RoumanianBiotechnologicalLetters Copyright2008BucharestUniversity RoumanianSocietyofBiologicalSciences

Vol.13,No.5,2008,pp.39073913 PrintedinRomania.Allrightsreserved ORIGINALPAPER

Thereleaseoflasparaginasefromhydrogelmagneticnanoparticles
Receivedforpublication, August22,2008 Accepted,September25,2008
1 1 2 EUGENIATEODOR ,GEORGIANATRUICA ANDIRINALUPESCU 1 National Institute for Biological SciencesCentre of Bioanalysis, Bucharest 6, 296, Spl. Independentei 2CentreofMicrobialBiotechnology BIOTEHGEN, Bucharest1,59,Marasti Correspondingauthor:tel/fax:+4021.2200900email:eu_didu@yahoo.com

Abstract
Lasparaginasewasimmobilizedbyentrapmentinhydrogelmagneticnanoparticlesobtained from magnetic nanoparticles and successive layers of chitosan and hyaluronic acid. The obtained nanostructures, with dimensions suitable for penetration cell/tissues, especially tumor tissues, were evaluated for swelling behavior, degradation behavior and the release of Lasparaginase in neutral mediaandslightacidmedia(pH4.5).Theexperimentsshowedagoodswellingbehaviorforallthe tested samples, but a high stability of nanostructures. Lasparaginase has an initial burst from hydrogel,attwohours,andothertworeleasesat24and between50and72hours.

Keywords:hydrogelmagneticnanoparticles,Lasparaginaserelease,drugdelivery.

Introduction
Lasparaginase (Lasparagine amidohydrolase (E.C. 3.5.1.1.) is a chemotherapy agent,usedintreatmentofacutelymphoblasticleukemia(ALL),butinsomecasesitpresents toxicity, the main side effect is an allergic or hypersensitivity reaction. Enzyme immobilizationintoapolymericshellcouldimprovetheirdeliveryandeliminatetheallergic reaction[1]. ThemostextensivelystudiedasparaginaseistheEC2from E.coliwhichdiffers fromEC1byitsbroadpHactivityprofileanditshighersubstrateaffinity. In the last decade, nanosize materials have been widely used as support for immobilizationofproteins,peptides,enzymes[27]antibodiesandnucleicacids[6],because of their unique properties [27]. Among these materials, magnetic nanoparticles are very popularwhentheyareusedincombinationwithbiologicalmaterials.Magneticnanoparticles are employed as contrast agents for magnetic resonance imaging (MRI) and as carriers for drugdeliveryduetothemuniquemagneticproperties,lowtoxicityandtheabilitytofunction atthecellularandmolecularlevelofbiologicalinteractions. In previous works we presented the synthesis and characterization of hydrogel magnetic nanoparticles, biocompatible, capable to penetrate cell and tissues and to embed active principle (protease inhibitor, Lasparaginase) [8,9]. The obtained nanostructures are nanometric dimensions (below 30 nm) and could bind or load protease inhibitor and L asparaginase.InthisworkisevaluatedthereleaseofLasparaginasefromhydrogelmagnetic nanoparticleswithLasparaginaseentrapped.Thereleasewasevaluatedbyproteinassayand by Lasparaginaseactivityassay.

