Magnetic Nanoparticles with Covalently Bound Self-AssembledProtein Corona for Advanced Biomedical ApplicationsRina Venerando,† Giovanni Miotto,† Massimiliano Magro,‡,§ Marco Dallan,‡ Davide Baratella,‡

Emanuela Bonaiuto,† Radek Zboril,§ and Fabio Vianello*,‡,§

†Department of Molecular Medicine, University of Padua, 35121 Padua, Italy‡Department of Comparative Biomedicine and Food Science, University of Padua

, 35020 Padua, Italy§Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University in Olomouc, 77147 Olomouc, Czech Republic

*S Supporting Information

ABSTRACT: Novel surface active maghemite nanoparticles (SAMNs) possess-ing peculiar colloidal properties and surface characteristics are able to covalentlybind biomolecules. The interactions of SAMNs and rhodamine derivatizedSAMNs (SAMN@RITC) with proteins from cell culture medium were studiedby gel electrophoresis and mass spectrometry. Among the 3000 proteins presentin fetal calf serum, SAMNs and SAMN@RITC give rise to the formation of aself-assembled corona shell with 22 selected proteins, representing minor plasmaproteins, among which α-2-HS- glycoprotein stands out. Bovine serum albumin(BSA), representing 80% of the total serum proteins, shows negligible absorptionon the SAMN surface. Nevertheless, SAMNs are able to bind BSA, uponincubation in pure BSA solutions. The interaction between SAMNs and BSA wasinvestigated by optical spectroscopy, circular dichroism, Fourier transforminfrared spectroscopy, and transmission electron microscopy. BSA bindingresulted a time-consuming process, nevertheless experimental results showed the interaction of 6 ± 2 BSA molecules pernanoparticle, and optical spectra indicate remarkable changes in SAMN optical features, as well as circular dichroism provedsecondary structure alteration of bound BSA, suggesting that the protein needs to adapt its structure to adhere to nanoparticlesurface. The selectively bound protein corona shell, formed upon SAMNs incubation in calf serum, was responsible for thecharacteristic behavior when SAMNs were tested for cell internalization and cytotoxicity on HeLa cells. Cytotoxicity of SAMNpreparations was extensively studied, and was negligible up to 100 μg mL−1. Moreover, nanoparticle uptake proceeded for longtimes, suggesting a correlation between internalization and stability of covalently bound self-assembled protein corona,representing a unique example of magnetic nanoparticle opsonization via covalent binding. We suggest that SAMN basednanobiocomposites can be employed for the preparation of self-assembled opsonized nanoparticles as future candidates forbiomedical applications.

1. INTRODUCTION

Many different kinds of magnetic nanoparticles have alreadydemonstrated their potential in biomedical applications,1,2 asthese nanostructures properly labeled with bioactive moleculescan serve in magnetic separations,3−5 drug delivery systems,6 orto generate heat by exposition to an alternating electromagneticfield, thus increasing the temperature of tumor tissues anddestroying pathological cells.7 Moreover, their use in magneticresonance imaging as contrast agents is common,8,9 owing totheir unique magnetic properties and biocompatibility,10 andcommercial preparations are available (Nanocs Inc., New York,NY; Nanoimmunotech SL, Vigo, Spain; MK Impex Corp.,Missisauga, ON, Canada; and many others). However, for mostiron oxide nanoparticles, little is known about their potentialadverse effects on health due to prolonged exposure inbiological systems, representing a hindrance to the develop-

ment of novel applications of magnetic nanoparticles innanobiology, nanomedicine, and nanotoxicology. The meta-bolic and immunological responses induced by these particleshave been rarely understood so far.11

An essential prerequisite for the implementation ofbionanotechnological applications is to obtain nanoparticleswith a hydrophilic surface able to maintain colloidal stabilityunder physiological conditions.12 Among magnetic nano-particles, magnetite (Fe3O4) appears to be an interestingcandidate, owing to its low toxicity, high saturation magnet-ization, and susceptibility. Unfortunately, upon exposure tophysiological environments, magnetite nanoparticles exhibit a

Received: July 10, 2013Revised: August 24, 2013

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tendency to aggregate, which considerably reduces theirefficiency, and more importantly, the magnetite surface tendsto oxidize, modifying its properties. Alternatively, maghemitenanoparticles appear very promising in theranostic applica-tions.13 However, obtaining monodisperse, colloidal suspen-sions of maghemite nanoparticles in aqueous media is achallenging task, due to the complexity of controlling thenucleation and growth processes in water.Recently, we developed a new simple wet method to

synthesize superparamagnetic nanoparticles constituted ofstoichiometric maghemite (γ-Fe2O3) in the dimension rangearound 10 nm, obtaining monodisperse preparations inaqueous media.14,15 This new category of magnetic nano-particles was called “surface active maghemite nanoparticles”(SAMNs), due to their specific surface chemical behavior,without any superficial modification or coating derivatization.They are freely stable in water for several months as colloidalsuspensions, present a high average magnetic moment and canbe easily derivatized to immobilize specific organic molecules insolution. SAMNs were already functionalized with ligands ofhigh biotechnological interest, such as biotin and avidin, bysimple incubation in aqueous solution.3 Nevertheless, the studyof SAMN interactions with proteins is still meaningful, andtheir effect on protein function and structure is worth forfurther studies. Furthermore, the investigation of proteinadsorption and interactions at interfaces is important from amedical, biotechnological, and industrial point of view.16

