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Bovine Serum Albumin-Based Magnetic Nanocarrier for MRI Diagnosis and Hyperthermic Therapy: A Potential Theranostic Approach Against Cancer

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Bovine Serum Albumin-Based Magnetic Nanocarrier for MRI Diagnosis and Hyperthermic Therapy: A Potential Theranostic Approach Against Cancer
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  Nanomedicine Bovine Serum Albumin-Based Magnetic Nanocarrier for MRI Diagnosis and Hyperthermic Therapy: A PotentialTheranostic Approach Against Cancer** Mauro Comes Franchini,*  Giovanni Baldi,*  Daniele Bonacchi, Denis Gentili,Guido Giudetti, Alessandro Lascialfari,*  Maurizio Corti, Patrick Marmorato, Jessica Ponti, Edoardo Micotti, Uliano Guerrini, Luigi Sironi, Paolo Gelosa,Costanza Ravagli, and Alfredo Ricci  After the 2005 milestone in clinical application of paclitaxel–albumin nanoparticles (NPs) for the treatment of metastaticbreast cancer, [1] albumin NPs have been thoroughly investi-gatedaseffectivedeliverysystemsinnanomedicine. [2] Thishasled to increasing interest in the use of bovine serum albumin(BSA) as nanocarrier for applications in nanomedicine, [3a–c] duetothepossibilitytocoatlipophilicNPsandbecauseitcanbeconsidered a crucial step before moving to its human version,human serum albumin (HSA). [3d–f] The scientific communityis seeking to exploit the intrinsic properties of magneticnanoparticles (MNPs) to obtain medical breakthroughs indiagnosisandtherapy.OneofthemainadvantagesofmagneticNPs is that they can be visualized acting as magnetic contrastagents(CA)formagneticresonanceimaging(MRI).Heatedina high-frequency magnetic field they trigger drug release orproducehyperthermia/ablationoftissues,currentlyreportedasmagnetic fluid hyperthermia (MFH). Accordingly, the termtheranostic nanomedicine has been defined as an integratednanotherapeutic system, which candiagnose, provide targetedtherapy and monitor the response to therapy. [4] In particular,oxide-basedspinelferrites [5] areconsideredverypromisingforMRIandforhyperthermictreatmentandironoxidesaregoodcandidates thanks to their well-known biocompatibility. Theuse of different materials with larger magnetic anisotropy andlarger magnetic moment is envisaged since it could allow asignificantimprovementofthematerialefficiencyforMRIandMFH. For example, spinel cobalt ferrite NPs (CoFe 2 O 4 ) havealready been proposed for biomedical applications. [6,7] Sincethe magnetocrystalline anisotropy is strictly connected tothehyperthermicefficiency, [8] theuseofthehighlyanisotropicCoFe 2 O 4 couldleadtogreatlyimprovedhyperthermicproper-ties, allowing the introduction of several benefits in suchbiomedical applications as, for example, the reduction of theinorganic dose. The magnetic properties of iron and CoFe 2 O 4 as hyperthermic agents have been recently compared. [9] Gazeau et al. showed for the first time that quantitativemagnetic hyperthermia data could fit the theoretical predic-tions based on relevant parameters such as particle size,material viscosity, and field characteristics.Despite the interesting perspectives, biomedical applica-tions of MFH have been scarcely reported. Kobayashi andco-workers [10] showed that magnetite nanoparticle-loadedanti-HER2 immunoliposomes under an alternating magneticfield, resulted in a strong cytotoxic effect in vitro on SKBr3breast-cancer cells. The hyperthermia, generated by communications [  ] Dr. M. Comes Franchini, Prof. A. Ricci, D. GentiliDipartimento di Chimica Organica A. ManginiViale Risorgimento 4, 40136 Bologna (Italy)E-mail: mauro.comesfranchini@unibo.itDr. G. Baldi, Dr. D. Bonacchi, Dr. C. RavagliCERICOL, ColorobbiaVia Pietramarina 123, 50053 Sovigliana, Firenze (Italy)E-mail: baldig@colorobbia.itProf. A. LascialfariDipartimento di Scienze Biomolecolariapplicate ai BiosistemiUniversita` degli studi di Milano20134 Milano (Italy)E-mail: lascialfari@fisicavolta.unipv.itProf. M. Corti, Dr. E. MicottiDipartimento di Fisica A. VoltaUniversita` degli