only 26%.

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Both polymers exhibited a small weight loss of<10% during the first 3 weeks of degradation,whereas after 3 weeks of degradation the 50/50PDLGA exhibited a fast weight loss while the 75/25PDLGA did not erode during the measuredtime period (Fig. 2b), as expected. These resultsindicate that most of the FTS is released from ourporous coatings before they undergo massive weightloss.

The changes in the shell microstructures of bothcopolymers during exposure to the aqueous mediumare presented in Figure 3a (50/50 PDLGA) andFigure 3b (75/25 PDLGA). The starting point (day 0)shows highly porous delicate structures with roundpores for both samples. The pore size of the 50/50PDLGA shell (Fig. 3a) is significantly smaller thanthat of the 75/25 PDLGA (Fig. 3b) and its porosity ishigher (Tab. 1). Also, the 50/50 PDLGA microstruc-ture is highly interconnected compared to the 75/25PDLGA, which enables more surface area for diffu-sion. After 7 days of degradation in the aqueousmedium, the 50/50 PDLGA shell exhibited a rougherstructure (Fig. 3a), while after 14 days of degradationit exhibited a totally dense structure at day 28(Fig. 3a). The 75/25 PDLGA also underwent a similarchange in microstructure (Fig. 3b) while however, inthis case the entire process was slower and tookapproximately 126 days, due to the more hydrophobicnature of the copolymer, which is rich in lactic acid.The changes in microstructure are caused by earlyswelling and water uptake and subsequent degrada-tion and erosion.

The water uptake measurements indicate anincrease of 40% in the 50/50 PDLGA’s weight duringthe first 24 h, after which it stabilizes, whereas the 75/25 PDLGA exhibited a slower water uptake, whichlasts for 200 h (Fig. 2c). This further supports ourhypothesis that the early structural changes are dueto water uptake rather than degradation or erosion.Since the FTS diffusion through the fiber’s shelloccurs in an aqueous swollen phase, a relatively highwater uptake, such as that of our 50/50 PDLGAporous shell, enables faster diffusion of the FTSmolecules.

It can therefore be concluded that higher GAcontent in the copolymer, that is, a less hydrophobiccopolymer, enables a greater initial surface areafor diffusion and higher FTS release from thefibers mainly due to early swelling. Higher wateruptake affects the microstructure and results in ahigher burst release and a higher degradation rateof the host polymer, which assists diffusion. Thecontribution of the swelling and the resultingmicrostructural effects are more significant for theFTS-eluting systems than for the paclitaxel-elutingsystems. Also, our paclitaxel release study anddegradation results show that this bulky and highly


Figure 3. SEM fractographs of shell structures showing their microstructuralchanges with time: (a) 50/50 PDLGA at days 0, 7, 14, and 28, (b) 75/25 PDLGA at days0, 7, 14, 28, 56, and 126.

142 KRAITZER ET AL.

hydrophobic drug is released mainly from very shortpolymer chains.

The effect of the coating’s microstructure(porous vs. dense) on the release profile of FTSfrom both polymers, 50/50 PDLGA and 75/25


PDLGA, is presented in Figure 4. The drugrelease rate from both polymers was faster for aporous coating compared to dense coating, due tolarger surface area for diffusion of the hydrophobicdrug.

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Figure 4. Effect of porous coating on FTS release profilefrom core/shell fiber structures (a) 50/50 PDLGA matrix(~- should be more pink, X - should be green &- (should bered)-porous structure, & dense structure-porous structure,& dense structure.


Qualitative Model Describing the Release ofAntiproliferative Drugs from Our Composite Fibers

A qualitative model describing the process!structure!drug release profile effects in our fiberswith controlled release of antiproliferative agents canbe described as follows: there are two routes by whichthe process affects the drug-release profile: direct andindirect.

Direct route: The emulsion formulation (especiallythe host polymer) affects the water uptake andswelling of the structure and therefore also the burstrelease of antiproliferative drugs such as FTS (earlymechanism). In such cases degradation of the hostpolymer affects the release rate at a later stage. Whena relatively big and extremely hydrophobic drug suchas paclitaxel is incorporated into the shell, itsdiffusion through the host polymer is much slowerand massive degradation and erosion of the hostpolymer must occur in order to enable it.