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Matherialsandmethods
SynthesisofhydrogelmagneticnanoparticleswithimmobilizedLasparaginase Waterdispersible magnetic nanoparticles were obtained using an adapted Massart method[10].The magnetic nanoparticles(MP)wereencapsulatedaccordingtoourprevious work [11], using layerbylayer technique in two different biopolymeric materials, chitosan (SIGMA) from crab shells (Chit.) and hyaluronic acid (HA) extracted by us from bovine vitreous [12]. Asparaginase was immobilized by entrapment in hydrogel layer of obtained nanostructures. Lasparaginase was obtained by biosynthesis from Escherichia coli using a recombinantstrainofE.coliwithimprovedcapacityofproducingisoenzymeEC2withanti tumor activity (kindly supplied by BIOTEHGENCentre of Microbial Biotechnologies, Bucharest) [13]. The synthesized hydrogelmagnetic nanoparticles were characterized in a previous work using DLS (Dynamic Light Scattering), TEM (Transmission Electron Microscopy) and FTIR (FourierTransformed Infrared) spectroscopy [9]. From our previous studies,thesynthesizedhydrogelmagneticnanoparticleswithLasparaginaseentrappedhave dimensionsbelow30nm,arebiocompatibleandrelativeinerttomicroorganisms. Swellingtest Theobtained hydrogelmagneticnanoparticles(~10mgdryweight)weresuspended 0 intubescontaining50mLofPBS(pH7.2at20 C).Atanappropriatetimeinterval,theexcess buffer was removed carefully using a magnetic separator and the hydrogelmagnetic nanoparticleswereweightedimmediately.Theswellingcapacitywascalculatedaccordingto thefollowingequation[14]: m - m i % S = w x 100, (1) m i where %S is swelling ratio, mw is the weight of samples after immersion and mi is initialweight. Invitrodegradationstudy HydrogelmagneticnanoparticlesweresuspendedintoFalcontubescontaining50mL 0 PBS (pH 7.2 at 37 C). At predetermining time points, hydrogels were collected with a magneticseparatorandexcessbufferwasremovedfromthetubes.Sampleswereweightedby usingananalyticalbalancewith0.1mgaccuracy.Aftertheequilibrationtimeofswellingin PBS,thedegradationratiowascalculatedaccordingtothefollowingequations[15]: m - m t % D= 0 x 100, (2) m 0 where %D represent degradation ratio, m0 is the original weight after equilibration timeofswellinginPBSandmt istheweightattimet. Asparaginasereleasestudies 0.2 g of the asparaginasehydrogelmagnetic nanoparticles were immersed in 1 mL 0 phosphatebufferedsolutionandkeptat37 Cwithoutagitationforvarioustimeperiods.The bufferedsolutionsusedwereaphosphatebufferedsalinesolutionofpH7.00andaphosphate buffer of pH 4.5. At the end of each immersion period, 1mL of the buffered solution was removed and analyzed for asparaginase activity and protein content (Bradford, [16]), spectrophotometrically on a Jasco UVVis spectrophotometer. The amount of BSA was 2 determinedusingastandardcurve,(m=0.005,R =0.995). An identical volumeof freshphosphatebuffered saline solutionwasaddedback into thecontaineraftereachsampleremoval. Asparaginaseassay
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TheresidualactivityoftheimmobilizedandreleasedLasparaginasewasdetermined spectrometricallyat 450 nm with Nessler reagent method (essentially thatof Mashburn and Wriston, 1963) where the rate of hydrolysis of asparagine is determined by measuring released ammonia [17], spectrophotometrically on a Jasco UVVis spectrophotometer. One unit releases one micromole of ammonia per minute at 37C and pH 5 under the specified conditions). The amount of ammonia was determined using a standard curve, (m=0.182, 2 R =0.998).

Resultsanddiscussions
The hydrogelmagnetic nanoparticles were obtained from magnetic particles covered with three successive layers of different proportions of chitosan and hyaluronic acid. According to our previous studies [8,9], the three layers of successive chitosan/hyaluronic acid/chitosanensurethefinalnanometricdimensionsandstabilityofobtainednanostructures (see table 1). Lasparaginase was immobilized by entrapment in hyaluronic acid (middle) layerorinexternalchitosanlayer(table1).Thesynthesizedhydrogelmagneticnanoparticles with Lasparaginase entrapped have dimensions suitable for penetration cell or tissue, respectivelybelow300nminswelledstageandbelow30nmindriedstage,asitcouldseein figure1andintable1.Theobtainednanostructuresweretestedforswellingcapacity,figure 2,anddegradationbehavior,figure3,toevaluatethebehaviorofnanoparticlesinbiological fluids.The hydrogelmagnetic nanoparticles had agoodswellingcapacitytheyretain fluids up to 10 times their weight. The samples have similar composition, so they have a similar swellingbehavior(figure2).
Table1.CompositionandLasparaginaseresidualactivityofobtainednanostructures D(nm) Initial Samp (Swelled immobilization mmoliNH3 le SampleType(composition) Stage) /sample U/mL yield(%) MP 25.21 0 20mlMP +1.5mlChit. 349.5 +5mlHA(and5mg asparaginaseBIOTEHGEN) 320 1 +1.5mLChit. 0.450.34 1.51.13 43.02 274 20mlMP +3mlChit. 418.7 +3mlHA(and5mg asparaginaseBIOTEHGEN) 539.2 2 +3mlChit. 0.530.35 1.771.16 50.73 261.8 20mlMP +3mlChit. 768.2 +3mlHA +3mlChit.(and5mg 835.5 4 asparaginaseBIOTEHGEN) 1.130.95 3.763.17 90.76 324.9 20mlMP +1.5mlChit. 304.4 +3mlHA(and5mg asparaginaseBIOTEHGEN) 676.9 5 +1.5mLChit. 0.640.55 2.131.83 61.05 289.1

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Figure1 a)TEMimagesofuncovered(bare)MP(d=15.97nm)b)MPcoveredwithhydrogel(sample1) (d=23.29nm)

3

swellingcapacity(%)