studi di Pavia27100 Pavia (Italy)E-mail: corti@fisicavolta.unipv.itDr. U. Guerrini, Dr. L. Sironi, Dr. P. GelosaCentro Cardiologico Monzino IRCCSvia Parea 4, 20138Dipartimento di Scienze FarmacologicheUniversita` di Milanovia Balzaretti 9, 20134 Milano (Italy)Dr. J. Ponti, Dr. P. Marmorato, Dr. G. GiudettiEuropean Commission, Joint Research CentreInstitute for Health and Consumer ProtectionNanobiosciencesvia Fermi 2749, 21027 ISPRA (Va), TP 203 (Italy)[  ] Financial support (Seventh Framework Programme, grant agree-ment number CP-IP 213631-2 NANOTHER) from the EuropeanCommission is gratefully acknowledged. :  Supporting Information is available on the WWW under http://www.small-journal.com or from the author. DOI: 10.1002/smll.200901689  366    2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  small  2010,  6,  No. 3, 366–370  submission of an aqueous suspension of   g -Mn  x Fe 2–  x O 3  usingthe polymer Acrypol 934 to an alternating magnetic field,caused HeLa cell death [11] proportional to the quantity of theparticlesandtothedurationofapplicationofthemagneticfield.Very recently, Cheon demonstrated that Zn-doped metalferrite NPs display values of contrast and hyperthermic effectmuch higher than conventional NP agents such as Feridex. [12] Having recently reported [13] monodisperse and stablelipophilic CoFe 2 O 4  NPs as a first step toward obtaining novelsystems for biomagnetic applications, we now suggest a novelBSA-based nanocarrier containing CoFe 2 O 4  NPs as a promis-ing nanosystem to perform theranostic treatment. In this workwe performed MRI in brain and liver of normal rats andhyperthermic treatments in standard human tumor cell lineHela cells.CoFe 2 O 4  NPs with an average size of about 6.7nm asmeasured by X-ray diffraction (XRD), were synthesizedaccording [13] to our polyol method in diethylene glycol(DEG). The XRD diffractograms were analysed by theScherrer formula. The size distribution was also assessed bydynamic light scattering (DLS), showing a uniform sizedistribution with average diameter of 22.3nm and withpolydispersity index (PDI) of 0.063. The magnetostaticproperties of the as-synthesized CoFe 2 O 4  particles weredefined by magnetization measurements (see SupportingInformation); the sample showed a saturation magnetizationvalue( M  s )at300Kof78AmKg  2 andablockingtemperature( T  B ) of 298K. It is worth noting that at  T  ¼ 300K  > T  B  thesample shows the typical behavior of superparamagneticmaterials with neither coercivity nor remanence. Thanks tothe large value [14] of the anisotropy constant of cobalt ferriteNPs (K  1.2MJm  3 ), even for small NPs the magnetizationrelaxation time is greatly increased, in such a way that the  T  B nearlyreachesroomtemperature.TheCoFe 2 O 4 NPswerethenstabilized by capping the surface with a hydroxamic acid, ethyl12-(hydroxyamino)-12-oxododecanoate(EHO) 1 , [15] tohomo-geneously disperse them in acetone (Scheme 1).DLS analysis showed a uniform size distribution of CoFe 2 O 4 - 1  with an average diameter of 26.3nm and with aPDI of 0.219 (see Supporting Information). The XRD patternrevealed that the srcinal crystallite size was preserved duringfunctionalization. In addition, the particles’ main magneticcharacteristics were found to be unaffected by the superficialcoating,thussuggestingthatthechemicalstateofsurfaceionsisnot significantly modified.Later, we prepared the nanocarrier BSA-CoFe 2 O 4 - 1  inacetone with poly(lactic-co-glycolic acid) (PLGA) and BSAaccordingtoScheme1,usingPLGAforthecoatingofmagneticNPs,asalreadyreported. [16] DLSanalysisrevealed(Figure1)auniform distribution of NPs with narrow size distribution(PDI ¼ 0.135) and average diameter of 106.0nm of the BSA-CoFe 2 O 4 - 1 . In addition, scanning transmission electronmicroscopy (STEM) images confirmed DLS investigationsshowing cobalt ferrite inside the nanocarrier. Interestingly,based on the low PDI value, we excluded any aggregationphenomenon of hybrid particles inside the suspension.Elemental analysis assessed energy-dispersive X-ray