Indirect route: The effect of the process on themicrostructure occurs also via an emulsion stabilitymechanism. The emulsion stability determines thesurface area for diffusion through the microstructure,for example, the surface area increases when porosityis high and pore size is low. For example, the 50/50PDLGA’s structure is finer than that of the 75/25

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PDLGA (Fig. 3a and b). These affect both the burstrelease and later release.

The most important parameter, which affects therelease behavior in our system is the copolymercomposition. It affects the water uptake and swellingand therefore the FTS release profile (early mechan-ism). The copolymer composition affects the degrada-tion rate of the polymer and therefore also thepaclitaxel release profile (late mechanism). Hence,the copolymer composition plays a very important rolein the drug release profile through the direct route.

The Fabrication Process Does Not Affect thePharmacological Activity of the Drugs

The fabrication process of a drug-release device mayaffect the activity of the incorporated drug. In ourprevious publications,20 we postulated that our freezedrying of the emulsion process should not have anegative effect on the incorporated drug molecules. Toprove this claim we examined the activity of post-fabrication paclitaxel and FTS on cell growth. Bothdrugs were extracted from the matrices as describedin the Materials and Methods Section and were thenused in cell culture experiments. In all experimentswe compared the action of the extracted drug, namelythe drug that was extracted from designated filmsusing our fabrication process (herewith postfabrica-tion drug), with that of the control drug, which wasnot exposed to any fabrication process (herewithcontrol drug). Drug activities were tested on threedifferent types of cancer cell lines: H-Ras-trans-formed Rat-1 fibroblasts (EJ), U87 glioblastoma, andA549 lung cancer cells, all of which were previouslyshown to be sensitive to FTS.29,11,21,22 The cells wereincubated with FTS (25, 50, or 100 mM) or with asingle dose of paclitaxel (50 nM) or with the vehiclecontrol. The FTS-treated and paclitaxel-treated cellswere counted after 4 days and 1 day, respectively.Figure 5 shows that postfabrication paclitaxel andcontrol paclitaxel caused a marked decrease in thenumber of EJ, A549, and U87 cells relative to thecontrol (60–90% decrease). The decrease in cellnumber was attributed to cell death, in line withthe known cytotoxic action of paclitaxel. Importantly,there was no difference between the extent of activityof postfabrication paclitaxel and control paclitaxel(p> 0.8; Fig. 5), indicating that the fabricationprocess did not affect paclitaxel’s pharmacologicalactivity.

Figure 6a–c shows the results obtained withpostfabrication FTS and control FTS. As shown, bothFTS preparations inhibited the growth of A549(Fig. 6a), EJ (Fig. 6b), and U87 (Fig. 6c) cells in adose-dependent manner. The range of FTS concen-trations that caused 50% inhibition of cell growth iscompatible with previous reports.29,11,21,22 Also inagreement with previous studies, FTS did not induce


Figure 6. Both control FTS and postfabrication FTSinduce a significant decrease in the cell count, in a dose-dependent manner. Cells (EJ, A549, and U87) were platedat a density of 8� 103 cell/well in a 24-well plate in tetra-plicate (n¼ 2). One day after plating, 0.1% DMSO (control)or the indicated FTS concentrations were added for 3 days.The cells were then counted using a hemocytometer.FTS dose-response effect on: (a) A549, (b) U87, and (c) EJis presented in terms of mean cell viability� standarddeviation.

Figure 5. Both control paclitaxel and postfabricationpaclitaxel induce a significant decrease in the cell count.Cells (EJ, A549, or U87) were plated at a density of 10�103 cells/well in a 24-well plate in tetraplicate (n¼ 2). Oneday after plating, 0.1% DMSO (control), 50 mM controlpaclitaxel (black columns), or 50 mM postfabrication pacli-taxel (white columns) were added for 24 h. The cells werethen counted using a hemocytometer (�p< 0.01 compared tothe control well). Paclitaxel’s effect on the above-mentionedcell lines is presented in terms of mean cell viability�standard deviation.