2,5 2 1,5 1 0,5 0 0 50 100 150 200 250

hours

sample1

sample2

sample4

sample5

MP

Figure2. Swellingbehaviorofcoveredmagneticnanoparticles

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The degradation ofobtained nanostructures is insignificant.Apparent degradation in first23hoursisdeterminedbythereleaseofLasparaginase,asitcouldseeinfigure4.The 0 hydrogelmagnetic nanoparticles are stable in neutral media at 37 C, the degradation ratio being<1(figure3). ThestabilityofobtainednanostructureswasconfirmedbypreviousFTIR studies [9] the spectra obtained showed that between magnetic nanoparticles and hydrogel layersofchitosanandhyaluronicacidareformedhydrogenbonds(oreven covalentbonds).
1,6 1,4 1,2
%degradation

1 0,8 0,6 0,4 0,2 0 0 20 40 60 80 time(days) 100

MP

Figure3. Degradationbehaviorofhydrogelmagneticnanoparticles

ThereleaseofLaspraginasewastestedinneutralmedia,atpH7,forthecaseofusing nanoparticles in parenteral delivery and at pH 4.5 (considered the pH of tissues). The experiments were repeated two times, and the delivery of Lasparaginase was measured by two methods (protein assay and Lasparaginase activity assay). The results were similar. In figures 4 and 5 are presented results obtained by protein assay (the method is shorter and easier, so is recommended instead of enzyme activity assay). All the samples, showed a similar release of Lasparaginase. As it could see in figure 4, at pH 7, the enzyme has an initial, major burst after two hours and a second release after 24 hours and a final smaller releasebetween50and72hours. AtpH4.5,thebehaviorofsamplesisdifferenttheenzyme is released slowly and in reduced quantities (figure 5).All the samples obtained present L asparaginaseactivity,butsamples4and5hadthebetterimmobilization yieldandthebetter stabilityatstoring.Sample4,which hastheenzyme inexternal layerof hydrogelpresented thebestLasparaginaseactivity,evenafter6monthssinceimmobilization.
14 12 10 4 5 1 2

mgprot/mL

8 6 4 2 0 2 0 20 40 60 80 100

time(hours),pH7

Figure4. ReleaseprofileofLasparaginasedeterminedatpH7byproteincontent 3911

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4,5 4 3,5 3

1 4

2 5

mgprot/mL

2,5 2

1,5 1

0,5 0 0,5 0 1 10 20 30 40 50 60 70 80 90

time(hours),pH4.5

Figure5. ReleaseprofileofLasparaginasedeterminedatpH4.5byproteincontent

Magnetic nanoparticles continue to receive attention from scientists. Recent studies describeanapproachtosynthesizeferrogelswithaprecisecontroloftheopeningandclosure of pore configuration, which allows a burst release or norelease action of therapeutically active agent to be controlled externally and magnetically [18]. This type of ferrogel can be consideredasaclassofnovelmagneticallytunabledrugdeliverysystem.Nanomedicinesare definedasdeliverysystemsinthenanometersizerange(preferably1to100nm)containing encapsulated,dispersed,adsorbed,orconjugateddrugsandimagingagents[19]. The hydrogelmagnetic nanoparticles with Lasparaginase entrapped previously obtainedbyus,havesizesbelow300nminswelledstage, andbelow30nmindriedstageand are capable to penetrate cells and tissues (especially tumortissues,which exhibit a vascular porecutoffsizebetween380and780nm[20]).Theresidualactivityofthe immobilized L 0 asparaginase remains the same after 6 months from immobilization, stored at 4 C. The immobilization yield of the Lasparaginase in different samples was between 4390%. The obtainednanostructuresarebiocompatibleandcouldbeused fordeliveryofLasparaginase. The release of Lasparaginase from hydrogelmagnetic nanoparticles is greatly longlasting compare with administration of free enzyme. In neutral conditions, Lasparaginase has an initialburstfromhydrogel,attwohours,andothertworeleasesat24andbetween50and72 hours.

Conclusions
The hydrogelmagnetic nanoparticles were obtained from magnetic particles covered with three successive layers of different proportions of chitosan and hyaluronic acid. L asparaginasewasimmobilizedbyentrapmentinhyaluronicacid(middle)layerorinexternal chitosan layer. The obtained nanostructures, with dimensions suitable for penetration cell/tissues, especially tumor tissues, were evaluated for swelling behavior, degradation behavior and the release of Lasparaginase in neutral media and slight acid media (pH 4.5). The experiments showed a good swelling behavior for all the tested samples, but a high stability of nanostructures. Lasparaginase has an initial burst from hydrogel, at two hours, andothertworeleasesat24andbetween50and72hours. Theseresultsarepromisingforusingtheobtainedhydrogelmagneticnanoparticlesfor deliveryforawideclassoftherapeuticagents.

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AKNOWLEDGMENT:WorksupportedbytheNationalResearchandDevelopmentAgency ofRomania,theProgramofExcellenceinResearch(No.129/2006CEEX).

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