spectros-copy (EDS) on BSA-CoFe 2 O 4 - 1  agglomerates revealed ironand cobalt presence confirming the effective entrapment of magnetic particles.Surface charge characterization, by Zeta potential mea-surement, has been also performed. The starting coated cobaltferrite NPs present a positive value of   þ 20.0-30.0mVdepending on the solvent (ethanol or water) [15b] while aPLGA solution in acetone dropwise added into water gave azeta potential of   24.0mV. The value obtained for the PLGAgrafted onto the inorganic particles CoFe 2 O 4 - 1  nanoprecipi-tated in water, without addition of the BSA, was þ 25.2mV atpH ¼ 7.0 indicating that the nanoparticles are in the outersurface of the negatively charged PLGA. On the other hand,once prepared and purified, the whole nanocarrier BSA-CoFe 2 O 4 - 1 ,gaveazetapotentialof   23.6mV(BSAalonegavea value of   19.1mV), thus indicating that the coated NPs andPLGA are surrounded by the BSA, which indeed act as astabilizer. These results led to the conclusion that electrostaticforcesbindtheproteinonthepolymershell. [17] Cobaltleakageof BSA–CoFe 2 O 4 - 1  was tested after two months by precipitat-ing the particles with a CaCl 2  solution 0.5% w/w and analyzingthe cobalt content of the supernatant solution by ion-coupledplasma atomic emission spectroscopy (ICP-AES). The cobaltsignalwasbelowtheinstrumentalsensitivity,indicatingthatnocobaltleakageoccursafterparticleinclusioninBSA-CoFe 2 O 4 - 1 .Thedispersionstabilityinbuffersolutionandinserum,testedby DLS, indicates only a slightly variation in the sizedistribution (see Supporting Information). Based on theseresults we concluded that the suspension was stable for at leasttwo months. Scheme 1.  CappingofCoFe 2 O 4 withEHO 1 andsynthesisofthemagneticnanocarrier BSA-CoFe 2 O 4 - 1 . Figure 1.  STEM and DLS of BSA-CoFe 2 O 4 - 1 . small  2010,  6,  No. 3, 366–370    2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  www.small-journal.com  367  For the evaluation of the effectiveness of the diagnosticabilities we investigated the typical MRI quantities, that is, thenuclear relaxivities [18] r  1  (longitudinal) and  r  2  (transverse). InFigure2wereport  r  1 and r  2  dataonourbiocompatiblesamplescompared to the commercial Endorem (Feridex in the USA).From Figure 2b it can be deduced that in the compound BSA-CoFe 2 O 4 - 1  r  1  is slightly lower and  r  2  is approximately doublethanthecorrespondingquantitiesofEndorem.ThismeansthanBSA-CoFe 2 O 4 - 1  is a very good negative CA.To assess the real efficiency of BSA-CoFe 2 O 4 - 1  in terms of image contrast at low frequencies (  8MHz), we performedsometests byplacing intheArtoscan Imager4vials containingBSA-CoFe 2 O 4 - 1 andEndorematthesametwoconcentrations, c .AscanbeseenfromFigure3intheimagesobtainedbymeansof gradient echo (GE) and spin echo (SE) sequences (seeSupportingInformation),thevialscontainingBSA-CoFe 2 O 4 - 1 at  c 1 ¼ 0.03125mgmL  1 appear much darker than Endorem atthe same concentration, while for  c 2 ¼ 0.0625mgmL  1 Endorem is still brilliant and BSA-CoFe 2 O 4 - 1  shows no signal(void in the left upper part of the image). The SE sequenceconfirmed this conclusion.By means of animager operating at 4.7Tesla (Avance-200,see previous paragraph), we collected in vivo images of brain(notshown)andliverofnormalrats,atdifferenttimesfromthebolus injection of Endorem and/or BSA-CoFe 2 O 4 - 1 . Theimages at different times refer to slices in the same spatialpositions. In Figure 4 we report the images corresponding toliver before injection and at  t  ¼ 0, 1, and 6 days. As is wellknown, [19] Endorem (Figure 4, left) tends to accumulate in theliver and stay there for some days before complete washout.Very interestingly, BSA-CoFe 2 O 4 - 1  (Figure 4, right) showssimilarorslightlybetterresultsthanEndorem,givingdarkwideregions where it is delivered. Also the time behavior of BSA-CoFe 2 O 4 - 1  is similar to Endorem, whereas for much longertimes the definition and the extension of the regions with pooror no signal is better for BSA-CoFe 2 O 4 - 1 .As far as the hyperthermic behavior was concerned wemeasured the specific loss power (SLP) value of the startingmagnetic material in diethylene glycol and the final BSA-CoFe 2 O 4 - 1  in water under the same irradiating condition. Wemeasured the heating rate by calculating the slope of thethermalprofileat t  ¼ 0.IntheformulausedtoevaluateSLP,  m is the mass in grams of the different materials (solvent, cobaltferrite, etc.), c pi  theirspecificheatcapacity (inJ/g  K)and  m Me is the total amount of metal in the sample. SLP ¼ P i  m i C   pi m Me  D T  = D t   (1) The values measured at a frequency of 168kHz and with afieldintensityof21kAm  1 are238Wattg  1 ,215Wattg  1 ,and215Wattg  1 forthenakedcobaltferriteparticlesindiethyleneglycol, the EHO- 1 -functionalised cobalt ferrite in  n -butanol,and the final nanocarrier BSA-CoFe 2 O 4 - 1  in water, respec-tively. The minor decrease observed after nanoparticlefunctionalization can be addressed mainly to solvent conduct-ibility effects rather than to the surface effect induced by thefunctionalization, as suggested by magnetic measurements, orto the increase of the particles’ hydrodynamic radius; howevertheSLPdiscrepancieswerewithinexperimentalerror( þ 10%),thus confirming that any unhomogeneity of magnetic particleencapsulation did not affect the average properties of thesamples. Consequently, to evaluate thetoxicological profile that could affect thepotential therapeutic effects of BSA-CoFe 2 O 4 - 1 ,wedecided touseHeLa cancercells.Theevaluationofthecytotoxicitywasdetermined using the standard XTT test(see Supporting Information). First of allthepersetoxicitywasevaluatedfor0.169%(1.69mgmL  1 of cobalt ferrite) of BSA-CoFe 2 O 4 - 1  for a treatment period of 4h.Data revealed that under this experimentalcondition, approximately 36% of the cellsweredeadduetotheNPtoxicity(FigureS3of Supporting Information). This 4h incu-bation time is the final time point, afterseveral preliminary experiments, for whichthe BSA-CoFe 2 O 4 - 1  incubation triggered atoxicitytobeusedforthefurtherirradiationstep. Finally, the final design of the communications Figure 2.  Relaxivities r  1 (a)and r  2 (b)ofbiocompatibleliquidnanocarrier BSA-CoFe 2 O 4 - 1 , compared with commercial compound Endorem. Figure 3.  MRI images of vials containing BSA-CoFe 2 O 4 - 1  and Endorem at differentconcentrationsofmagneticcentre,obtainedwiththeArtoscanImager(  H ¼ 0.2T);sequencesareGE-PDW (a) and SE-PDW (b).  368  www.small-journal.com    2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  small  2010,  6,  No. 3, 366–370  experimental procedure, BSA-CoFe 2 O 4 - 1  treatment andirradiation steps, that was applied to the all cells batches wasthe following: the petri dishes were placed at the center of thecoil of the high-frequency magnetic field (168kHz, 21kAm  1 )and cells were treated with NPs for 4h and then irradiated for1h.AftertheincubationthecellswerewashedthreetimeswithHank’s buffer solution and their viability was measured after24h.Analysisofthedatarevealedthatbyaddingtheirradiationstep, the cell mortality increased up to 82% compared to the36% previously found without irradiation (see SupportingInformation). It is likely that the hyperthermic effect of BSA-CoFe 2 O 4 - 1  took place in conjunction with the toxic effect atthat concentration of the cobalt ferrite NPs, or possibly to theCo 2 þ ions leaching, as observed in the not-irradiated experi-ment, resulting in a significant increase of cell mortality. In thecase of lower concentration (0.84mgmL  1 of cobalt ferrite)thereisnoevidenceofanyhyperthermiceffectwhileinthecaseof middle concentration (0.42mgmL  1 ) the toxicity effect of nanoparticles (or better, the error associated with themeasurement masks a possible hyperthermic effect. On theother hand, as a control experiment, irradiation of the HeLacells with the same magnetic field and time used for the BSA-CoFe 2 O 4 - 1  did not show any cell mortality. Therefore, asconfirmed by other authors, both in vitro and in vivo, we couldexcludethepossibility