144 KRAITZER ET AL.

cell death under these conditions.29,11,21,22 However,FTS can induce cell death in U87 cells,29,30 asdiscussed below. Importantly, there was no signifi-cant difference between the activity of the postfab-rication FTS and the control FTS (p> 0.5; Fig. 6).

Postfabrication FTS Did Not Lose RasInhibition Activity

Growth inhibition or induction of cell death by FTS isattributed to its action on Ras. FTS competes withRas binding to Ras chaperons in the cell membrane,thereby dislodging the active Ras-GTP from themembrane.31 The reduction in the level of activeRas or in the amount of total Ras in the cell membraneresults in an inhibition of Ras signaling that origi-nates from the cell membrane and is required for cellgrowth.31 We therefore examined whether the growthinhibition induced by the postfabrication FTS wasindeed associated with a reduction in total Ras andRas-GTP. We used EJ cells in these experiments,since the effects of FTS on H-Ras12V have beenwell characterized.21,32 As shown in Figure 7, bothpostfabrication FTS and control FTS caused a markedreduction in total Ras and Ras-GTP in EJ cells. Itshould be pointed out that in these Ras-transformedcells, the major expressed Ras protein is the H-Ras12V,which is constitutively GTP-bound.21 This explainswhy we observed a FTS-induced reduction in total Rasand in Ras-GTP. These results demonstrate that ourfabrication procedure does not affect the anti-Rasactivity of FTS.


FTS-Eluting Fibers Exhibit a Concentration-Gradient anda Dose-Dependent Effect on Cells in Culture

The experiments described above showed that post-fabrication FTS extracted from the core shell fiberis as active as control FTS. However, these studiesdid not examine the impact of FTS that elutes fromthe fibers directly onto the cells as would be the casein vivo. Moreover, we could not tell whether theamount of drug incorporated into the fiber is highenough to inhibit cell growth. To provide answers forthese questions we designed a set of experiments in

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Figure 7. EJ cells were plated at a density of 1� 106 cells/10 cm dish. One day afterplating, 0.1% DMSO (control) or 50 mM control FTS or 50 mM postfabrication FTS wereadded for 24 h. Ras total proteins were then determined in cell lysates by immunoblot-ting with Pan-ras Ab. Ras activity in cell lysates was determined by GST-RBD pull-downassay followed by immunoblotting with Pan-Ras Ab and ECL as described in theMaterials and Methods Section. The immunoblots visualized by ECL were then sub-jected to densitometry. The left panel (a) shows blots of a representative experiment.The right panel (b) shows the results of the densitometric analysis (n¼ 3).


which cells were directly exposed to the FTS-core-shell fibers (see Figs. 8a–c and 9a and b). Both slowand fast release fibers (Fig. 1) were used so as to allowa relatively slow and fast accumulation of FTS in thewells. A relatively slow release rate was obtained byshells based on 75/25 PDLGA, while relative fasterFTS rate was obtained by shells based on 50/50PDLGA. We used U87, A549, and EJ cells in theseexperiments in order to document inhibition of cellgrowth and induction of cell death. Cells were imagedat various time points after being exposed to control(no drug) or FTS fibers. We chose not to test paclitaxelfibers since paclitaxel release fibers have a well-established documentation in the literature. Further-more, paclitaxel’s cytotoxic mechanism of action wasnot damaged during fabrication, and the effect of thepaclitaxel fibers is therefore predictable.

Typical images of U87 cells exposed to control, slowand fast release fibers for 5 days are presented inFigure 8a. A relatively homogenous distribution ofcells with approximately the same density wasobserved around the control and the slow releasefibers (Fig. 8a). In the slow release fiber well, the celldensities were slightly higher at a significant distancefrom the fiber than around the fiber, indicating thatthe amounts of FTS that diffused out of the slowrelease fiber during 5 days were not high enough toinduce the death of U87 cells. In contradistinction,cell density around the fast release fibers was alreadyreduced after 5 days, whereas their density wassimilar to that observed in the control fiber at arelatively far distance from the fiber. Three additionalobservations strongly suggested that a FTS concen-tration gradient was formed in the well as a result