The present report describes a detailed investigation onSAMNs binding to a model biological host, namely bovineserum albumin (BSA), leading to a stable complex (SAMN@BSA). BSA has been one of the most extensively studiedproteins, particularly for its structural homology with humanserum albumin.17,18 It is a single polypeptide chain protein of66 kDa, which is cross-linked by 17 disulfide bonds.19,20 TheSAMN@BSA complex was prepared and studied with differentspectroscopic technique and transmission electron microscopy.Therefore, the behavior of SAMNs was studied in physiologicalcell culture media, evidencing the high nanoparticle colloidalstability under physiological conditions. Protein absorption onSAMNs, mimicking in vivo opsonization, was analyzed by massspectrometry, showing that SAMNs interact selectively withdifferent minor serum proteins, among which α-2-HS-glycoprotein stands out. These results evidenced that SAMNsdisplay specific binding behavior toward protein molecules andthat their behavior in real systems is not easily predictable.Furthermore, we studied SAMNs cytotoxicity and their effecton HeLa cells, demonstrating that they can be safely used atconcentrations up to 100 μg mL−1. Moreover, these uniquemaghemite nanoparticles, exhibiting excellent colloidal behaviorwithout any additional organic or inorganic surface coating,were exploited for the immobilization of rhodamine Bisothiocyanate (RITC) acting as a fluorescent label and, atthe same time, as spacer arm, useful to immobilizeenzymes.21,22 As a result, a magnetically drivable rhodamine-based fluorescent nanocomposite has been synthesized. In ourprevious works we demonstrated that SAMNs@RITCrepresents a friendly environment for immobilized enzymes,and we proposed this fluorescent magnetically drivablenanocarrier as an universal tool for creating fluorescentmagnetic drivable enzymatic nanosystems. We already reportedon two different enzymes used to prepare magnetic drivable,fluorescent nanocatalysts: bovine serum amine oxidase21 and

glucose oxidase.22 In both cases, immobilized enzymes on thenanomaterial retained their catalytic activity.In the present work, we combined the advantages of

immobilized fluorescent probes on nanomaterials and theeasy drivability of magnetic nanoparticles to propose a lowtoxicity system for cells, providing a novel bimodal nanodevice,which can be incorporated in cells, for future applications intargeted drug delivery and theranostics.

2. EXPERIMENTAL SECTION

2.1. Materials. Chemicals were purchased at the highestcommercially available purity and were used without furthertreatment. Iron(III) chloride hexahydrate (97%), sodiumborohydride (NaBH4), rhodamine B isothiocyanate (RITC),tetramethylammonium perchlorate, and ammonium hydroxidesolution (35% in water) were obtained from Sigma-Aldrich(Italy), as well as trypan blue, resazurin, and RNase. HeLa cellswere form ATCC (Middlesex, U.K.). Sterile filtered fetal bovineserum (FBS) was from Sigma-Aldrich (cod. F9665).Propidium iodide, tetramethyl-rhodamine, CM-H2DCFDA

(general oxidative stress indicator), MTT (3-[4,5-dimethylth-iazolyl-2]-2,5-diphenyltetrazolium bromide), penicillin (10 000U mL−1), streptomycin (10 000 μg mL−1), glutamine solutions,and acetomethoxy calcein (calcein AM) were from MolecularProbes-Invitrogen (U.K.). Buffers were prepared according tostandard laboratory procedures using Milli-Q reagent gradewater (Merck Millipore, Billerica, MA).Intrumentation and methods are reported in the Supporting

Information.

3. RESULTS

3.1. SAMN Surface Functionalization with BSA. SAMNsurface functionalization was accomplished with BSA by simpleincubation of reactants in 50 mM tetramethylammoniumperchlorate, at pH 7.0. In particular, SAMN colloidaldispersions (50 mg L−1) were incubated with BSA (100 mgL−1) under overnight end-over-end mixing at 4.0 °C. It shouldbe noticed that, in our previous work on SAMN@avidinpreparations, the binding process occurred within less than 1 h,preserving avidin biological activity upon immobilization onSAMN surface, and suggesting that protein tertiary structurewas unaltered.3 Conversely, in the present case, BSA bindingresulted negligible for short incubation times (1 h) and theincubation period was prolonged overnight. After theincubation period, nanoparticles were separated by theapplication of an external magnetic field and the presence ofthe biomolecule in the supernatant was checked byspectrophotometry. SAMN@BSA complex was magneticallyisolated and washed several times with 50 mM tetramethy-lammonium perchlorate, pH 7.0, and water. Biomoleculecoverage on SAMNs was stable, without any loss in solutionof BSA, as checked by spectrophotometry.UV−vis absorption is a simple and easily applicable

technique to explore structural changes on the surface ofnanomaterials and to prove complex formations.3 Theelectronic absorption spectrum of bare SAMNs, acquired inwater, shows a wide band with a maximum at about 400 nm(Figure S1 in the Supporting Information) characterized by anextinction coefficient of 1520 M−1 cm−1, expressed as Fe2O3molar concentration. The interaction of BSA with SAMNsurface produced an alteration of nanoparticle optical proper-ties (see Figure S1). Upon BSA binding, both the shape and

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position of maximum absorption were altered with respect tobare SAMNs, confirming the influence of SAMN surfaceproperties on optical characteristics, as already extensivelydescribed in our previous work.3 In water, the spectrum ofSAMN@BSA is characterized by a peak at 436 and a shoulderat 500 nm (see Figure S1).The amount of bound BSA was determined by measuring the

disappearance of unbound BSA in solution at 280 nm (ε280nm =43 800 M−1cm−1), as a function of increasing nanoparticleconcentration, in the range 200−1800 mg L−1, in 50 mMtetramethylammonium perchlorate, pH 7.0. Experimental datashowed the presence of 6 ± 2 BSA molecules per nanoparticle,corresponding to 3 ± 1 × 10−2 mg of BSA per milligram ofnanoparticles, in agreement with previous work on avidinbinding.3 BSA molecules, in water, have a prolate ellipsoidalshape, with the axes being 11.6 and 2.7 nm.23 As a comparison,the possible theoretical number of BSA molecules on SAMNsurface, considering proteins positioned on the SAMN surfacealong their 11.6 nm axis, gives 10 BSA molecules pernanoparticle.The Fourier transform infrared (FTIR) spectra of SAMN@