oftoxicityinducedbythemagneticfielditself. [20] In addition, to verify that the NPs interacted withHeLa cells BSA-CoFe 2 O 4 - 1  was labeled with the fluorescentdye BODIPY 505/515 (see Supporting Information). Underourexperimental conditions, we observedNP interaction after4h of incubation in 0.23mgmL  1 , but the fluorescence signalwasnotstrongenoughtoberecordedbyimaginganalysis.After24h the cellular interaction was well defined. In comparisonwith negative and filtrate controls (see SupportingInformation), in which no green spot was visible, we observedgreen fluorescent nanoparticles mainly in the peri-nuclearregion of the exposed cells. It is possible to hypothesise, on thebase of the distribution and shape of the fluorescence signal,that the nanoparticles could be associated with vesicular-likestructures such as endosome- or lysosome-like vesicles, inagreement with previous observations of the uptake of magnetic or gold nanoparti-cles in in vitro models. [21] Moreover, toclearlyverifythattheirradiationinducedanincrease in the in situ temperature, theheatingprofileofthepetridishwascarefullymeasured with a FLIR E65 thermocameraand a maximum temperature of 48 8 Crecorded. Other concentrations gave lowervalues of cell inhibition (32% and 9% for0.84 and 0.42mgmL  1 of Co, repsectively),thusconfirmingthatfurtherinvestigationtofind the right concentration of the nanocar-rier combined with the mode of irradiationis needed.In conclusion, experimental results forour theranostic nanomedicine BSA-CoFe 2 O 4 - 1  led to the following considera-tions: from the relaxometry data thetransverse  r  2  relaxivity of BSA-CoFe 2 O 4 - 1 ishigherthanEndorem,suggestingourcompoundwouldmakeagoodnegativeMRIcontrastagent.MRIexperimentsonvialsatlowfield, H  ¼ 0.2T,confirmedthatBSA-CoFe 2 O 4 - 1 isbetterthan Endorem in the negative contrast of the images, whileMRI experiments in vivo showed that BSA-CoFe 2 O 4 - 1  andEndorem have very similar efficiency in contrasting images of liver atshort times frombolus injection ofCA. Withthe aim toemploy these nanocarriers as heating mediators in biomedicalapplications, our highly anisotropic materials based on Co,according to these results, allow enhanced hyperthermicefficiency even with reduced fields and frequencies applied. [22] Concerning the destruction of cancer cells, although theseresults must be treated with caution, there is clear evidence of the therapeutic potential of BSA-CoFe 2 O 4 - 1  and it is useful torealisethatasignificantchallengewhenworkingattheinterfacebetween biology and engineering is to ascertain the toxicologyand biocompatibility profiles of the materials being used. Keywords: cancer  . cobalt ferrite . hydroxamic acids . hyperthermictherapy . magnetic nanoparticles [1] a)N. K.Ibrahim,N.Desai,S.Legha,P.Soon-Shiong,R.L.Theriault,E. Rivera, B. Esmaeli, S. E. Ring, A. Bedikian, G. N. Hortobagyi, J. A. Ellerhorst,  Clin. Cancer Res.  2002 ,  8 , 1038–1044;b) S. Rhaese, H. vonBriesen, H. Rubsamen-Waigman, J. Kreuter,H. Langer,  J. Controlled Release  2003 ,  92 , 199–208.[2] O.M.Koo,I.Rubinstein,H.Onyuksel, Nanomedicine 2005 , 1 ,193–212.[3] a) W. Jiang, S. P. Schwendeman,  Pharm. Res.  2001 ,  18 , 878–885;b) M. Jahanshahi, Z. Zhang, A. Lyddiatt,  IEE Proc. Nanobiotechnol. 2005 ,  152 , 121–126; c) M. Rahimnejad, M. Jahanshahi,G. D. Najafpour,  Afr. J. Biotechnol.  2006 ,  5 , 1918–1923;d) N. Shamim, L-Hong, K. Hidajat, M. S. Uddin,  J. Colloid InterfaceSci.  2006 ,  304 , 1–8; e) L. Xianqiao, M. D. Kaminski, H. Chen,M.Torno,L.Taylor,A.J.Rosengart,  J.ControlledRelease 2007 , 119 ,52–58; f) K. J. Widder, G. Flouret, A. E. Senyei,  J. Pharm. Sci.  1979 , 68 , 79–82. Figure 4.  Images of the liver at different times from the injection of Endorem (left) and BSA-CoFe 2 O 4 - 1 (right).a)Pre-CAinjection;b) t  ¼ 0;c) t  ¼ 1day;d) t  ¼ 6days.Theredarrowssignalroughly the region where Endorem or our compound reaches the liver. small  2010,  6,  No. 3, 366–370    2010 Wiley-VCH Verlag GmbH & Co. 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