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of the release from the fiber. First, as shown inFigure 8a (fast release, near the fiber), the number ofcells very near to the direction orthogonal to the fiberaxis is smaller than that recorded near the directionof the fiber axis. This is clearly in accord with ourknowledge that the perimeter of the fiber, not itscross-section, contains the FTS coat. Second, and inline with this notion, a small effect of FTS could havebeen expected if the FTS concentrations were indeedlow near the fiber’s cross-section. As shown inFigure 8a (fast release), U87 cells located near thefiber’s cross-section or those located further from thefiber gained a flat morphology and became larger thanthe control cells. This is in agreement with previousreports, which demonstrated that changes in U87 cellmorphology occur already at low FTS concentrations,lower than those that inhibit cell growth.11 On day 5about 50% of the FTS is released from the fast releasefiber (50/50 PDLGA; Fig. 1b). When reachingequilibrium, the amount of FTS in the mediumaccounts for approximately 10 mM FTS. This concen-tration may induce a change in U87 cell morphol-ogy.11 Third, we could demonstrate a decrease in celldensity as a function of increased distance from thefiber; cell density was relatively low near the fiberand relatively high close to the edge of the well(Fig. 8b, panoramic image and cell counts of a slowrelease fiber). This correlation is consistent withstrong and weak cell growth inhibition (and celldeath) near and at a greater distance from the fiberattributable to high and low FTS concentrations,respectively.

The presumed FTS gradient is formed by its initialburst release that occurs within the first 24 h and the


Figure 8. FTS loaded core/shell fiber structures inhibit growth or induce cell death ofglioblastoma cells by a gradient effect and dose-dependent manner. U87 cells wereplated at a density of 8� 103 cells/well in a 24-well plate in tetraplicate (n¼ 2). One dayafter plating, control fibers (not loaded with FTS), slow or fast FTS release fibers wereadded to each well (two fibers, each with a length of 1 cm, as described in the Materialsand Methods Section). a: Images taken after 5 days of incubation with FTS fibers, inlocations, which are near and distant to the fiber (magnification 100�). b: A single welledge to edge panoramic view shows a gradiential increase in the cell concentration withthe increase in the distance from a slow release fiber (magnification 100�, cells werecounted using an image analysis software). c: Images were taken after 7 days ofincubation with FTS fibers; the control fiber well presents high cell viability whilethe well containing the fast FTS fiber exhibited cell death (magnification 100�). Notethat the dark shape is the actual fiber.

146 KRAITZER ET AL.

subsequent slower release (see Fig. 1b). Accordingly,the cells near the fiber were transiently exposed to thehighest FTS concentration, whereas those furtheraway were exposed to a lower drug concentration. Theeffect recorded after the 5th day of release reflectsboth the impact of the early burst of FTS release andthe late equilibrium level of the drug in the entirewell, which was achieved when most of the drug wasreleased from the fiber. Finally, we found that morethan 90% of the cells died after a longer periodof exposure to the fast release core–shell fiber (after7 days, see Fig. 8c). The difference between the


amount of FTS released from the fast release fiber(50/50 PDLGA) on the 5th and 7th day (Fig. 1b) is nothigh enough to induce cell death. However, cell deathoccurred since more regions were transiently exposedto relatively high FTS concentrations during thelonger incubation period, which triggers death of U87cells.29,30 Fung et al.16 reported a similar gradientconcentration effect using either a Carmustine,4-hydroperoxycyclophosphamide (4-HC), or paclitaxel-loaded polyanhydride pellet implanted intracraniallyin cynomolgus monkeys as a new modality ofchemotherapy delivery in primates.