BSA show the presence of typical protein IR bands, attributableto vibrational frequencies of C−C, C−O, and CH2− groups, inthe 1000−1700 cm−1 range, confirming the presence of BSAbound on nanoparticle surface (see Figure 1). In particular, thecharacteristic protein amide I and amide II IR bands at 1654and 1544 cm−124 are present in the SAMN@BSA FTIRspectrum. SAMN@BSA was also studied by transmissionelectron microscopy (TEM) and FTIR. TEM microscopyimages of the SAMN@BSA complex, reported in Figure 2,indicate the presence of an organic matrix shell, of about 3 nmthickness around the iron oxide nanoparticles, characterized bya lower electron density. The aggregation phenomenon showedby the coated nanoparticles is probably due to TEM samplepretreatment and drying on the copper grid. In watersuspensions SAMN@BSA still maintain their stable colloidalbehavior. The shell thickness is in good agreement with BSAshort axis (2.7 nm),23 suggesting that the protein adheres toSAMN surface by laying on its long axis. Considering that BSA

long axis is 11.6 nm, thus, protein should bend to adapt itself tonanoparticle curvature, and as a consequence causing analteration of its tertiary structure. In fact, it is well-known byliterature that the interactions between proteins and solidsurfaces could lead to significant changes in proteinstructure,25,26 possibly altering protein functionality andbiological activity. Conformational changes on BSA inducedby SAMN binding were detected using circular dichroismspectroscopy (CD). CD spectra of BSA and SAMN@BSA arereported in Figure 3. CD spectrum of BSA resulted consistentwith a previously published measurements,27 while thespectrum of BSA adsorbed onto SAMN surface was markedlyaltered, confirming the interaction occurrence. Native BSAclearly shows a sharp absorption at 190 nm and two negativeabsorption bands: one is attributed to carbonyl excitation inpolypeptide chains due to π−π* transition at 209 nm, andanother one at 222 nm corresponding to an n−π* transition.28

The characteristic positive sharp peak at 190 nm and negativedouble humped peaks suggest a high proportion of α-helices.The comparison of the CD spectra of native BSA and SAMN@BSA indicates a degree of attenuation in the α-helical peakssimilar to those observed upon binding on silica coatednanoparticles27 confirming a substantial alteration in α-helical

Figure 1. FTIR spectrum of SAMN@BSA complex. Nanoparticles samples were lyophilized, homogenized with KBr powder, and pelleted via an 8.0ton hydraulic press. (black line) SAMN; (red line) BSA; (blue line) SAMN@BSA.

Figure 2. TEM image of maghemite nanoparticles coated with BSA.

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structure of the protein in direct contact with surface groups onthe magnetic particles.Differently from the case of avidin binding, occurring in short

times (1 h) and without protein structure alterations,3 thebinding of BSA to SAMNs was time-consuming (at least 12 h),leading to dramatic changes in the protein tertiary structure. Apossible explanation can be due to the presence of properly

positioned anchor groups on avidin for a multiple point bindingon SAMN surface, while BSA needs to adapt itself for asuccessful absorption. This second process reasonably takesmore time and causes dramatic structure modifications.

3.2. Mass Spectrometry Analysis of Protein Bound toSAMNs. It is well-known that iron oxide nanoparticles are ableto bind plasma proteins and that proteins tend to coat the

Figure 3. Far UV circular dichroism spectra of BSA free and immobilized on SAMN surface. BSA samples (0.25 mg mL−1) were in 10 mMpotassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA. CD spectra were obtained using a 0.2 cm path length cell, and the data are theaverage of 16 scans. (black line) BSA; (red line) SAMN@BSA.

Table 1. Serum Protein Bound to Bare SAMNs and SAMN@RITCa

SAMN score SAMN@RITC Score Total removed proteins (NH4OH) gel band

TSP1 thrombospondin-1 1280 TSP1 thrombospondin-1 1088 yes 3TETN tetranectin 1185 TETN tetranectin 916 yes 11FETUA α-2-HS-glycoprotein 841 FETUA α-2-HS-glycoprotein 1081 yes 7, 8KNG1 kininogen-1 188 KNG1 kininogen-1 590 yesAPO-B100 apolipoprotein-B100 1335 APO-B100 apolipoprotein-B100 1335 no 1APOA1 apolipoprotein-A1 617 APOA1 apolipoprotein-A1 1116 yes 9APO-E apolipoprotein E 430 APO-E apolipoprotein E 550 yes

APOA2 apolipoprotein-A2 285 yes 13PPIB peptidyl-prolyl isomerase B 381 PPIB peptidyl-prolyl isomerase B 381 no 10GPX3 glutathione peroxidase 3 384 GPX3 glutathione peroxidase 3 384 no 10CO3 complement C3 658 CO3 complement C3 201 yes 4CFAB complement factor B 294 CFAB complement factor B 672 no 5

CFAH complement factor H 334 yes 4CO9 complement C9 365 yes 6HBBF fetal hemoglobin sub. β 920 HBBF fetal hemoglobin sub. β 1694 yes 12HBA hemoglobin sub. α 274 HBA hemoglobin sub. α 1031 yes 12A1AT α-1-antiproteinase 571 A1AT α-1-antiproteinase 722 yes 7SPP24 secreted phospho-prot.24 545 SPP24 secreted phospho-prot.24 457 yes 11

A2MG α-2-macroglobulin 458 yesMYH9 myosin-9 1093 MYH9 myosin-9 1250 yes 2ALBU serum albumin 755 ALBU serum albumin 546 yes 6HSPB1 heat shock protein beta-1 536 HSPB1 heat shock protein beta-1 536 no 10

aSAMN and SAMN@RITC (2 mg) were incubated for 1 h in DMEM medium (40 mL), supplemented with 10% FBS. Then nanoparticles werewashes four times, under stirring, for 15 minutes in PBS buffer, in order to remove loosely bound proteins. Proteins bound to SAMN surface wereidentified by mass spectrometry (LC/MS/MS) of trypsin digest of (i) total proteins removed by 0.5 M ammonium hydroxide (total removedproteins), and (ii) most intense bands obtained by SDS-PAGE (see figure 5) after SAMN extraction by TRIS/SDS buffer (gel band). Theidentification was carried out by MASCOT software against the SwissProt database, setting, as a minimum condition, the identification of at leastthree unique peptides and a score above 250.

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nanoparticle surface upon their entrance into biologicalenvironments.29,30 Moreover, the function and fate of nano-particles in biological systems are not only related to theproperties of nanoparticle surface, but also to boundproteins.31,32 Actually, living cells recognize the proteinmodified surface of nanoparticles.Generally, bare or functionalized iron oxide nanoparticles

reported in literature are able to absorb serum proteinsexclusively by electrostatic interactions.33 Conversely, bareSAMNs bind molecules by complexation to undercoordinatediron(III) sites or, in the case of SAMN@RITC, by nucleophilicaddition to the isothiocynate moiety. As a consequence, nosignificant contribution of electrostatic interactions should beexpected in the protein binding to SAMNs or SAMN@RITC.In the present case, bare SAMNs and SAMN@RITC were

introduced in cell culture medium containing 10% fetal bovineserum (FBS) and the amount and nature of absorbed proteinson nanoparticle surface was determined by gel electrophoresisand mass spectrometry. Considering that temperature couldinfluence the formation of a protein corona on nanoparticlesurface when incubated in cell culture medium,34 in the presentcase serum protein binding was studied at 37 °C.Briefly, after nanoparticle (SAMNs or SAMN@RITC)

incubation in 10% FBS, followed by several washes in PBS(see Methods in the Supporting Information), proteinsassociated with nanoparticle surface were released byincubation in 0.5 M ammonium hydroxide, which was alreadytested for the removal of bound molecules from SAMNsurface,21 or 0.15 M TRIS buffer, pH 8.5, containing 2% SDS.The quantification of removed proteins was carried out by a

spectrophotometric assay (BCA assay kit by Thermo FisherScientific Inc., Rockford, IL). Bound proteins resulted 42.4 and82.8 mg g−1 nanoparticles in the case of bare SAMNs andSAMN@RITC, respectively, in agreement with previousstudies on avidin binding on SAMNs.3

Protein samples removed by ammonium hydroxide weredried at reduced pressure, resuspended in 40 mM ammoniumbicarbonate, containing of 10% acetonitrile, and were digestedovernight at 37 °C by trypsin treatment. The resulting peptideswere analyzed by LC/MS/MS to identify associated proteins,and results are reported in Table 1 (as total removed protein).Alternatively, the dried extracts were resupended in TRIS/SDSbuffer and analyzed by gel electrophoresis, along with samplesobtained from protein removal by TRIS/SDS treatment. Mostintense electrophoretic bands (see Figure 4) were excised anddigested by trypsin, and proteins were identified by massspectrometry as above (see “gel band” column in Table 1).Notwithstanding SAMNs and SAMN@RITC have differentsurface chemistries, electrophoretic profiles of proteinsremoved from both these nanoparticles are quite similar(Figure 4). Indeed, as reported in Table 1, among the 22identified plasma proteins, only 4 were not detected in bothSAMNs and SAMN@RITC, and none of these proteinsrepresents a major serum component. This suggests that thepresence of available anchoring groups on protein structure isnecessary but not sufficient for effective binding. Proteinsshould present a proper conformation in order to guarantee anefficient multiple point binding on SAMNs surface, and, asconsequence, protein tertiary structure should play a main rolein discriminating protein binding efficiency. In addition, thesmall number of protein associated with SAMNs (only 22proteins, among about 3000 identified in plasma, as reported byOmenn et al.35) indicates that protein binding to nanoparticle

surface is selective. Bound proteins (Table 1) can be dividedinto six main groups: histidine-rich glycoproteins (thrombo-spondin, fetuin-A, tetranectin, and kininogen); variousapolipoproteins and associated proteins (apo-B100, Apo A1,Apo A2, Apo-E PPIB, and GPX3); proteins belonging to thecomplement system (C3, C9, and factors B and H);hemoglobin subunits α and β; protease inhibitors (α1-antiproteinase, SPP24, and α2-macroglobulin) and the notfunctionally related BSA, myosin-9, and HSPB1. The mostabundant protein, evidenced by intensity in the electrophoreticprofile, was α-2-HS-glycoprotein, followed by hemoglobinsubunits, thrombospondin, myosin-9 and Apo-B100. Therelative protein abundance in FBS does not seem to play amajor role in the proteins association with SAMNs. In fact,while α-2-HS-glycoprotein and Apo-B100 are present in FBS inthe milligrams per milliliter range (≈15 and 1 mg mL−1,respectively), thrombospondin, myosin-9, and hemoglobinsubunits are at the micrograms per milliliter level (between 5and 40 μg mL−1). Moreover, BSA, which is the most abundantFBS protein (≈25 mg mL−1), resulted to poorly bind onSAMNs (see band 6 in Figure 4) and transferrin, the thirdplasma protein for abundance (2−3 mg mL−1), wasundetectable. These results recall the comparison betweenavidin and BSA binding on SAMNs: the first one tends to bindefficiently, in short times, possibly because it does not need toalter its tertiary structure for stable multiple point binding onSAMN surface. Differently, BSA binding requires long times,probably because the protein must modify its structure to adapt

Figure 4. Electrophoretic profile of proteins bound to SAMN andSAMN@RITC. Proteins bound to SAMNs, bare or rhodamine coated,were released by 0.25 M TRIS/2% SDS buffer (lanes A and C) or 0.5M NH4OH (lanes B and D). SDS-PAGE was performed with aNOVEX 4−12% gel (Invitrogen) in MOPS buffer. The most intensebands were analyzed by LC/MS/MS after tryptic digestion. Bandidentification, corresponding to the numbering reported on theelectrophoretic profile, is reported in Table 1.

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to nanoparticle surface. As in the case avidin, the 22 adsorbedmedium proteins coat efficiently on SAMNs and SAMN@RITC surface in a short time, despite the competition withother 3000 kinds of proteins, some of them (BSA) highlyconcentrated. It is noteworthy that Mahmoudi et al. reportedon transferrin interaction with magnetite SPIONs, involvingirreversible denaturation,26 working in a system devoid ofcompetition with other plasma proteins.The comparison of lanes A vs B and C vs D in Figure 4