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Figure 9. FTS loaded core/shell fiber structures inhibit growth of H-Ras-transformedRat-1 fibroblasts (EJ), and lung cancer (A549) by a gradient effect and dose-dependentmanner. Cells (EJ and A549) were plated at a density of 5� 103 cells/well in a 24-wellplate in tetraplicate (n¼ 2). One day after plating, control fibers (not loaded with FTS), orfast FTS release fibers were added to each well (two fibers, each fiber with a length of1 cm, as described in the Materials and Methods Section). a: Images were taken after7 days incubation of EJ or A549 cells with fast FTS release fibers, at a magnificationof 400� and 100�, respectively. Note that the dark shape is the actual fiber. b: Aquantitative hemocytometer cell count was performed in tetraplicates (n¼ 2) after8 days of incubation of A549 cells with either control, slow, or fast FTS release fibers.Results are presented as mean� standard deviation relative to control (�p< 0.01compared to control).


Additional experiments were performed with bothfast and slow FTS release core-shell fibers using twoother transformed cell lines that are driven in part byoncogenic Ras. One is Rat-1 fibroblasts transformedby H-RasG12V (EJ cells) and the other is A549 cellsexpressing the oncogenic K-RasG12V Ras (Fig. 9a).The results obtained with these cell lines were similarto those obtained with U87 cells. Thus, in both EJ andA549 cells, the fast release fibers exhibited a strongergrowth inhibition effect near the fiber than at agreater distance from the fiber. Here too a change incell morphology was demonstrated at FTS concentra-tions, which are lower than those that inhibit cellgrowth. It should be noted that the FTS release fibershad a stronger effect on A549 than on EJ and U87cells, as may be inferred by the cell count (Fig. 9b).Both the fast and slow release fiber wells exhibited asignificantly lower cell count after 8 days of incuba-tion compared to the control.

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SUMMARY AND CONCLUSIONS

In this study, we developed and studied bioresorbablecore/shell fiber structures loaded with antiprolifera-tive agents. These structures were composed of apolyglyconate core and a porous PDLGA shell loadedwith paclitaxel or FTS, prepared using freeze dryingof inverted emulsions. In addition to the usage of theshell as coating for stents, the entire unique core/shellfiber platform can be used as a basic element ofbioresorbable vascular stents as well as for localtreatment of cancer. Longer periods of drug releaseare probably favorable for the latter application.

In general, the porous shell structures (porosity of67–85% and pore size of 2–7 mm) exhibited a relativelyfast FTS release within several weeks and a slowerpaclitaxel release within several months. The co-polymer composition was found to be the mostimportant parameter affecting release behavior in


148 KRAITZER ET AL.

our systems. Its direct effect can be described asfollows: an increase in the GA content of the PDLGAcopolymer resulted in an increase in the burst effectand release rate of FTS during the first 2 weeks,mainly due to higher water uptake, swelling, andchanges in microstructure. Higher GA also enabledfaster paclitaxel release, mainly due to a fasterdegradation rate of the host polymer. In addition, anindirect effect of the microstructure on the releaseprofile occurs via an emulsion stability mechanism,that is, a higher diffusion rate of the hydrophobicantiproliferative agents can be achieved when highporosity is combined with a fine structure of lowerpore size. The direct effect is more significant than theindirect effect.

The FTS-eluting fibers exhibited a concentrationgradient effect in the treatment wells of all tested celllines, namely U87, EJ, and A549, as indicated bythe gradiential increase in cell density with theincrease in distance from the fiber. When thecumulative release of FTS reached high FTS con-centrations, induced cell death was achieved in A549and U87 cells. However, a lower cumulative FTSrelease-induced growth inhibition as well as a changein cell morphology. This is in agreement withprevious reports, which demonstrated that a changein cell morphology occurs already at low FTSconcentrations. FTS-eluting composite fibers provedto effectively induce growth inhibition or cell death bya gradient effect and a dose-dependent manner. Thecombined effect of the targeted mechanism of FTSas a Ras inhibitor together with the localized andcontrolled release characteristics of fiber is anadvantageous antiproliferative quality.

ACKNOWLEDGMENTS

The authors are grateful to the Israel Science Foun-dation (ISF, grant number 1312/07) and to the SlezakFoundation, Tel-Aviv University, for supporting thisresearch. We would like to thank Concordia Pharma-ceuticals for kindly providing us with farnesylthiosa-licylate (FTS, Salirasib). We would like to thank AnatKantarovich, and Shani Katz for the drug releasemeasurements.

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