indicates that TRIS/SDS treatment induced a more efficientprotein removal from SAMN surface than ammoniumhydroxide. Data reported in Table 1 shows that SAMNdissociation of Apo-B100, PPIB, GPX3, CFAB, and HSPB1occurred only in SDS containing buffer. SDS is a powerfulprotein denaturing agent, whose interactions with polypeptideslead to the complete loss of tertiary structures. Thus, it isconceivable that high affinity interactions with SAMNs dependon specific tertiary structure of the polypeptide amino acidsequence. Clearly, our observations suggest the coexistence ofseveral types of interactions between proteins and SAMNs,attributable to multiple point binding,21 and deserving in-depthinvestigations. On the other hand, a correlation betweenprotein tertiary structure and affinity for the nanoparticlesurface is pointed out. In the present case, a shell of biologicallyrecognizable proteins was formed on the nanoparticle surface,suggesting that, as a consequence of the spatially governedmultiple point binding, the best absorbable proteins are thosepresenting correctly positioned anchoring groups and which donot need to adapt their structure to nanoparticles surfaces.3.3. Bioactivity of Bare SAMN and SAMN@RITC. With

the aim to develop further uses of SAMNs and SAMN@RITCcomplexes for biomedical applications, the interaction, internal-ization processes and cytotoxicity of both the preparations wereevaluated on HeLa cell cultures. Hela cells were incubated withbare and RITC coated SAMNs (30 μg mL−1) and analyzed atdifferent times by confocal fluorescence microscopy, whichallows the accurate signal selection from specific focal planesand, consequently, the precise fluorescence localization withinthe cells. The serial acquisition of different planes along the Zaxis permitted the reconstruction of three-dimensionaldistribution of the fluorescence signal. In the present study,in addition to the red fluorescence signal of RITC associatedwith SAMN (excitation at 542 nm, emission at 590−620 nm),the signal of acetomethoxy derivative of calcein (calcein AM)was simultaneously recorded (excitation at 480 nm, emission at515−530 nm). In fact, HeLa cells were incubated with calceinmethyl ester few minutes before the observations. Thisderivative of calcein was used due to its ability to freelypermeates cell membranes, and therefore to spread in allcellular compartments, where, as a result of esterase hydrolysis,becomes fluorescent. Calcein is an excellent marker of cellvolume and is useful to precisely define the cell membraneboundary.36 Thus, the colocalization with calcein allows theprecise identification of nanoparticles position within the cells.Furthermore, acidic cell compartments, such as lysosomes, arehighlighted by negative staining since the fluorescence ofcalcein is pH-sensitive, disappearing at pH below 5.0.SAMN@RITC internalization in HeLa cells, following 24 h

incubation, is shown in Figure 5 A. Both fluorescent signals(SAMN@RITC and calcein) are present (see panel 1),indicating that SAMN@RITC are internalized into HeLacells, where they appear with various degrees of aggregation(panels 1 and 3). Most of SAMN associated fluorescence

results to colocalize with green signal of calcein (see arrows inpanels 1, 2 and 3), suggesting a possible compartmentalizationin nonacidic cellular structures, like pino/endocytotic vacuoles.This inference is supported by the high concentration ofSAMN@RITC observed in acidic vacuolar (lysosomal)structures, as shown in reported images (letters L panels 1and 2), evidencing a strong activity of the vacuolar trafficking,as expected in the case of endocytotic uptake. In the sameFigure 5A, a large SAMN@RITC aggregate, located in a plasmamembrane invagination outside the cell (indicated by the letterI in panels 1 and 2), can be noted. This observation clearlyindicates that large aggregates cannot enter into cells, but thatcan induce modifications of plasma membrane topology. The

Figure 5. (A) SAMN@RITC localization within HeLa cells byconfocal microscopy. HeLa cells were incubated in the presence of 25μg mL−1 SAMN@RITC for 24 h and treated with 1 μM calceinacetoxymethyl ester, 15 min before the observation by 3D confocalfluorescence microscopy. Thirty scans on Z axis (250 nm depth) wereacquired at 520 nm (calcein AM) and 610 nm (rhodamine). XZ andYZ planes represent the projection on Z axis of vertical sectionsindicated by the two orthogonal lines on the XY plane. Panel 1:acquisition of signals at 520 and 610 nm. Arrows indicate examples ofcolocalization of SAMN and Calcein AM; L (lysosome) indicates anacidic vacuolar area in which the signal at 610 nm is present but at 520nm is absent; I (invagination) indicates an area, external with respectto the cell membrane, in which the signal at 610 is present. Panel 2:Imagine acquired at 520 nm. Panel 3: imagine acquired at 610 nm. (B)Internalization kinetics of SAMN@RITC in HeLa cells. HeLa cellswere incubated in the presence of 25 μg mL−1 SAMN@RITC andtreated with 1 μM calcein AM, 15 min before the observation byconfocal fluorescence microscopy, acquiring fluorescence signal at 520nm (calcein AM) and 610 nm (rhodamine). Top panels (1): signal at610 nm after 0, 2, 7, and 24 h incubation. Bottom panels (2):simultaneous signal acquisition at 520 and 610 nm after 0, 2, 7, and 24h incubation.

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internalization process results continuous with time, as seen inFigure 5B, where SAMN@RITC are already detectable aftertwo hours incubation and gradually accumulate during thefollowing 22 h. The progressive accumulation of SAMNs withinthe cells, leading to the formation of macro-aggregates, can benoted. These aggregates are likely located in the lysosomalcompartment, as shown in Figure 5A.Qualitative observations obtained by confocal microscopy

were accompanied by quantitative analysis by flow cytometry.Hela cells (150,000 cell/12 wells microplate) were incubatedfor different times (up to 48 h) with increasing concentrationsof SAMN@RITC (0 - 200 μg mL−1), and the associatedfluorescence was analyzed. The analysis confirms theinteraction of cells with SAMN@RITC: the intensity ofRITC fluorescence is proportional to nanoparticle concen-tration and incubation times (see Figure S2 in the SupportingInformation). The kinetics of SAMN@RITC fluorescenceincorporation by HeLa cells showed a saturation behavior,suggesting that the phenomenon was not due to simplediffusion. However, fluorescence analysis cannot discriminatethe contribution of SAMNs simply adsorbed on the outer cellmembrane surface or internalized within cell cytoplasm. Forthis aim experiments at different temperatures were carried out.The entrance of nanoparticle within the cells is usually

mediated by endocytosis, a set of energy-dependent processes,which can be blocked at low temperatures or by ATP depletion.Therefore, a series of experiments, in which HeLa cells wereincubated for 1 h with increasing SAMN@RITC concen-trations (0 − 200 μg mL−1) were carried out at 4 °C,temperature at which the almost complete arrest of endocytosisoccurs.37 Control experiments were carried out at 37 °C. Asdepicted in Figure S3, a negligible increase of incorporatedSAMN@RITC fluorescence was detected at low temperature,while, at 37 °C, the increase was significant and dose-dependent.We cannot a priori exclude the release of RITC from SAMN

surface, and it is known that rhodamine accumulates in amembrane potential manner inside the mitochondria.38 Inorder to exclude this possibility, cell samples were incubated for30 min with 1 μM carbonyl cyanide m-chlorophenylhydrazone(CCCP), a proton ionophore, which is able to uncouple themitochondrial membrane potential. No differences in terms ofSAMN@RITC fluorescence between HeLa cells populationsincubated with or without the uncoupling agent, both at 4 and37 °C, were observed, indicating that fluorescence inside Helacells depended only on energy dependent SAMN@RITCincorporation and not on mitochondrial potential, ruling outthe presence of free RITC (see Figure S4).Unfortunately, fluorescence cannot be used for the

evaluation of the cellular uptake of unlabeled (bare) nano-particles. For this purpose, an alternative method, consisting inthe determination of nanoparticle effect on cellular lightscattering properties, was utilized.39 In the case of cellularuptake, the intensity of side scattered light (which providesinformation on internal structure) increases with theconcentration of nanoparticles inside the cells.Figure 6A shows biparametric graphs reporting the

morphological cytometric parameters related to granulosity(side scattering) and cell volume (forward scattering) in HeLacells, as function of temperature and bare SAMN concentration.At 4 °C, no appreciable differences of forward and sidescattering signals as a function of nanoparticle concentration,were observed, indicating that nanoparticles were not

internalized. Conversely, the incubation at 37 °C led to adramatic increase of side scattering as a function of SAMNconcentration, while no relevant modification of forwardscattering was detected. At the highest tested concentration(200 μg mL−1) a 600% increase of side scattering was observedat 37 °C, compared to control, while at 4 °C only a 6% increasewas determined (see Figure 6B).Control experiments were carried out with fluorescent

SAMN@RITC, evidencing that cell morphological changes,in terms of side and forward scattering, were superimposablewith those observed with bare SAMNs (data not shown).These results clearly show that bare and rhodamine coatedSAMNs share the same energy dependent mechanism ofcellular uptake, and exclude any role of rhodamine in theinternalization process.It was documented that the presence of nanoparticles within

cells can induce a detectable damage in terms of reduction ofmetabolic activity.40 In order to assess if SAMNs are able toproduce the same phenomenon, the resazurin reduction assaywas carried out on SAMN treated HeLa cells. Resazurin is avital nonfluorescent dye, which is reduced to the fluorescentresorufin by cellular oxido-reductases, active only in livingcells.41 Since this assay constitutes a direct measurement of cellmetabolic state, well correlating with living cell density, it canbe used for an indirect evaluation of cell viability. Indeed, in our

Figure 6. Biparametric analysis of bare SAMN uptake by HeLa cells.(A) Cells were incubated in the presence of increasing concentrationsof bare SAMN at 4 and 37 °C. After 1 h incubation, cells wereanalyzed by forward scatter (FSC) and side scatter (SSC). (B) Datasummarized with respect to controls (0 μg mL−1 SAMN). Results arethe means of three experiments carried out in triplicate ± SD.

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experimental conditions, resazurin reduction rate was foundproportional to cell density in a wide range of cellular density(5000−80 000 cells/well). Preliminary experiments on theinfluence of nanoparticles on the resazurin assay were carriedout in culture medium ±10% FBS, without cells or with cellularlysates, and the results altogether excluded interferences in theassay (see Figure S5 in the Supporting Information). The effectof increasing concentrations of bare and rhodamine coatedSAMNs (0 - 250 μg mL−1) on Hela metabolic activity wastherefore assayed. After 24 h treatment, a significant decrease inresazurine reduction rate (metabolic activity) appeared atnanoparticle concentrations above 100 μg mL−1, being morepronounced with SAMN@RITC (see Figure S5A in theSupporting Information), but no differences were observedbelow this concentration, indicating negligible nanotoxicolog-ical effect of SAMNs.32 After 48 h incubation, the most evidentcell damage was found with bare nanoparticles, while cellstreated with SAMN@RITC tended to recover the resazurinreduction rate with the prolonging of incubation time (seeFigure S5B in the Supporting Information). This differentbehavior may derive from the different nature of the insult

produced by the two types of nanoparticles, observed only atthe highest concentrations. Probably an acute cell damage isexerted by bare SAMNs, while a chronic damage was evidentwith SAMN@RITC. Finally, the high variability of cellmetabolic state, observed at SAMN concentration of 50 μgmL−1, can be interpreted as the threshold SAMN concentrationat which HeLa cells are unaffected (see Figure S5B in theSupporting Information).Further information about SAMN effect on HeLa cells was

obtained from the determination of the activity of lactatedehydrogenase (LDH), a cytoplasmic enzyme, in the culturemedium, due to cellular death and lysis. LDH concentration inthe culture medium represents a good indication of cellmortality by necrosis.42 Experiments were performed byexposing cells to increasing concentrations of bare and RITCcoated SAMNs (0−250 μg mL−1). The LDH release in theculture medium and LDH amount still present within the cells,were determined at 12, 24, and 48 h. No toxic effects on HeLacells were observed at SAMN concentrations below 100 μgmL−1, both for SAMNs and SAMN@RITC. SAMNs induced adose dependent LDH release, which was 21.6% vs control at

Figure 7. Effect of bare and rhodamine coated SAMNs on HeLa cell cycle. Cells were incubated in the presence of increasing concentrations ofSAMNs and SAMN@RITC for 24 and 48 h and stained with propidium iodide for cytofluotimetric analysis of the DNA content. Experiments werecarried out in triplicate ± SD. In the lower panel, it is reported, for example, the analys of the DNA content concerning Hela cells incubated withbare SAMN. Cells in the G0/G1 step have half the DNA of a G2/M phase cell while the DNA content is in between these two during the synthesisphase.

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the highest concentration (250 μg/mL), at 12 h incubation (seeFigure S6A in the Supporting Information). At longerincubation times, the behavior was confirmed: SAMN toxicitywas evident only at the highest concentration (250 μg mL−1),independently of the presence of the rhodamine coating (seeFigure S6B in the Supporting Information). Surprisingly, cellmortality, measurable only at 250 μg mL−1 SAMN, was reducedat 48 h incubation. This apparent mortality reduction wasprobably due to cell replication, which for HeLa cells isapproximately 24 h, leading to nanoparticle dilution withincells. Overall, results on cell toxicity rated by LDH releaseconfirmed the data obtained by the determination of resazurinreduction.Nanoparticles, in particular those based on iron oxides, can

cause cell damage by induction of oxidative stress.43 Theproduction of reactive oxygen species (ROS) may occursdirectly on nanoparticle surface, by spontaneous release of ironions or by enzymatic nanoparticle degradation withinlysosomes.44 Moreover, an increase of ROS production (andof reactive nitrogen species) may derive from the uncontrolledactivation of the anti-inflammatory cell response.44 Since ROSproduction represents an early event, preceding cell damageand death, short incubation times (90 and 180 min) in thepresence of SAMNs and SAMN@RITC were chosen to tracktheir possible toxic effects. ROS production by HeLa cells wasdetermined by a specific probe, namely 6-carboxy-2,7′-dichlorodihydrofluorescein diacetate (DCFHDA), and flowcytometric analysis.45 We found that bare SAMNs did notstimulate ROS production, at least in the explored concen-tration range (0−100 μg mL−1). Conversely, a significant ROSproduction was observed at the highest concentration ofSAMN@RITC (100 μg mL−1) after 180 min incubation (seeFigure S7 in Supporting Information). It is well-known thatcationic rhodamine derivatives can induce mitochondrialdamage, both in vivo and in vitro.46 Even if free rhodaminewas undetectable within HeLa cells treated with SAMN@RITCunder our experimental conditions, we cannot exclude that tinyamount of rhodamine could be released from SAMN surface,interfering with mitochondria metabolism and causing atransient increase of ROS.Proper cell function requires the maintenance of mitochon-

drial membrane potential, supported by the electron transportchain. Oxidative stress and/or mitochondrial dysfunction areconsidered to be causes of cellular damage and cell death byapoptosis or necrosis. The mitochondrial membrane potentialcan be assessed in viable cells by flow cytometry and specificfluorescent probes. In our experiments, tetramethyl-rhodamine(TMR), a lipophilic fluorescent cationic probe, accumulatingwithin the mitochondria as a function of membrane potential,was used. In this case, SAMN@RITC were not tested, due tothe superimposition of fluorescent emission by RITC andtetramethyl-rhodamine. Thus, experiments were performedonly with bare SAMNs. Incubation of HeLa cells with SAMNsup to 100 μg mL−1, for 90 and 180 min, indicated no effect ofnanoparticles on the mitochondrial potential, further demon-strating the safety of SAMNs (see Figure S8 in the SupportingInformation).Moreover, it was reported that nanoparticles, depending on

their composition, surface coating, size, and so forth, mightinterfere with the regulation of cell cycle.32 Cell cycle is a highlycontrolled process, and in each phase consensus checkpointsare present to ensure that the cell is ready to address the crucialstep of cell division. The duration of the process normally lasts

from 12 h up to a few days, depending on the cell type. HeLacells present a cell cycle of about 30 h.47 Generally, in anonsynchronized cell culture, about 65% cell population is inthe G0/G1 phase, 15% in the S phase, and 20% in G2/M phase.Flow cytometry was used to study the effects of bare SAMNsand SAMN@RITC (0−250 μg mL−1) on cell cycle progressionin HeLa cells, for 12, 24, and 48 h. Results indicated that therewere no significant changes in the distribution of the cellpopulations among the various cell cycle phases due to SAMNtreatment (see, as an example, Figure 7A). Only at the highestconcentration (250 μg mL−1), with both SAMNs and SAMN@RITC, a biologically negligible reduction in the number G2/Mcells, with the concomitant increase of S phase cells, wasobserved. As can be seen from Figure 7B, cell incubation withSAMN and SAMN@RITC led to a change in the shape (an“enlargement”) of the cytofluorimetric peaks, corresponding tothe cells in G1 and G2 phases. It can be supposed that theobserved peak shape modification was due to scatteringphenomena, caused by the presence of nanoparticles insidethe cells.

4. DISCUSSIONAs described in our previous works, both SAMNs and SAMN@RITC show an efficient binding capability toward proteins andenzymes and, moreover, the surfaces of these nanoparticlesexpose friendly environments for protein immobilization, astested molecules preserved their biological activity afterabsorption.3,21,22 Generally, bare or functionalized iron oxidenanoparticles reported in literature are able to absorb serumproteins exclusively by electrostatic interactions.33 Conversely,bare SAMNs bind molecules by complexation on under-coordinated iron(III) sites and SAMN@RITC by nucleophilicaddition to the isothiocynate moiety. As a consequence, nosignificant contribution of electrostatic forces in the opsoniza-tion of SAMNs and SAMN@RITC are expected.Albumin (BSA) is the predominant protein in mammalian

blood serum and is one of the most adsorbed proteins on ironoxides nanoparticles via electrostatic interactions.33 BareSAMNS and SAMN@RITC were not able to significantlybind albumin in a complex system, such as fetal bovine serum.Albumin binding on SAMN surface is possible only followingincubation in pure BSA solutions under controlled conditions,as demonstrated in the present work, for a long incubation timeand in the absence of competitors. The need of a prolongedincubation time may be attributable to low collision efficiency,most likely due to sparse suitable binding sites on nanoparticlessurface. Nevertheless, when set in the proper orientation, BSAeventually binds on SAMN surface leading to a monomolecularlayer. In this case bovine serum albumin effectively interactswith nanoparticle surface forming a stable monomolecularlayer, comprising 6 ± 2 molecules per nanoparticle.Unfortunately, BSA seemed to be denatured by this prolongedincubation, as evidenced by circular dichroism. It should beremembered that other proteins, which effectively bind onSAMNs within few tens of minutes, are not subjected todramatic alteration of their tridimensional structure uponimmobilization, as demonstrated by the maintenance of theirbiological activity.3,21,22

Within fetal bovine serum (containing 80% BSA), albumincompetes with many other proteins for SAMNs and SAMN@RITC surface binding on iron(III) undercoordinated sites andisothiocynates moieties, respectively, as suggested by Thieleand coauthors on other nanoparticles.48 Interestingly, the two

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different surface chemistries led to the formation of the sameprotein shell, mainly constituted by different proteins, such asAPO-B100, fetal hemoglobin α and β subunits, trombospondin-1 and, among which α-2-HS-glycoprotein stands out.The presentation of surface bound ligands may be used to

target nanoparticles to cells or tissues, and promotingnanoparticles uptake by cells.49 The present study on Helacells evidenced that the proposed nanoparticles (SAMNs andSAMN@RITC) show the same behavior in terms of internal-ization efficiency and cytotoxicity, suggesting that they aresimilarly recognized by cells, indeed they resulted coatedpredominantly by the same proteins.Among the different proteins involved in the binding with

nanoparticle surface, as evidenced by gel electrophoresis andmass spectrometry results, α-2-HS-glycoprotein was the mostrepresented and APO-B100 the most tightly bound. Theseproteins could play an important role in nanoparticleinternalization in cells,50 as already evidenced by Thiele andcoauthors.48 They also reported that serum protein binding onnanoparticle surface (opsonization) is lost after 2 h incubation.In the present case, SAMNs and SAMN@RITC coated withserum proteins, mainly with α-2-HS-glycoprotein, can beinternalized even after 24 h incubation, confirming a correlationbetween binding efficiency and stability of protein-nanoparticlecomplexes. Only proteins presenting correctly positionedanchoring groups on their surface can quickly and efficientlyadsorb on SAMNs and SAMN@RITC. For these kinds ofproteins, the nanoparticle surface represents a friendlyenvironment, leading to a stable protein shell, easilyrecognizable by cells for a long time. Moreover, conversely torecent findings,51,52 in which the spontaneous formation of aprotein corona led to a loss of targeting capabilities ofnanoparticles, SAMNs and SAMN@RITC are available forprojecting a synthetic, functional, and stable protein corona,thus avoiding further uncontrolled serum protein binding. Thisapproach can be exploited for the preparation of properlytargeted magnetic nanoparticles for different cell lines,overcoming the role of culture media on spontaneous proteinabsorption on nanoparticle surface, as evidenced by Laurentand co-workers,53 and allowing the selective recognition bydifferent cell lines. This cell recognition property was recentlydefined as “cell vision”,54 emphasizing that what the cell “sees”,when it is faced with nanoparticles, is most likely dependent onthe cell type.The present report evidences the importance of studying

protein adsorption on nanoparticle surfaces in complex proteinmixtures for further biomedical applications. In the presentcase, protein absorption experiments, on two maghemitenanoparticle formulations, carried out in fetal fetal bovineserum led to the preparation of iron oxide nanoparticlescharacterized by a suitable and stable protein coating for cellinternalization. Moreover, this study allows one to gatherinformative data on SAMN internalization and cytotoxicity andgives important indications for future preparations of selectivelyopsonized nanoparticles. SAMN based nano-biocomposites,due to their stability and ability to stimulate cell uptake, arepossible future candidates for a wide range of biomedicalapplications, from drug delivery, cancer therapy, andtheranostics.

5. CONCLUSIONSIn the present case, our nanoparticles form stable colloidalsuspensions without any preliminary polymeric coating and,

when introduced in cell culture medium, spontaneously bindcovalently to a small group of specific proteins, producing aself-assembled corona, representing a unique example ofmagnetic nanoparticle opsonization via covalent binding.Thus, the prolonged nanoparticle uptake indicates a correlationbetween internalization and stability of covalently boundprotein corona. We suggest that SAMN based nanobiocompo-sites can be employed for the preparation of self-assembledopsonized nanoparticles as future candidates for diagnostic andtherapeutic applications in biomedicine.

■ ASSOCIATED CONTENT*S Supporting InformationIntrumentation and methods, comprising synthesis of ironoxide nanoparticles (SAMN), preparation of rhodamine boundnanoparticles (SAMN@RITC), immobilization of bovineserum albumin on SAMNs, opsonization of SAMNs by fetalbovine serum (FBS), detachment of FBS proteins bound toSAMNs, SDS-PAGE electrophoresis, in gel protein digestion,protein identification by mass spectrometry, cell culture,confocal microscopy imaging, cytofluorimetric analysis, deter-mination of lactate dehydrogenase activity, resazurin fluori-metric assay, cytofluorimetric analysis of mitochondrial activityand reactive oxygen species production (ROS) and cell cycleanalysis. Figures regarding the optical spectrum of SAMNsuspension, the SAMN interaction with HeLa cells determinedby flow cytometry, SAMN@RITC uptake by HeLa cellsdetermined by flow cytometric analysis of fluorescence,SAMN@RITC uptake by HeLa cells determined by flowcytometry analysis of fluorescence, metabolic activity of HeLacells following SAMN treatment, HeLa cell viability determinedby LDH activity measurements in the culture medium, reactiveoxygen species production by bare and RITC coated SAMNson HeLa cells, and effect of SAMNs on mitochondrial potentialof HeLa cells. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 0039-049-8276863. E-mail: fabio.vianello@unipd.it.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe present experimental work was partially funded by anItalian Ministry grant (60A06-0587/12). The authors gratefullyacknowledge the support by the Operational Program Researchand Development for Innovations - European RegionalDevelopment Fund (project CZ.1.05/2.1.00/03.0058) and bythe Operational Program Education for Competitiveness -European Social Fund (project CZ.1.07/2.3.00/20.0155) of theMinistry of Education, Youth and Sports of the Czech Republic.The Foundation for Advanced Biomedical Research and VIMMare grateful to the “Veneto Banca” Holding for funding theacquisition of the MALDI-TOF/TOF mass spectrometer. TheUniversity of Padova is grateful to the “Cassa di Risparmio diPadova e Rovigo” Holding for funding the acquisition of theLTQ-Orbitrap XL mass spectrometer.

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